Multi-gram synthesis of a hyaluronic acid subunit and synthesis of fully protected oligomers

Article abstract of DOI:10.1002/adsc.201000008

Fully protected tetra-, hexa- and octasaccharides of hyaluronic acid were synthesized on a scale of several 100 mg up to gram quantities using allyl (methyl 2-O-benzoyl-3-O-benzyl-4-O-levulinoyl-β-D-glucopyranosyluronate)- (1→3)-4-O-acetyl-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-D- glucopyranoside as a key building block. This disaccharide was subjected to deprotection, then glycosylation via the trichloroacetimidate method was employed to achieve the formation of the oligosaccharides.

Full text of DOI:10.1002/adsc.201000008

UPDATES
DOI: 10.1002/adsc.201000008
Multi-Gram Synthesis of a Hyaluronic Acid Subunit and Synthesis
of Fully Protected Oligomers
Mickaꢀl Virlouvet,a,b Michael Gartner,a,b Katarzyna Koroniak,a
Jonathan P. Sleeman,b,c,* and Stefan Brꢁsea,*
a
Institute for Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
Fax : (+49)-721-608-8581; e-mail: braese@kit.edu
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1,
b
76344 Eggenstein-Leopoldshafen, Germany
Fax : (+49)-7247-82-3354; e-mail: jonathan.sleeman2@kit.edu
Universitꢁtsmedizin Mannheim, University of Heidelberg, Ludolf-Krehl-Str. 13–17, 68167 Mannheim, Germany
c
Fax : (+49)-621-383-9955; e-mail: sleeman@medma.uni-heidelberg.de
Received: January 5, 2010; Revised: September 11, 2010; Published online: October 7, 2010
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201000008.
Abstract: Fully protected tetra-, hexa- and octasac-
charides of hyaluronic acid were synthesized on a
scale of several 100 mg up to gram quantities using
allyl (methyl 2-O-benzoyl-3-O-benzyl-4-O-levulino-
yl-b-d-glucopyranosyluronate)-(1!3)-4-O-acetyl-6-
O-benzyl-2-deoxy-2-trichloroacetamido-b-d-gluco-
pyranoside as a key building block. This disacchar-
ide was subjected to deprotection, then glycosyla-
tion via the trichloroacetimidate method was em-
ployed to achieve the formation of the oligosacchar-
ides.
wide variety of biological processes, such as cell mi-
gration, proliferation, adhesion, recognition,[2] tumor
invasion[3] and tumor inhibition.[4] Degradation of HA
during inflammation and tumor growth results in the
accumulation of fragments of HA (sHA).[5] These
fragments are potent activators of angiogenesis,[5] den-
dritic cells[6] and the expression of matrix metallopro-
teases by tumors.[7]
sHA is commonly prepared by partial enzymatic
degradation of HA followed by fractionation using
size exclusion chromatography. Heterogeneous mix-
tures of sHA are often isolated by this method. How-
ever, the size of sHA is critical for its biological activ-
ity, so it would be highly desirable to produce defined
lengths of sHA through organic synthesis. Organic
synthesis would also avoid potential problems associ-
ated with lipopolysaccharide contamination of sHA
preparations.
Keywords: carbohydrates; glycosaminoglycans; gly-
cosylation; hyaluronic acid; oligosaccharides
Introduction
Since the pioneering work[8] of Jeanloz et al. in
1964, several research groups have developed the syn-
Hyaluronic acid (HA), first isolated from the bovine thesis of disaccharide subunits[9] but only three groups
vitreous body,[1] is a linear, high molecular weight (up have recently reported the synthesis of oligosacchar-
to 2ꢂ104 kDa) glycosaminoglycan assembled from b- ides.[10] Here we report the multigram scale-up of the
1,3-linked-2-acetamido-2-deoxy-d-glucose-b-(1!4)-d-
disaccharide unit and the synthesis of fully protected
glucuronic acid repeating units (Figure 1). As an ex- tetra-, hexa- and octasaccharides.
tracellular matrix component, HA is involved in a
Starting from d-glucosamine hydrochloride 1 and
1,2,4,6-diisopropylidene-d-glucofuranose 14 we syn-
thesized the disaccharide 26, originally proposed by
Seeberger et al. as a key building block,[9b] on a multi-
gram scale. The key step in our synthesis is the glyco-
sylation of glucosamine acceptor 13 employing the
glycosyl donor 23 using N-iodosuccinimide and cata-
lytic amounts of TMSOTf. The formed disaccharide, a
key intermediate, was further elaborated by selective
Figure 1. Hyaluronic acid disaccharide repeating unit.
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UPDATES
has to be protected with a trichloroacetyl group[9a]
since glycosylation of the N-acetylated glucosamine
derivative does not proceed with high yields.[11] In
principle, tetraacetate 5 could be prepared using a
two-step procedure as described in the literature,[9a]
where the glucosamine hydrochloride 1 was first se-
lectively acetylated at the amino group. However, the
obtained N-trichloroacetylglucosamine was difficult to
recrystallize and the yields for large-scale preparation
were rather poor (<20%). We therefore decided to
prepare the N-trichloroacetylglucosamine using a
four-step sequence.[12] Tetraacetate 5 was prepared in
this way with an overall yield of 51% (4 steps from 1)
on a 0.1 mol scale. The glycosyl bromide 7 was
formed by the action of 33% HBr in acetic acid, and
subsequent glycosylation with allyl alcohol and
indium chloride[13] as activating reagent. Formation of
the b-glycoside 7 proceeded with very good selectivity
(a/b, 1:13). The obtained mixture of anomers could
be separated by column chromatography. The use of
tin tetrachloride was also tested as an activating re-
agent at this stage but the reaction proceeded unse-
lectively and a 1:1 mixture of a/b anomers was ob-
tained. A further 6 steps (see Supporting Informa-
tion) were necessary for selective protection of the
OH-4 and OH-6 and freeing of the OH-3. With this
route the glycosyl acceptor 13 was synthesized with a
22% yield over twelve steps in multigram quantities
(Scheme 2).
Synthesis of d-Thioglucoside Donor 23
The synthesis of donor 23 is depicted in Scheme 3.
Due to the incompatibility of the glucuronic methyl
ester moiety with many reactions, we chose to start
with a glucose derivative and to oxidize the C-6 posi-
Scheme 1. Retrosynthesis of oligosaccharides 30, 32 and 34
via the disaccharide acceptor 26.
deprotection and subsequent glycosylation via the tri- tion at a late stage of the synthesis. 1,2,4,6-tetra-O-
chloroacetimidate method. (Scheme 1).
acetyl-3-O-benzyl-b-d-glucopyranose 17 was elaborat-
ed in a three-step sequence[15] from 1,2,4,6-diisopropy-
lidene-d-glucofuranose 14. This sequence could be
performed on an 80 mmol scale with good yields (75–
83%).
Result and Discussion
Synthesis of d-Glucosamine Acceptor 14
The glucose derivative 17 was further converted
into thioglycoside 22 using a procedure described by
In our strategy, the glucosamine building block bears Nilsson et al.[16] Thioglycoside 18 was formed using
three orthogonal O-protecting groups at the positions ethanethiol and boron trifluoride etherate as promot-
C-1, C-4 and C-6, respectively, and the 2-amino group er. The glycosylation proceeded with moderate selec-
Scheme 2. Synthesis of the glycoside acceptor 13.
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Multi-Gram Synthesis of a Hyaluronic Acid Subunit and Synthesis of Fully Protected Oligomers
Scheme 3. Synthesis of the thioglycoside donor 23.
tivity (a/b, 1:2.5) and 53% yields for the desired b- and the formation of by-products. This observation
product. A further 5 steps (see Supporting Informa- was strongly dependent on the quality of the reagent
tion) were needed to selectively protect the OH-2, and may have been caused by traces of trifluorome-
OH-4 and OH-6. With this route the thioglycoside thansulfonic acid in the reagent. The PMB protecting
donor 23 was synthesized with a 26% yield over ten group of disaccharide 24 was then removed with
steps and on a 10-gram scale.
cerium ammonium nitrate (CAN) and the resulting
primary alcohol 25 was oxidized with periodic acid
and a catalytic amount of chromium trioxide, as de-
veloped by Seeberger et al.[9b] This reaction proceeded
smoothly without the formation of by-products but
Synthesis of Key Intermediate 26
Glycosyl acceptor 13 and thioglycoside donor 23 were only with partial conversion. The remaining starting
reacted with N-iodosuccinimide and catalytic amounts material could be recovered. The carboxylic acid in-
of
trimethysilyl
trifluoromethansulfonate termediate was directly converted into the methyl
(Scheme 4).[17] The reaction was fast and only minor ester with a solution of diazomethane in diethyl ether.
amounts of by-products were formed. The use of Disaccharide building block 26 was obtained with a
methyl trifluoromethansulfonate[18] as promoter was yield of 45% over four steps on a multigram scale.
also tested but longer reaction times (about 20 h) and
concomitant deprotection of the p-methoxybenzyl
(PMB) group was observed, resulting in lower yields Synthesis of Oligomers 30, 32 and 34
For the further glycosylations of building block 26 to
tetra-, hexa- and octasaccharides 30, 32 and 34 respec-
tively, the trichloroacetimidate method[19] was used
(Scheme 5). On one hand the allyl protecting group
of 26 was removed with palladium chloride[20] and
sodium acetate in wet acetic acid to give the hemiace-
tal 27, from which the trichloroacetimidate donor 28
was formed by the use of trichloroacetonitrile and
catalytic amounts of DBU as a base with 45% yields
over two steps. On the other hand the levulinoyl
group of 26 was cleaved with hydrazine acetate to
give the disaccharide acceptor 29 in 90% yields. After
glycosylation of 29 with 28, fully protected tetrasac-
charide 30 was obtained in 51% yield in amounts of
up to 1.5 g (Scheme 5). Trichloroacetimidate donor 28
was also coupled with the deprotected tetrasaccharide
31 and hexasaccharide 33 to give hexasaccharide 32
and octasaccharide 34 with 45% and 32% yields over
the two steps, respectively.
Deprotection of Dimer 31
Deprotection was performed on the tetrasaccharide
31 as a model for higher oligomers (Scheme 6). First,
the TCA group had to be changed into an acetyl
group. We explored an indirect transformation in
which the TCA group was cleaved with sodium meth-
Scheme 4. Synthesis of the disaccharide 26. Reaction condi-
tions: a) NIS, cat. TMSOTf, CH2Cl2, ꢀ308C, 72%; b) CAN,
CH2Cl2, room temperature; c) H5IO4, cat. CrO3, CH3CN/
H2O, 0 8C; d) diazomethane, CH2Cl2, room temperature,
62% (3 steps).
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Mickaꢀl Virlouvet et al.
UPDATES
Scheme 5. Synthesis of the tetra- 30, hexa- 32 and octasaccharides 34. Reaction conditions: a) PdCl2, NaOAc, AcOH/H2O,
room temperature, 79%; b) Cl3CCN, cat. DBU, CH2Cl2, 08C, 52%; c) N2H4·AcOH, CH2Cl2/MeOH, room temperature, 90%;
d) 28, cat. TMSOTf, CH2Cl2, 08C, 32–51%.
anolate and acetylated with acetic anhydride. Howev-
er, both the one-pot and the two-step procedures led
to decomposition of 31. Importantly, the reduction
with the couple Zn/AcOH in 1,4-dioxane gave rise to
the formation of the dimer 35 but only in poor yields
(20%). Since the reaction was very slow at room tem-
perature, we also tried different approaches. First, we
performed this step at 508C but unfortunately more
decomposition products were formed, which caused a
significant drop in yields. The direct reduction of the
trichloromethyl group into a methyl group with tribu-
tyltin hydride and catalytic amounts of AIBN as a
radical starter under reflux conditions also resulted in
the decomposition of the tetrasaccharide 31. In con-
trast, the reduction of the TCA group of the disac-
charide 26 under the same conditions led to a rapid
conversion in 78% yields. During the deprotection of
tetrasaccharide 31 we observed in MALDI mass spec-
trometry a decomposition product in the range of a
disaccharide subunit. This allowed us to conclude that
the TCA group was reduced but also the b-1,4 glyco-
syl bond of the tetrasaccharide was cleaved. The b-1,4
glycosyl is quite labile and the high temperatures
might cause its cleavage. We tried to perform this re-
action at lower temperatures of 708C but unfortu-
nately, also in this case we observed decomposition to
disaccharides subunits. Further optimization studies
Scheme 6. Deprotection of dimer 31. Reaction conditions: a)
are ongoing to improve the yields of the reduction.
Next, the allyl group and benzyl groups were cleaved
respectively with PdCl2 in a mixture of acetic acid and
Zn/AcOH, 1,4-dioxane, room temperature, 15%; b) PdCl2,
NaOAc, AcOH/H2O, room tempeature; c) H2, cat.
Pd(OH)2/C, MeOH, room temperature, (37%, 2 steps).
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Multi-Gram Synthesis of a Hyaluronic Acid Subunit and Synthesis of Fully Protected Oligomers
aqueous phase was extracted with THF (3ꢂ10 mL). The
water and subsequently with catalytic amount of
Pd(OH)2 on charcoal in the presence of hydrogen to
give 36.
combined organic phases were collected, dried over sodium
sulfate and evaporated to yield compound 35. The crude
product was purified by preparative HPLC (yield: 26 mg,
15%). MS-MALDI for C79H88N2O27 [M+Na]: m/z=1519.8
Methyl 2-O-benzoyl-b-d-glucopyranosyluronate)-(1!3)-
(4-O-acetyl-2-deoxy-2-acetamido-b-d-glucopyranosyl)-(1!
4)-(methyl 2-O-benzoyl-b-d-glucopyranosyluronate)-(1!3)-
4-O-acetyl-2-deoxy-2-acetamido-b-d-glucopyranoside (36):
Allyl (methyl 2-O-benzoyl-3-O-benzyl-b-d-glucopyranosyl-
Conclusions
Herein we describe the synthesis of the modular dis-
accharide subunit of hyaluronic acid on a multi-gram
scale, as well as the synthesis of the corresponding,
fully protected, tetra-, hexa- and octamers on a scale
of several 100 mg. The first deprotection strategy was
demonstrated on the tetrasaccharide and gave promis-
ing results to achieve the full deprotection.
AHCTUNGTRENNUNG
benzyl-b-d-glucopyranosyluronate)-(1!3)-4-O-acetyl-6-O-
benzyl-2-deoxy-2-acetamido-b-d-gluco-pyranoside
(35)
(0.015 g, 0.01 mmol) was dissolved in AcOH (1 mL). Water
(0.2 mL) was added, followed by addition of NaOAc
(0.0125 g, 0.15 mmol) and PdCl2 (0.0035 g, 0.02 mmol), and
the mixture was stirred for 12 h at room temperature. The
reaction mixture was diluted with EtOAc and water, and
aqueous NaHCO3 was added until neutralization. The aque-
ous layer was extracted with EtOAc (3ꢂ10 mL), dried over
Na2SO4, filtered and concentrated under vacuum to yield
the product 36. The crude product was used without further
purification in subsequent reaction. MS-MALDI: for
C76H84N2O27 [M+Na]: m/z=1479.8.
Experimental Section
General Remarks
All reactions were performed in oven-dried glassware under
an argon atmosphere. Solvents and chemicals used were pur-
chased from commercial suppliers. Solvents were dried
under standard conditions. All materials were used without
further purification. Thin-layer chromatography (TLC) was
carried out on silica gel plates (silicagel 60, F254, Merck)
with detection by UV and visualized by Seebach solution.
Purification was performed with preparative chromatogra-
phy using normal-phase silica gel (silica gel 60, 230–400
mesh, Merck). IR spectra were recorded on a Bruker IFS88.
Crude methyl 2-O-benzoyl-3-O-benzyl-b-d-glucopyrano-
syluronate)-(1!3)-(4-O-acetyl-6-O-benzyl-2-deoxy-2-acet-
amido-b-d-glucopyranosyl)-(1!4)-(methyl 2-O-benzoyl-3-
O-benzyl-b-d-glucopyranosyluronate)-(1!3)-4-O-acetyl-6-
O-benzyl-2-deoxy-2-acetamido-b-d-glucopyranoside
(36)
was subsequently dissolved in MeOH (1 mL) and catalytic
amounts of Pd(OH)2 were added. The reaction mixture was
stirred at room temperature under a hydrogen atmosphere
(hydrogen balloon) overnight. After completion, the catalyst
was filtered and washed with MeOH. The filtrate was
evaporated to give the product 36. The crude product was
purified by preparative HPLC (4 mg, 37% over 2 steps).
MS-MALDI measurement for C48H60N2O27 [M+Na]: m/z=
1119.4.
1
Absorption is reported as the n value in cmꢀ1. H NMR and
13C NMR were recorded on a Bruker AM 400 spectrometer.
Chemical shifts are reported as d values (ppm). High-resolu-
tion mass spectra (HRMS) were determined on a Finnigan
MAT90.
Detailed experimental procedures and characterization
data for compounds 2–34 are given in the Supporting Infor-
mation.
Deprotection of Dimer 31
Allyl (methyl 2-O-benzoyl-3-O-benzyl-b-d-glucopyranosyl-
uronate)-(1!3)-(4-O-acetyl-6-O-benzyl-2-deoxy-2-acetami-
Acknowledgements
do-b-d-glucopyranosyl)-(1!4)-(methyl
2-O-benzoyl-3-O-
This work was supported by a grant to JPS from the Deut-
sche Forschungsgemeinschaft under the auspices of the
Schwerpunkt Program SPP1190 (tumor-vessel interface).
benzyl-b-d-glucopyranosyluronate)-(1!3)-4-O-acetyl-6-O-
benzyl-2-deoxy-2-acetamido-b-d-glucopyranoside (35): Allyl
(methyl
2-O-benzoyl-3-O-benzyl-b-d-glucopyranosyluro-
nate)-(1!3)-(4-O-acetyl-6-O-benzyl-2-deoxy-2-trichloroace-
tamido-b-d-glucopyranosyl)-(1!4)-(methyl 2-O-benzoyl-3-
O-benzyl-b-d-glucopyranosyluronate)-(1!3)-4-O-acetyl-6-
O-benzyl-2-deoxy-2-trichloroacetamido-b-d-glucopyranoside References
(31) (0.20 g, 0.13 mmol) was dissolved in 1,4-dioxane (5 mL)
and AcOH (0.37 mL, 50 equiv.) and activated zinc powder
(0.42 g, 50 equiv.) were added. The reaction mixture was
stirred at room temperature until complete conversion
(TLC/MALDI control). In those cases where the reaction
was not completed, again the same amounts of AcOH and
activated zinc powder were added. This procedure was re-
peated several times until complete conversion. After com-
pletion, the reaction mixture was diluted with water and
neutralized with saturated sodium bicarbonate solution. The
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