Subsequent enzymatic galactosylation and sialylation towards sialylated Thomsen-Friedenreich antigen components

Article abstract of DOI:10.1002/adsc.200606063

Sialyloligosaccharides of the type Neu5 Acα2-3Galβ1-3GalNAc and a range of corresponding motifs play an important role in nature and are found in gangliosides, Lewis type I structures and the sialyl-Thomsen-Friedenreich antigen occurring in higher animals, viruses, bacteria, protozoa and pathogenic fungi. There is considerable interest to evaluate the significant functionalities of these glycostructures, and here we present a chemoenzymatic approach by a facile synthesis of such motifs. Employing chemoenzymatic methods, several modified Galβ1-3GalNAc derivatives were synthesized. Modifications were introduced at the stage of the monomeric building blocks prior to formation of the disaccharides by means of four different β-galactosidases from bovine testes, Bacillus circulans and Xanthomonas manihotis as well as the phosphorylase from Bifidobacterium bifidum. Finally, the modified disaccharide derivatives could be efficiently sialylated using the recombinant trans-sialidase from Trypanosoma cruzi.

Full text of DOI:10.1002/adsc.200606063

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DOI: 10.1002/adsc.200606063
Subsequent Enzymatic Galactosylation and Sialylation Towards
Sialylated Thomsen–Friedenreich Antigen Components
Lars Krçger,a Agnes Scudlo,a and Joachim Thiema,*
a
Institut of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
Fax : (+49)-40-42838-4325; e-mail: thiem@chemie.uni-hamburg.de
Received: February 20, 2006; Accepted: May 11, 2006
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: Sialyloligosaccharides of the type fications were introduced at the stage of the mono-
Neu5Aca2-3Galb1-3GalNAc and a range of corre- meric building blocks prior to formation of the disac-
sponding motifs play an important role in nature and charides by means of four different b-galactosidases
are found in gangliosides, Lewis type I structures and from bovine testes, Bacillus circulans and Xanthomo-
the sialyl-Thomsen–Friedenreich antigen occurring nas manihotis as well as the phosphorylase from Bifi-
in higher animals, viruses, bacteria, protozoa and dobacterium bifidum. Finally, the modified disacchar-
pathogenic fungi. There is considerable interest to ide derivatives could be efficiently sialylated using
evaluate the significant functionalities of these glyco- the recombinant trans-sialidase from Trypanosoma
structures, and here we present a chemoenzymatic cruzi.
approach by a facile synthesis of such motifs. Em-
ploying chemoenzymatic methods, several modified Keywords: carbohydrates; enzyme catalysis; glycosy-
Galb1-3GalNAc derivatives were synthesized. Modi- lation; regioselectivity; sialic acid
Introduction
and maintenance of the myelin membrane was pro-
posed.[4] In the growth of neurites MAG has a bifunc-
More than 40 different sialic acid derivatives occur in tional effect. On the one hand, neurite outgrowth of
nature on carbohydrate residues, often masking the neurons in newborns will be promoted, on the other
non-reducing terminus of the oligosaccharide chains. hand, it strongly inhibits neurite outgrowth of neurons
Thus they participate in recognition processes,[1] and in the adult species.[5] In this way the adult nervous
it is well known that pathogens such as bacteria and system is protected from wild branching of neurons.
viruses like the influenza virus are able to recognize However, the drawback is that, after injury, the cen-
and bind selectively sialic acids on cell surfaces.[2] The tral nervous system cannot be repaired. Substrates
Thomsen–Friedenreich antigen (T-antigen) Galb1- binding strongly to MAG prevent their interaction
3GalNAc is the central component of the core A with the native receptor and in this way prevent
structure in O-glycoproteins and ubiquitously present MAGꢀs inhibition of neurite outgrowth. In conjunc-
in organisms. It does not only present the preliminary tion with respective growth factors the recovery of in-
stage of complex oligosaccharides of the mucin type jured nerve tissue might be achievable in this way.
but also appears unmasked or preliminarily sialylated MAGꢀs change of function originates from a different
on the surface of cancer cells. For this reason com- constitution of gangliosides: in the neonatalic nervous
pounds with such motifs are important in cancer re- system the trisaccharide GD3 prevails without having
search as potential leads en route to vaccines or anti- the Neu5aAc2-3Galb1-3GalNAc motif (Figure 1) but
metastatically active cancer drugs.[3] The T-antigen
determinant is a part of ganglio-glycosphingolipides,
and the sialylated derivative Neu5Aca2-3Galb1-
3GalNAc (sialyl-T-antigen determinant) is present on
the non-reducing end of the gangliosides GQ1b, GD1a
and GT1b
.
Since the first observation of myelin-associated gly-
coprotein (MAG), its key function for the formation Figure 1.
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with the beginning of myelination the hexasaccharide Results and Discussion
GD1a containing that motif takes the lead.
In vitro MAG prefers binding to glycoconjugates Synthesis of Monosaccharide Acceptors
containing the Neu5Ac2-3bGal1-3GalNAc motif[6,7]
for example, the gangliosides GQ1b, GD1a and GT1b Radical dehalogenation of 2-acetamido-3,4,6-tri-O-
found in membranes of nervous cells.[8] Any modifica- acetyl-2-deoxy-a-d-glucopyranosyl chloride[21] accord-
tions in the N-acetylneuraminic acid moiety will pre- ing to Bramford et al. followed by deprotection yield-
vent recognition by MAG, but for a systematic recog- ed 2-acetamido-1,5-anhydro-glucitol 1.[22] Regioselec-
nition a facile synthesis of the above mentioned tive acylation furnished the 3,6-dipivaloate 2, the
motifs is required (Figure 1). So far the octasacchar- structure of which was confirmed by HMBC NMR.
ide ganglioside GQ1ba is the most potent antagonist of Synthesis of the corresponding triflate succeed quanti-
MAG identified.[9]
tatively in situ in dichloromethane/pyridine (20:1),
The parasitic trans-sialidase from Trypanosoma followed by intramolecular attack and migration of
cruzi, the pathogen responsible for Chagasꢀ disease, the pivaloyl groups under inversion at position 4.[23]
catalyzes an unusually efficient sialyltransfer, transfer- The galacto-configured 4,6- (3) and 3,4-dipivaloates 4
ring terminal Neu5Ac units from sialo-glycoconju- in a ratio of 9:1 were deprotected and yielded the 2-
gates to sialylate its own surface mucins and thus acetamido-1,5-anhydro-galactitol 5 in a total yield of
mask its own epitopes.[10–13] The exact catalytic mech- 48% over six steps based on the glycosyl chloride
anism of trans-sialylation is presently under (Scheme 1).
study.[14–16] Correct binding of sialic acid donors to
Starting with allyl 2-acetamido-a-d-glucopyranoside
trans-sialidase (TS) may trigger a conformational 6[24] pivaloylation in positions 3 and 6 could be ach-
change in the enzyme that induces the conditions for ieved to give crystalline 7 in 77% yield with only 6%
formation of a ternary complex. Acceptor binding of the tripivaloate as side product. Substitution of a
would displace the water molecules from the TS cata- triflyl group by NaBH4 allows the deoxygenation of
lytic cleft before formation of the oxocarbonium ion primary and secondary positions.[25] Thus the triflyl
or sialyl-enzyme intermediate takes place, facilitating group was introduced quantitatively at position 4 of
the selective transfer reaction. By using NMR spec- compound 7, and subsequent deoxygenation with
troscopy it was demonstrated that inactive T. cruzi TS NaBH4 in acetonitrile gave compound 9 in only 48%
has a carbohydrate recognition domain for b-Gal resi- yield. Consequently, the Barton–McCombie deoxyge-
due that is formed only after a conformational switch nation of thiocarbonyl compounds with Bu3SnHwas
triggered by prior sialoside binding.[17]
selected.[26] Employing tetrahydrofuran as solvent the
with 1,1’-thiocarbonyldiimidazole
In this work, we present chemoenzymatic methods acylation of
7
for the synthesis of the sialyl T-antigen determinant (TCDI)[27] yielded compound 8 in very good yield
Neu5Aca2-3Galb1-3GalNAc and derivates thereof. (97%), and the radical reduction with tributyltin hy-
Employing b1-3-galactosidases from various sources dride without a radical starter furnished the 4-deoxy
or a phosphorylase, the building block C, being compound 9 in high yields. The subsequent deprotec-
GalNAc or a modification of it, was glycosylated with tion of the pivaloyl groups to give 10 was unusually
building block B. The disaccharides obtained were sia- slow and required considerable amounts of catalyst
lylated in the final step utilizing trans-sialidase of T. (Scheme 2).
cruzi. Previously sialylated T-antigen determinant
structures were efficiently synthesized by Kunz
et al.[18,19] as well as by Schmidt et al.[20] Our enzy- Synthesis of Disaccharides
matic reaction sequence presented here is straightfor-
ward, fast and efficient, affording the desired products The modified N-acetyl-galactosamines needed to be
in absolute regio- and stereoselectivity.
galactosylated chemoenzymatically in order to obtain
compounds corresponding to the T-antigen structure
Scheme 1.
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Scheme 2.
Galb1-3GalNAc. Thus, modified monosaccharide ac- ditions as well.[36–38] Optimal reaction conditions
ceptors were glycosylated using p-nitrophenyl b-d-gal- proved to be 20% DMF in potassium phosphate
actopyranoside (pNP-Gal) and a-d-galactopyranosyl buffer (100 mM, pH6.0) at an incubation temperature
phosphate (Gal-P) donors, respectively. The glycosidic of 508C. During incubations of relatively sensitive
linkages were established by means of b-galactosidas- GalNAc derivatives decomposition products were ob-
es from bovine testes, Bacillus circulans (bgac-gen) served, thus reactions were conducted at 378C with-
and Xanthomonas manihotis, as well as by the phos- out considerably slowing down the reaction.
phorylase from Bifidobacterium bifidum.
Bifidobacterium bifidum located in the human in-
Gyçrgy et al. carried out first synthetic investiga- testinal tract encloses several enzymes digesting
tions employing a cell-free extract from bovine testes. mucins. From the mutant B. bifidum DSM 20082 the
N-Acetyl-d-glucosamine was galactosylated using lac- non-Leloir glycosyl transferase b1-3-galactosyl-N-ace-
tose as donor at positions 3 and 4 in a ratio of 10:1 in tylhexosamine phosphorylase was isolated which spe-
a yield of 2.3%.[28] Since the b-galactosidase is quite cifically cleaves a galactose residue from position 3 of
expensive, an extract from bovine testes was isolated GlcNAc and GalNAc to give galactose 1-phosphate
according to a modified procedure by Distler and and the corresponding N-acetyl-hexosamine. So far,
Jourdian.[29] This crude extract contained a b-hexos- this is the only known phosphorylase recognizing a
aminidase as impurity, thus any b-glycosidic linkage in terminal galactose moiety.[39] In the reverse reaction
the acceptor structures such as N-acetyl-d-glucos- N-acetyl-d-galactosamine and glucosamine could be
amine or galactosamine would be hydrolyzed, pre- galactosylated with galactose 1-phosphate at position
venting their galactosylation.[30]
3.[40] The enzyme used was a cell-free crude extract
The Gram-negative bacteria Xanthomonas maniho- containing a b-hexosaminidase. Due to the small
tis contains eight exo-glycosidases, including a b-galac- amount of the enzyme available, no attempts were
tosidase which preferably cleaves the b1-3 link- undertaken to optimize the reaction conditions.
age.[31,32] Whereas the acceptor GlcNAc was galacto- Therefore previously elaborated conditions with 1.2
sylated only in position 3 in a yield of 22%, GalNAc equivalents of the acceptor in imidazole buffer
furnished both the b1-3 and b1-6 regioisomers in a (25 mM) at pH6.8, 37 8C and 20 h were employed.
ratio of 7:3 in a similar yield of 25%.[33,34] The
enzyme displays an activity maximum at a pHvalue
The reaction conditions optimized during hydroly-
ses were employed with allyl 2-acetamido-a-d-galac-
between 5.0 and 6.5. The addition of DMSO, DMF or topyranoside (a-Allyl-GalNAc) as a sample acceptor
acetone was not tolerated, whereas a content of 10% to examine the conditions for transglycosylation. In
acetonitrile or 20% tert-butanol had no noteworthy all hydrolase reactions an excess of donor led to some
effect on the activity of the enzyme. The best temper- extent of auto-galactosylation of the donor pNP-Gal
ature for the incubation proved to be 378C, and a and galactosylation of the hydrolysis product d-galac-
positive effect was observed by addition of 0.05% of tose. These side reactions were not observed using an
bovine serum albumin (BSA). The optimal conditions excess of acceptor.
employed in this study were sodium acetate buffer
(100 mM, pH5.5, 0.05 % BSA) with 10% acetonitrile testes and X. manihotis showed a maximum of prod-
and a temperature of 378C. uct formation after 48–55 h incubation time, whereas
Reactions catalyzed by b-galactosidase from bovine
In addition to the two well known and commercial- the optimum yield using b-galactosidase from B. cir-
ly available galactosidases I and II, Ito and Sasaki ob- culans was reached after 4.5 h. The reactions were
served in Bacillus circulans (mutant ATCC31382) a stopped by heating to 908C, lyophilization of the sol-
novel b-galactosidase. They successfully cloned the re- vent followed by purification using size exclusion
sponsible bgaC-gen and expressed it in Escherichia chromatography.
coli.[35] In contrast to the commercially available b-
The galactosylation of allyl-GalNAc employing the
galactosidase, the new b-galactosidase favourably hy- b-galactosidases from X. manihotis and B. circulans
drolyzed a b1-3-linked galactose and established this did not only give the desired b1-3-disaccharides, but a
linkage regioselectively under transglycosylation con- further major product was observed. The hydrolyzed
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donor and excess acceptor were removed by size ex- gave the b1-3-linked products. The yields for the gal-
clusion chromatography on Biogel P2 and the disac- actosylation could be increased from 22%[41] to a
charide fraction subsequently acetylated under stan- convincing 67%, proving this method to be superior
dard conditions. Purification on silica gel yielded both to previously published methods in efficiency and
the b1-3 product and the b1-6 derivative in yields of overall yield. The regioselective galactosylation of 12
33% and 22%, respectively.
was successfully performed in a yield of 66%
The chemoenzymatic galactosylation of the allyl (Scheme 3).
(11) and methyl a-glycopyranosides (12) of GalNAc
The chemoenzymatic galactosylation of 11 and 12
in the presence of galactosidase from bovine testes with galactose 1-phosphate as donor in the presence
Scheme 3. Transglycosylations with b1-3-galactosidases from bovine testes, Xanthomonas manihotis, Bacillus circulans and
the phosphorylase from Bifidobacterium bifidum.
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Towards Sialylated Thomsen–Friedenreich Antigen Components
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of phosphorylase from Bifidobacterium bifidum, entation of the acceptor towards a galactosylation at
yielded solely the b1-3-linked products in yields of position 3. The chemoenzymatic galactosylation of
43% and 44%, respectively. The yields obtained were the 4-deoxy derivative 14 occurred exclusively at posi-
lower than when using galactosidase from bovine tion 3 in yields comparable to the glycosylation of 11
testes. It may be assumed that steric effects in the or 12. In general, the b-galactosidases from X. mani-
active center between the substrate and enzyme could hotis and B. circulans glycosylated the primary posi-
be responsible. Using b-galactosidase from Xantho- tion of galacto-configured acceptors, however not
monas manihotis and Bacillus circulans, the yields of those of the respective gluco-configured components.
the desired b1-3-linked products were comparable. In In conclusion, the equatorial orientation of the
the presence of X. manihotis the b1-6-linked byprod- hydoxy group in position 4 of GlcNAc was proven to
uct was observed in small amounts, whereas in the be not responsible for the regioselective galactosyla-
presence of B. circulans a considerable amount of the tion at position 3. In contrast, apparently the axial hy-
same byproduct was found. A comparable regioselec- droxy group of GalNAc derivatives seems to disturb
tivity was observed by Ajisaka et al. employing B. cir- the recognition by the enzyme leading to a mixture of
culans with d-GalNAc and d-Gal-O-Me acceptors.[42] regioisomers.
For facile purification and isolation the b1-6 byprod-
uct could be hydrolyzed by b-galactosidase from Es-
cherichia coli without affecting the b1-3 products 15 Synthesis of Sialylated Oligosaccharides
and 16, respectively.
It was expected that galactitol 13 missing an agly- Employing pNP-N-acetyl-neuraminic acid, the disac-
cone structure would provide insight into the role of charide derivatives obtained (Scheme 3) were used as
the aglycone concerning yield and regioselectivity. acceptors in enzymatic reactions catalyzed by the
Apparently, the aglycone did not show any effect or trans-sialidase derived from T. cruzi. It is assumed
consequence for the binding of the acceptor in the that the enzyme has two subsites in the active center:
enzyme pocket. The galactosylation employing the the sialic acid binding site and the galactose binding
enzymes from bovine testes or Bifidobacterium bifi- site, the latter being absent in bacterial and viral siali-
dum did not show any response to the missing agly- dases. Binding of the sialic acid moiety of the donor
cone structure regarding yield or regioselectivity in may trigger a conformational switch, generating the
comparison to 11 and 12. Using galactosidase from b-galactosyl acceptor binding site.[43] The transfer ac-
Xanthomonas manihotis the yield was comparable to tivity depends strongly on incubation conditions, such
the other acceptors investigated, however, the forma- as pH, temperature and time. Trans-glycosylation with
tion of a by-product was observed in higher amounts. T. cruzi took place with equimolar amounts of sub-
Possibly stabilizing steric or hydrophobic interactions strate and in eight hours incubation time as developed
of the aglycone residue in the binding pocket of the previously.[44] Higher enzyme activities were achieved
enzyme are absent in 13, which might favour the ori-
Scheme 4. Sialylation of GalNAc-modified disaccharides.
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Scheme 5. Trans-sialylation with trans-sialidase of Galb1-3GalNAc derivatives modified in the terminal Gal residue.
in a buffer system of Tris-HCl buffer at pH 7.5 and a Conclusions
temperature of 238C.
Sialylation of GalNAc-modified disaccharides em- A series of modified N-acetyl-galactosamine accept-
ploying trans-sialidase led to modified determinants ors has been prepared and galactosylated employing
shown in Scheme 4 and Scheme 5 in fairly good yields three b-galactosidases from bovine testes, Bacillus cir-
of 30 to 46%. In reactions catalyzed by trans-sialidase culans, Xanthomonas manihotis as well as the phos-
of T. cruzi a galactose linked at position 1 is a prereq- phorylase from Bifidobacterium bifidum. Their subse-
uisite. No significant differences were observed in the quent sialylation was performed with trans-sialidase
synthesis of trisaccharides 19, 21 and 22, showing no (T. cruzi) in good yields to give building block C-
influence of the aglycone structure in the recognition modified sialylated Thomsen–Friedenreich antigen
by trans-sialidase of T. cruzi. In contrast, the yield of components. Furthermore, Galb1-3GalNAc structures
trisaccharide 20, with a methyl residue as aglycone, modified in the terminal building block were convinc-
turned out to be somewhat lower.
ingly sialylated to give the corresponding Sia-T anti-
Previous work reported enzymatic sialylations with gen with modified B building units.
trans-sialidase (T. cruzi.), employing a Gal residue in
the acceptor site.[44] In other studies reactions with
the immobilized enzyme were investigated.[45] In our Experimental Section
further work we could use 2-deoxy- (25) and 6-deoxy
derivatives (24) of d-galactose as well as l-arabinose General Remarks
(23) as building block B[46] to investigate the influ-
ence of the hydroxy groups in positions 2 and 6 or a
missing CH2OHgroup on the transfer employing
trans-sialidase.
Commercially available starting materials were used without
further purification. Solvents were dried according to stan-
dard methods. Purifications of the products were carried out
by column chromatography using Merck silica gel 60 (230–
400 mesh). The enzymatic reactions were incubated in a
thermomixer Comfort (Merck) at 600 rpm. The NMR spec-
tra were recorded on a Bruker AMX-400 (100.62 MHz for
13C) or DRX-500 (125.83 MHz for 13C) spectrometer. All
chemical shifts are quoted in ppm downfield from TMS or
referred to the characteristic signals of the used solvents
CHCl3 in CDCl3 (7.24 ppm), MeOH-d3 in [D4]MeOH
(3.35 ppm) or HDO in D2O (4.63 ppm). Mass spectra were
recorded on a Bruker MALDI-Tof Biflex III.
Apparently, as deduced from the respectable yields
of the trisaccharide 28 modified in position 2 of build-
ing block B, it can be assumed that the lack of the hy-
droxy group in this position does not influence the
transfer. Further, use of l-arabinose 23 and 2-deoxy
derivative 25 as building blocks B again does not
change the sialylation to give average yields of 46%
for 26 and 43% for 28.
In contrast, the terminal fucose residue, being less
polar than arabinose and the 2-deoxy-galactose, can
be considered to show a lower binding affinity to the
active center of the enzyme, and thus the yield of 27
is somewhat lower than that of 26 or 28.
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Trans-sialylation with Recombinant Trans-sialidase
from Trypanosoma cruzi (Method 1)
Enzymatic Transglycosylation with b-Galactosidase of
Bacillus circulans (Method 4)
A solution of 10 mg pNP-Neu5Ac and 10 mg acceptor in
1.0 mL degassed incubation buffer (100 mM Tris/HCl,
pH7.5, 50 mg BSA, 0.02% NaN3) was incubated with
100 mL recombinant TS (5.7 mg/1 mL) at 238C for 8 h. The
reaction was monitored by TLC (butanol/acetic acid/water,
5:2:2). After completion of the reaction, the enzyme was de-
natured and centrifuged before the supernatant was lyophi-
lized. The dry residue was dissolved in water and purified by
biogel chromatography (P-2 Biogel, 16”900 mm) with
water.
A solution of donor pNP-galactopyranoside and acceptor (3
equivalents) in potassium phosphate incubation buffer
(100 mM, pH6.0, 20% N,N-dimethylformamide) was incu-
bated with b-galactosidase of Bacillus circulans (bgaC-Gen,
5.9 U/mmol) at 378C for 4.5 h. After completion of the reac-
tion, the enzyme was denatured by heating to 908C. The so-
lution was diluted with the ten-fold volume of sodium phos-
phate buffer (50 mM, pH7.0, 1 m m magnesium chloride). To
this solution the b-galactosidase of Escherichia coli (250 U/
mmol) was added and the mixture incubated for 4 h at
378C. The pHvalue was adjusted to 4.3 using citric acid and
the enzyme was denatured. The liberated pNP-OHwas ex-
tracted with ethyl acetate and the aqueous layer containing
the product was lyophilized. The dry residue was dissolved
in water and purified by biogel chromatography (P-2 Biogel,
16”900 mm). First elution was with an ammonium hydrogen
carbonate buffer (25 mmol), and subsequently water was
used. Buffer salts were removed via a short mixed bed ion
exchange chromatography column.
Enzymatic Transglycosylation with b-Galactosidase of
Bovine Testes (Method 2)
A solution of donor pNP-galactopyranoside and acceptor
(10 equivalents) in incubation buffer (McIlvaine-buffer
50 mM, pH4.3) was incubated with b-galactosidase of
bovine testes (4.7 U/mmol) at 378C for 50 h. After comple-
tion of the reaction, the enzyme was denatured by heating
to 908C. The liberated pNP-OHwas extracted with ethyl
acetate, and the aqueous layer containing the product was
lyophilized. The dry residue was dissolved in water and puri-
fied by biogel chromatography (P-2 Biogel, 16”900 mm).
First elution was with an ammonium hydrogen carbonate
buffer (25 mmol), and subsequently water was used. Buffer
salts were removed using a short mixed bed ion exchange
chromatography column.
Enzymatic Glycosylation with Phosphorylase of
Bifidobacterium bifidum (Method 5)
A solution of the donor galactose-1-phosphate and acceptor
(1.2 equivalents) in imidazole incubation buffer (25 mM,
pH6.8 L) was incubated with phosphorylase of Bifidobacte-
rium bifidum DSM 20082 (30 mLmmolÀ1) at 378C for 4 h.
After completion of the reaction, the enzyme was denatured
by heating to 908C. After centrifugation the supernatant
containing the product was lyophilized. The dry residue was
dissolved in water and purified by biogel chromatography
(P-2 Biogel, 16”900 mm). First elution was with an ammo-
nium hydrogen carbonate buffer (25 mmol), and subse-
quently water was used. Buffer salts were removed via a
short mixed bed ion exchange chromatography column.
1H, 13C NMR data, MS data and combustion analyses for
all compounds are given in the Supporting Information.
Enzymatic Transglycosylation with b-Galactosidase of
Xanthomonas manihotis (Method 3)
A solution of donor pNP-galactopyranosid and acceptor (3
equivalents) and small amounts BSA in sodium acetate in-
cubation buffer (100 mM, pH5.5, 10% acetonitrile) was in-
cubated with b-galactosidase of Xanthomonas manihotis
(230 U/mmol) at 378C for 50 h. After completion of the re-
action, the enzyme was denatured by heating to 908C. The
solution was diluted with the ten-fold volume of sodium
phosphate buffer (50 mm, pH7.0, 1 mM magnesium chlo-
ride). To this solution the b-galactosidase of Escherichia coli
(250 U/mmol) was added and the mixture incubated for 4 h
at 378C. The pHvalue was then adjusted to 4.3 using citric
acid and the enzyme was denatured. The liberated pNP-OH
was extracted with ethyl acetate and the aqueous layer con-
taining the product was lyophilized. The dry residue was dis-
solved in water and purified by biogel chromatography (P-2
Biogel, 16”900 mm). First elution was with an ammonium
hydrogen carbonate buffer (25 mmol), and subsequently
water was used. Buffer salts were removed using a short
mixed bed ion exchange chromatography column.
2-Acetamido-1,5-anhydro-2-deoxy-3,6-di-O-piva-
loyl-d-glucitol (2)
A solution of 2-acetamido-1,5-anhydro-2-deoxy-d-glucitol
(1)[22] (1.60 g, 7.80 mmol) was dissolved in dry dichlorome-
thane (8 mL) and dry pyridine (16 mL). After cooling the
solution to À28C pivaloyl chloride (2.88 mL, 23.4 mmol, 3
equivalents) was added. The mixture was stirred at 08C for
5.5 h. The reaction was stopped with methanol and diluted
with dichloromethane. The organic layer was washed with
aqueous sodium hydrogen carbonate, aqueous sodium chlo-
ride, and the organic layer was dried over MgSO4. After re-
moval of the solvent under reduced pressure the product
was purified by column chromatography (silica gel, petrole-
um ether/ethyl acetate 1:1) to give compound 2 as an amor-
phous solid; yield: 2.38 g (6.37 mmol, 82%); [a]2D0: À248 (c
0.6, CHCl3).
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drofuran (40 mL) was refluxed for 7 h. The solvent was
evaporated and the residue was dissolved in dichlorome-
thane. The organic layer was washed with hydrochloric acid
(2M), aqueous sodium hydrogen carbonate and water. The
solvent was dried over MgSO4, filtered and then removed
under reduced pressure to give 8 as a yellow solid; yield:
2.72 g (5.04 mmol, 97%); mp 1078C; [a]2D0: +648 (c 0.55,
CHCl3).
2-Acetamido-1,5-anhydro-2-deoxy-4,6-di-O-pivaloyl-
d-galactitol (3) and 2-Acetamido-1,5-anhydro-2-
deoxy-3,4-di-O-pivaloyl-d-galactitol (4)
A solution of 2 (2.09 g, 5.60 mmol) in dry dichloromethane
(45 mL) and dry pyridine (2.4 mL) was cooled to À358C.
Trifluoromethanesulfonic anhydride (1.2 mL, 7.1 mmol, 1.3
equivs.) was added dropwise to the solution and warmed up
to 08C. After stirring for 8 h water (6.0 mL) was added and
the solution was refluxed for 42 h. The reaction mixture was
diluted with dichloromethane. The organic layer was washed
with aqueous sodium hydrogen carbonate and water. The
solvent was dried over MgSO4, filtered, and the organic
layer was removed at reduced pressure. The product was pu-
rified by column chromatography (silica gel, toluene/acetone
5:2) to give 3 and 4 as an amorphous solid; yield: 1.81 g
Allyl 2-Acetamido-2,4-dideoxy-3,6-di-O-pivaloyl-a-
d-xylo-hexopyranoside (9)
The reaction was carried out under an argon atmosphere.
Tributyltin hydride (3.30 mL, 12.5 mmol, 2.5 equivs.) was
dissolved in dry toluene (375 mL) and refluxed. Compound
8 (2.65 g, 4.91 mmol) in dry toluene (90 mL) was added
dropwise over 2 h to the solution. After 15 h reaction time
the solvent was removed under vacuum and the residue was
extracted with hot acetonitrile. The acetonitrile was extract-
ed with hexane and the solvent was removed under reduced
pressure. The residue was purified by column chromatogra-
phy (silica gel, petroleum ether/ethyl acetate 3:2) to give 9
as an amorphous solid; yield: 1.90 g (4.59 mmol, 94%);
[a]2D0: +828 (c 0.5, CHCl3).
1
(4.85 mmol, 87%); ratio 3:4=9:1 (from HNMR).
2-Acetamido-1,5-anhydro-2-deoxy-d-galactitol (5)
To a solution of 3 and 4 (1.68 g, 4.50 mmol) in dry methanol
(40 mL) sodium methoxide (40 mg) was added and stirred
at room temperature for 30 h. After neutralization with Am-
berlite IR 120, H+, the solution was filtered, dried over
MgSO4, and the solvent was removed under reduced pres-
sure. Recrystallization was from methanol gave 5 as a col-
ourless solid; yield: 855 mg (4.17 mmol, 93%): mp 1178C;
[a]2D0: +498 (c 1, MeOH).
Allyl 2-Acetamido-2,4-dideoxy-a-d-xylo-hexo-
pyranoside (10)
To a solution of 9 (1.76 g, 4.26 mmol) in dry methanol
(100 mL) a sodium methanolate solution (7.5 mL) was
added at 308C and stirred at room temperature for 8 days.
After neutralization with Amberlite IR 120, H+ the solution
was filtered and dried to give 10 (989 mg, 4.03 mmol, 95%)
as a colourless solid; mp 1538C; [a]2D0: +2398 (c 0.8, MeOH).
Allyl 2-Acetamido-2-deoxy-3,6-di-O-pivaloyl-
a-d-glucopyranoside (7) and Allyl 2-Acet-amido-2-
deoxy-3,4,6-tri-O-pivaloyl-a-d-glucopyranoside (7a)
A solution of allyl 2-acetamido-2-deoxy-a-d-glucopyrano-
side (6)[24] (17.5 g, 67.0 mmol) in dry dichloromethane
(70 mL) and dry pyridine (140 mL) was cooled to 08C. Piva-
loyl chloride (24.7 mL, 20 mmol, 3 equivs.) was added to the
solution. The reaction was stopped with methanol and dilut-
ed with dichloromethane (840 mL). The organic layer was
washed with aqueous sodium hydrogen carbonate, water
and sodium chloride, dried over MgSO4, filtered and then
the solvents removed at reduced pressure. The residue was
purified by column chromatography (silica gel, petroleum
ether/ethyl acetate 2:1) to give the products 7 and 7a.
Allyl 2-acetamido-2-deoxy-3,6-di-O-pivaloyl-a-d-glucopyr-
anoside (7): Yield: 22.2 g (51.5 mmol, 77%); colourless crys-
tals; [a]20: +608 (c 1, CHCl3); mp 498C.
Allyl 2-acetamido-2-deoxy-3-O-(b-d-galacto-
pyranosyl)-a-d-galactopyranoside (15)
Following method 2, pNP-Gal (80 mg, 266 mmol) and allyl 2-
acetamido-2-deoxy-a-d-galactopyranoside (11)[47] (695 mg,
2.66 mmol, 10 equivs.) were incubated in buffer (3.0 mL)
with b-galactosidase (25 mg, 1.25 U) to give 15; Yield:
75.1 mg (177 mmol, 67%).
Following method 3, pNP-Gal (28.8 mg, 96.6 mmol) and
11 (75 mg, 287 mmol, 3 equivs.) were incubated for 50 h in
buffer (800 mL) with BSA (40 mg), and b-galactosidase
(2.2 mL, 22 U). Dilution with hydrolysis buffer (8 mL), and
treatment with b-galactosidase (24 U) from E. coli gave 15;
yield: 8.6 mg (20.3 mmol, 21%).
Allyl D2-acetamido-2-deoxy-3,4,6-tri-O-pivaloyl-a-d-gluco-
pyranoside (7a): Yield: 2.06 g (4.01 mmol, 6%); amorphous
solid; C26H43NO9: 513.63; [a]2D0: +938 (c 1, CHCl3).
Following method 4, pNP-Gal (21.4 mg, 71.0 mmol) and
11 (55.4 mg, 212 mmol, 3 equivalents) were incubated for
4.5 h in buffer (1.2 mL) with b-galactosidase (20 mL, 420 U).
Dilution with hydrolysis buffer (12 mL), and treatment with
b-galactosidase from E. coli (18 U) gave 15; yield: 10.3 mg
(24.3 mmol, 34%).
Allyl 2-Acetamido-2-deoxy-3, 6-di-O-pivaloyl-
4-O-thiocarbonylimidazoyl-a-d-glucopyranoside (8)
A solution of 7 (2.24 g, 5.22 mmol) and 1,1-thiocarbonyldi-
imidazole (TCDI) (2.20 g, 12.3 mmol, 2.4 equivs.) in tetrahy-
1224
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Adv. Synth. Catal. 2006, 348, 1217 – 1227

Towards Sialylated Thomsen–Friedenreich Antigen Components
FULL PAPERS
Following method 5, Gal-1-P (14.0 mg, 41.6 mmol) and 11
(13.1 mg, 50.1 mmol, 1.2 equivalents) were incubated in
buffer (8.2 mL) with phosphorylase (1.2 mL) gave 15 as a
colourless solid; yield: 7.6 mg (17.9 mmol, 43%); mp 2248C;
Allyl 2-Acetamido-2,4-dideoxy-3-O-(b-d-galacto-
pyranosyl)-a-d-xylo-hexopyranoside (18)
Following method 2, pNP-Gal (80 mg, 266 mmol) and 14
(650 mg, 2.65 mmol, 10 equivs.) were incubated in buffer
(3 mL) with b-galactosidase (25 mg, 1.25 U) to give 18;
yield: 72.8 mg (179 mmol, 67%).
[a]20: +1048 (c 0.5, H2O); {ref.[49] mp 2318C; [a]2D0: +1208 (c
D
1, H2O)}.
Following method 3, pNP-Gal (20.0 mg, 66.4 mmol) and
14 (49.0 mg, 200 mmol, 3 equivalents) were incubated for
50 h in buffer (600 mL) with BSA (25 mg), and b-galactosi-
dase (1.5 mL, 15 U) to give 18; yield: 6.0 mg (14.7 mmol,
22%).
Following method 4, pNP-Gal (24.1 mg, 80.0 mmol) and
14 (59 mg, 241 mmol, 3 equivalents) were incubated for 4.5 h
in buffer (1.4 mL) with b-galactosidase (22 mL, 462 U) to
give 18; yield: 12.0 mg (29.5 mmol, 37%).
Following method 5, Gal-1-P (15.0 mg, 44.6 mmol) and 14
(13 mg, 53 mmol 1.2 equivalents) were incubated in buffer
(8.7 mL) with phosphorylase (1.3 mL) to give 18 as an amor-
phous, colourless solid; yield: 7.4 mg (18.2 mmol, 41%);
[a]2D0: +428 (c 0.1, H2O).
Methyl 2-Acetamido-2-deoxy-3-O-(b-d-galacto-
pyranosyl)-a-d-galactopyranoside (16)
Following method 2, pNP-Gal (57 mg, 189 mmol) and
methyl 2-acetamido-2-deoxy-a-d-galactopyranoside (12)[48]
(444 mg, 1.89 mmol, 10 equivs.) were incubated in buffer
(2.1 mL) with b-galactosidase (18 mg, 902 mU) to give 16;
yield: 49.5 mg (125 mmol, 66%).
Following method 3, pNP-Gal (28.8 mg, 96.6 mmol) and
12 (67 mg, 285 mmol, 3 equivs.) were incubated for 50 h in
buffer (800 mL) with BSA (40 mg), and b-galactosidase
(2.2 mL, 22 U). Dilution with hydrolysis buffer (8 mL), and
treatment with b-galactosidase from E. coli (24 U) gave 16;
yield: 9.1 mg (22.9 mmol, 24%).
Following method 4, pNP-Gal (21.4 mg, 71.0 mmol) and
12 (50.1 mg, 213 mmol, 3 equivs.) were incubated for 4.5 h in
buffer (1.2 mL) with b-galactosidase (20 mL, 420 U). Dilu-
tion with hydrolysis buffer (12 mL), and treatment with b-
galactosidase from E. coli (18 U) gave 16; yield: 9.3 mg
(23.4 mmol, 33%).
Following method 5, Gal-1-P (14.0 mg, 41.6 mmol) and 12
(11.8 mg, 50.1 mmol 1.2 equivs.) were incubated in buffer
(8.2 mL) with phosphorylase (1.2 mL) gave 16 as a colour-
less solid; yield: 7.6 mg (18.4 mmol, 44%); mp 2318C; [a]2D0:
+1178 (c 0.5, H2O); {ref.[50] mp 2358C); [a]2D0: +1288 (c 0.38,
H2O)}.
Allyl (5-Acetamido-3,5-dideoxy-a-d-glycero-
d-galacto-2-nonulopyranosylonic Acid)-(2–3)-
(b-d-galactopyranosyl)-(1–3)-2-acetamido-2-deoxy-
a-d-galactopyranoside (19)
Following method 1, acceptor disaccharide 15 (15 mg, 35
mmol) and pNP-aNeu5Ac (11 mg, 35 mmol) were incubated
with TS for 8 h to give 19 as a colourless solid; yield: 12 mg
20
546
(16 mmol, 46%); [a] : +238 (c 0.05, H2O).
2-Acetamido-1,5-anhydro-2-deoxy-3-O-(b-d-galacto-
pyranosyl)-d-galactitol (17)
Methyl (5-Acetamido-3,5-dideoxy-a-d-glycero-
d-galacto-2-nonulopyranosylonic Acid)-(2–3)-
(b-d-galactopyranosyl)-(1–3)-2-acetamido-2-deoxy-
a-d-galactopyranoside (20)
Following method 2, pNP-Gal (75.3 mg, 250 mmol) and 13
(513 mg, 2.50 mmol, 10 equivs.) were incubated in buffer
(2.8 mL) with b-galactosidase (23.4 mg, 1.17 U) to give 17;
yield: 63.9 mg (1174 mmol, 70%).
Following method 3, pNP-Gal (20.0 mg, 66.4 mmol) and
13 (40.9 mg, 199 mmol, 3 equivs.) were incubated for 50 h in
buffer (550 mL) with BSA (28 mg), and b-galactosidase (1.5
ml, 15 U). Dilution with hydrolysis buffer (5.5 mL), and
treatment with b-galactosidase from E. coli (16 U) gave 17;
yield: 4.6 mg (12.5 mmol, 19%).
Following method 1, acceptor disaccharide 16 (15.5 mg, 32
mmol) and pNP-aNeu5Ac (10.4 mg, 37 mmol) were incubat-
ed with TS for 8 h to give 20 as a colourless solid; yield:
20
546
8 mg (11 mmol, 30%); mp 1818C; [a] : +268 (c 0.05, H2O).
Following method 4, pNP-Gal (15.1 mg, 50.1 mmol) and
13 (30.9 mg, 151 mmol, 3 equivs.) were incubated for 4.5 h in
buffer (850 mL) with b-galactosidase (15 mL, 315 U). Dilu-
tion with hydrolysis buffer (8.5 mL), and treatment with b-
galactosidase from E. coli (12.5 U) gave 17; yield: 5.7 mg
(15.5 mmol, 31%).
(5-Acetamido-3,5-dideoxy-a-d-glycero-d-galacto-2-
nonulopyranosylonic Acid)-(2–3)-(b-d-galacto-
pyranosyl-2-acetamido-1,5-anhydro-2-deoxy-
d-galactitol (21)
Following method 5, Gal-1-P (14.0 mg, 41.6 mmol) and 13
(10.3 mg, 50.2 mmol 1.2 equivs.) were incubated in buffer
(8.2 mL) with phosphorylase (1.2 mL) to give 17 as a colour-
less, amorphous solid; yield: 6.3 mg (17.1 mmol, 41%); [a]2D0:
+638(c 0.2, DMSO/MeOH1:1).
Following method 1, acceptor disaccharide 17 (16.4 mg, 40
mmol) and pNP-aNeu5Ac (10 mg, 32 mmol) were incubated
with TS for 8 h to give 21 as a colourless solid; yield: 13 mg
(19 mmol, 44%); C25H47N2O19: 658.62; mp 1838C; [a]52406
+968 (c 0.05, H2O).
:
Adv. Synth. Catal. 2006, 348, 1217 – 1227
ꢁ 2006 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim
www.asc.wiley-vch.de
1225

Lars Krçger et al.
FULL PAPERS
References
Allyl (5-Acetamido-3,5-dideoxy-a-d-glycero-
d-galacto-2-nonulopyranosylonic Acid)-(2–3)-
(b-d-galactopyranosyl)-2-acetamido-2,4-dideoxy-
a-d-xylo-hexopyranoside (22)
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20
546
14.8 mg (21 mmol, 43%); mp 1728C; [a] : +448 (c 0.05,
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(a-d-fucopyranosyl)-(1–3)-2-acetamido-2-deoxy-
a-d-galactopyranoside (27)
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(24)[46] (13 mg, 32 mmol) and pNP-aNeu5Ac (10 mg, 32
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:
20
546
698.42; mp 1708C; [a] : + 628 (c 0.05, H2O).
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Acknowledgements
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