Selectively protected disaccharide building blocks for modular synthesis of heparin fragments

Article abstract of DOI:10.1002/1099-0690(200207)2002:13<2033::AID-EJOC2033>3.0.CO;2-W

A modular approach for the synthesis of heparin fragments is described. Levulinoyl esters were employed to protect those hydroxy groups intended to be sulfated in the final product, while acetyl esters and benzyl ethers were used as the permanent protecting groups. A highly efficient chemoenzymatic reaction sequence was used for the deprotection of an O-sulfated fragment, while the final stage of the synthesis entailed a selective oxidation of a primary alcohol of a glucoside with TEMPO/NaOCl to give a glucuronic acid moiety. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.

Full text of DOI:10.1002/1099-0690(200207)2002:13<2033::AID-EJOC2033>3.0.CO;2-W

FULL PAPER
Selectively Protected Disaccharide Building Blocks for Modular Synthesis of
Heparin Fragments[‡]
Michael F. Haller[a] and Geert-Jan Boons*[a]
Keywords: Carbohydrates / Glycosylation / Heparin / Protecting groups
A modular approach for the synthesis of heparin fragments is
described. Levulinoyl esters were employed to protect those
hydroxy groups intended to be sulfated in the final product,
while acetyl esters and benzyl ethers were used as the per-
O-sulfated fragment, while the final stage of the synthesis
entailed a selective oxidation of a primary alcohol of a gluc-
oside with TEMPO/NaOCl to give a glucuronic acid moiety.
manent protecting groups. A highly efficient chemoenzym- ( Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
atic reaction sequence was used for the deprotection of an
2002)
Introduction
which a set of presynthesized disaccharide building blocks
resembling the different disaccharide motives found in HSs
can be used for the assembly of a wide range of sulfated
oligosaccharides. The proposed methodology would thus
make it possible to synthesize any putative HS fragment in
an efficient way for biological studies.
Heparan sulfates (HSs) are highly N- and O-sulfated
polysaccharides that are important for a large number of
biological processes, and many enzymes, growth factors, en-
zyme inhibitors, chemokines and extracellular matrix com-
ponents, cell adhesion molecules, and microbial proteins re-
quire HSs for their functions. Among others, HS-protein
complexes have been implicated in modulation of embry-
onic development (by growth factor modulation), inhibition
of blood coagulation, organization of the extracellular mat-
rix, angiogenesis, anchorage of cells, the presentation of en-
zymes and cytokines on cell surfaces, and as ‘‘co-receptors’’
in viral infections.[1]
We have reported[4] a strategy for the synthesis of HS
fragments previously. A sulfated oligosaccharide containing
glucoside and idoside moieties was synthesized first, and
this compound could then selectively be oxidized to the cor-
responding uronic acids. This approach avoided problems
caused by the presence of uronic acids, such as epimeriz-
ation of the C-5 position, poor glycosyl donating properties,
and difficulties with protecting group manipulations. An-
other important feature of our approach is that the primary
hydroxy groups of glucosamine moieties were protected as
TBDPS ethers to avoid oxidation. Their removal, however,
could easily be accomplished by treatment with HF in pyr-
idine.
Here we describe a set of protecting groups that can be
employed in combination with the approach described
above for modular synthesis of HS fragments. In particular,
it is shown that levulinoyl esters can be used to protect
those hydroxy groups that should be sulfated in the end
product. Furthermore, it was possible to protect those C-2
hydroxy groups that did not need sulfation as acetyl esters,
and these esters could be enzymatically removed at the end
of the synthetic sequence in the presence of the acid- and
base-sensitive sulfate esters by use of Pseudomonas lipase
type B.
The biosynthesis of HSs involves partial N-deacetylation
of
a protein-linked polysaccharide composed of β-
GlcA(1Ϫ4)GlcNAc repeating units, followed by N-sulf-
ation, substrate-directed epimerizations of glucuronic acid
to iduronic acid moieties, and Ϫ finally Ϫ selective O-sulfa-
tions.[2] Although these enzymatic modifications result in a
mixture of very complex polysaccharides, detailed struc-
tural studies have shown that HSs are composed of only
19 distinct disaccharide subunits, differing in their sulfation
pattern and in the presence of either -glucuronic or -idu-
ronic acid.
It has often been difficult to determine the oligosacchar-
ide sequence and sulfation pattern of an HS fragment re-
quired for activation or deactivation of a given protein.[3]
In order to address this important issue, we are developing
a modular approach for the chemical synthesis of HSs, in
[‡]
Part 2. Part 1: Ref.[4]
Results and Discussion
A key issue for the development of a modular approach
for heparin synthesis is the selection of a set of protecting
groups that meet the following requirements:
[a]
Complex Carbohydrate Research Center, University of
Georgia,
220 Riverbend Road, Athens, GA 30602, USA
Fax: (internat.) ϩ1-706/542-4412
E-mail: gjboons@ccrc.uga.edu
Eur. J. Org. Chem. 2002, 2033Ϫ2038  WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1434Ϫ193X/02/0713Ϫ2033 $ 20.00ϩ.50/0
2033

M. F. Haller, G.-J. Boons
FULL PAPER
i. the C-2 hydroxy-protecting groups of the glucose or protecting groups at the C-6 positions should be removed
idose moieties should allow stereoselective introduction of to give the desired HS fragment. This selective oxidation[4]
β(1Ǟ4)-glycosidic linkages, whereas the C-2 amino group can be performed with a catalytic amount of 2,2,6,6-tetra-
of the glucosamine derivatives need to be derivatized in methyl-1-piperidinyloxy (TEMPO) in the presence of so-
such a way that α(1Ǟ4)-glycosidic linkages can be formed, dium hypochlorite as the regenerating co-oxidant[8] without
ii. a protecting group that can be selectively removed to affecting sulfate esters.
reveal those hydroxy groups that need sulfation is required,
Disaccharide 18 was prepared in order to examine
iii. the protecting group scheme should allow the selective whether the proposed protecting group scheme and manip-
oxidation of C-6 hydroxy groups to give glucuronic and idu- ulations would be suitable for a modular synthesis of HS
ronic acid moieties,
fragments. The selectively protected disaccharide 11, the
iv. the removal of the permanent protecting groups precursor of target compound 18, was prepared from the
should be compatible with the presence of base- and acid- readily available glycosyl acceptor 8, with a non-participat-
labile sulfate esters, and finally
v. the protecting-group scheme should be applicable to group at C-6 (Scheme 1), and the known glycosyl donors
the synthesis of each of the 19 targeted disaccharides.
9[9] or 10,[10] which each have an acetyl group at C-2
ing azido group at C-2, a Lev ester at C-3, and a TBDPS
Our proposed generic protecting-group scheme is shown (Scheme 2).
in Figure 1. Thus, levulinoyl esters (Lev)[5] should be em-
ployed for those hydroxy groups that need sulfation. In
HSs, the C-3 and C-6 hydroxy groups of the glucosamine
and the C-2 hydroxy groups of the hexuronic acid moieties
may be sulfated, and so, depending on the sulfation pattern
of a targeted disaccharide building block, one or more of
these positions should be protected by one or more Lev
groups. An important feature of the Lev ester is that, when
present at the C-2Ј position, it should be able to direct the
formation of 1,2-trans-glycosides through neighboring-
group participation. In cases in which the C-2Ј position of
a building block does not need sulfation, an acetyl group
should be employed as a permanent protecting group. This
ester can also enter into neighboring-group participation,
but is stable under the conditions used for the removal of
the Lev groups. It was anticipated that, after introduction
of sulfate esters, the permanent acetyl groups should be re-
movable without the sulfate esters being affected.[6] The lat-
ter was a concern because sulfate esters are hydrolyzed un-
der relatively mild acidic or basic conditions. Benzyl ethers
should be used as protecting groups for primary hydroxy
groups intended to be oxidized to the carboxylic acids and
Scheme 1. Reagents and conditions: (a) MeONa, MeOH, room
temp., 2 h; (b) PhCH(OMe)2, camphorsulfonic acid (CSA), DMF,
60 °C, 16 h; (c) Ba(OH)2·8H2O, H2O, MeOH, reflux, 16 h (87%);
(d) TfN3, DMAP, MeOH, room temp., 16 h (73%); (e) levulinic
acid, DCC, DMAP, pyridine, room temp., 5 h; (f) 10% TFA in
DCM, room temp., 3 h (81%); (g) TBDPSCl, imidazole, DMF,
room temp., 16 h (73%)
Glycosyl acceptor 8 was prepared from the known methyl
for secondary hydroxy groups that should remain unsul- 2-acetamido-2-deoxy-β--glucopyranoside 1 (Scheme 1).[11]
´
fated in the final product. Finally, a tert-butyldiphenylsilyl Thus, deacetylation of 1 under Zemplen conditions, fol-
(TBDPS)[7] ether should be employed for the protection of lowed by selective protection of the 4,6-diol of the resulting
the C-6 position of the glucosamine residues. Thus, after 2 as a benzylidene acetal by treatment with benzaldehyde
the assembly of an oligosaccharide and the introduction of dimethyl acetal[12] in the presence of catalytic camphor-10-
sulfate esters, the acetyl esters and benzyl ethers should be sulfonic acid in DMF, gave partially protected 3. N-De-
removed. Next, the free C-6 hydroxy groups of the glucos- acetylation of 3 with Ba(OH)2·8H2O[13] afforded the free
ides and idosides should be selectively oxidized to the cor- amine 4 in 87% overall yield, and this compound was
responding uronic acids, and finally, the remaining TBDPS treated with the diazo transfer reagent triflic azide (TfN3)
in the presence of 4-(dimethylamino)pyridine (DMAP) in
methanol[14] to give the corresponding 2-azido-2-deoxy-
glucopyranoside 5 in 73% yield. The free hydroxy group of
5 was protected as a levulinoyl ester by treatment with levu-
linic acid, 1,3-dicyclohexylcarbodiimide (DCC), and
DMAP in pyridine,[5] and the benzylidene acetal of the re-
sulting compound 6 was cleaved by treatment with trifluo-
roacetic acid[15] in dichloromethane (DCM) to afford diol 7
in 81% overall yield. Finally, the primary hydroxy group of
7 was selectively protected as a TBDPS ether by treatment
Figure 1. Proposed protecting groups for building blocks
2034
with tert-butyldiphenylsilyl chloride (TBDPSCl) in the pres-
Eur. J. Org. Chem. 2002, 2033Ϫ2038

Disaccharide Building Blocks for Modular Synthesis of Heparin Fragments
FULL PAPER
Reduction of the azido moiety with concomitant
acetylation of the amino group was easily accomplished by
treatment with thioacetic S-acid[18] to give the N-acetamido
derivative 13 in 76% overall yield. It also proved feasible to
convert the azido group into an acetamido moiety first and
then to remove the levulinoyl ester under standard condi-
tions. Next, the C-3 hydroxy group of 13 was sulfated with
SO3·NEt3 in DMF[19] to give the sulfate ester 14. Surpris-
ingly, the NMR spectrum of 14 showed that the GlcNAc
moiety had anomerized during sulfation to the more stable
α configuration, as was evident from the chemical shift of
C-1 (δ ϭ 97.8 ppm) and the JC-1,1-H coupling constant of
173.5 Hz. It is known[20] that glycosides can anomerize to
their more stable α anomers under acidic conditions, but to
the best of our knowledge no such reaction has been de-
scribed for the reaction conditions used for sulfation.
The benzyl ethers of 14 were removed by catalytic hydro-
genation in the presence of Pd/C, and the acetyl group was
´
subsequently cleaved under Zemplen conditions. Unfortu-
nately, apart from the expected compound 16, a substantial
amount of material that had lost its sulfate ester was also
isolated. Several other conditions for removal of the acetyl
ester were investigated, but each attempt resulted either in
partial desulfation or in recovery of starting material. To
address this serious problem, the use of a lipase or esterase
to accomplish the selective deacetylation was considered.
These enzymes have often been used for the resolution of
chiral alcohols by selective hydrolysis of chiral acetyl es-
ters,[21] but have only seldom been employed for the depro-
tection of acid- or base-sensitive compounds. A library of
lipases was screened to select an enzyme that could perform
the selective deacetylation, and these studies showed that
Pseudomonas lipase type B in a phosphate buffer could
cleave the acetyl ester of 15 efficiently, to give 16 in 78%
yield. It was also found that pig liver esterase also cleaved
the secondary acetyl ester, although longer reaction times
were required. The transformation of 15 into 16 showed
that it was possible to cleave a sterically hindered acetyl
ester enzymatically without the sensitive sulfate esters be-
ing affected.
Scheme 2. Reagents and conditions: (a) 9, 8, NIS, TfOH, DCM, 4
˚
˚
A mol. sieves, 0 °C, 15 min (56%); (b) 10, 8, DCM, TMSOTf, 4 A
mol. sieves, Ϫ40 °C, 30 min (72%); (c) hydrazine acetate, DCM,
MeOH, room temp., 45 min (85%); (d) CH3C(O)SH, pyridine,
room temp., 18 h, (89%); (e) SO3·NEt3, DMF, room temp., 2d,
(87%); (f) Pd/C, H2, EtOH, 4 bar, 10 d, (84%); (g) Pseudomonas sp.
Lipase type B, 0.1  phosphate buffer, 0.2  NaCl, 3 m CaCl2,
pH ϭ 7.1, 37 °C, 3 d, (78%); (h) TEMPO, NaBr, NaOCl, H2O,
0 °C, (76%); (i) HF·pyridine in pyridine, room temp., 8 h, (81%)
ence of imidazole in DMF[7] to yield the glycosyl acceptor
8 in 73% yield.
The primary hydroxy group of 16 was selectively oxidized
with a catalytic amount of 2,2,6,6-tetramethylpiperidin-1-
Coupling of glycosyl acceptor 8 with glycosyl donor 9[9] oxyl (TEMPO) and NaOCl as the co-oxidant[8] to give gluc-
in the presence of the promoter system N-iodosuccinimide uronic acid 17. In this reaction, the reduced TEMPO was
(NIS) and trifluoromethanesulfonic acid (TfOH)[16] gave regenerated by slow addition of NaOCl, and it proved im-
the desired disaccharide 11 in 56% yield (Scheme 2). Only portant to maintain the pH between 8.5 and 9.0 by careful
the β anomer was formed in this coupling, due to neighbor- addition of aqueous sodium hydroxide. Because of the lipo-
ing group participation by the acetyl group. The yield of philic TBDPS ether in 17, purification of the product could
the glycosylation was improved by use of the trichloroacet- easily be accomplished by C-18 reversed-phase column
imidate donor 10[10] in combination with trimethylsilyl tri- chromatography. Finally, the TBDPS ether of 17 could be
fluoromethanesulfonate (TMSOTf) as the promoter, and in removed by treatment with HF·pyridine[4] to give the target
this case disaccharide 11 was obtained in 72% yield.
With the requisite disaccharide 11 in hand, attention was
focussed on its conversion into the sulfated HS fragment
16. Firstly, removal of the levulinate ester of 11 by treat-
ment with hydrazine acetate[17] in DCM gave clean forma-
tion of compound 12, showing that the Lev could be re-
compound 18 in 81% yield.
Conclusion
A set of reaction conditions and protecting groups have
moved without the other protecting groups being affected. been established for the modular synthesis of HS fragments.
Eur. J. Org. Chem. 2002, 2033Ϫ2038
2035

M. F. Haller, G.-J. Boons
FULL PAPER
pyranoside, which was used without further purification. The crude
product was suspended in water (60 mL) and methanol was added
until a homogeneous solution was obtained. Ba(OH)2·8H2O (5.3 g,
17 mmol) was added, and the mixture was heated (100 °C) for 18
h. The reaction mixture was diluted with water (600 mL) and fil-
tered, and H2SO4 was added (1.6 g, 17 mmol, in 100 mL H2O). The
precipitate was centrifuged, and the combined aqueous layers were
concentrated in vacuo to yield compound 4 as a white solid (3.28 g,
It has been shown that levulinoyl esters can be employed as
a temporary protecting group for those hydroxy groups that
will ultimately need sulfation. Furthermore, C-2 hydroxy
groups not requiring sulfation could be protected as acetyl
esters, and these groups could be removed at the end of the
synthetic sequence in the presence of the acid- and base-
sensitive sulfate esters by use of a lipase. A primary hydroxy
group of a sulfated disaccharide could be selectively oxid-
ized to a carboxylic acid with a catalytic amount of
87%). Rf ϭ 0.12 (10% methanol in dichloromethane). [α]2D0
Ϫ152.2 (c ϭ 0.24, chloroform). 1H NMR (300 MHz, CD3OD): δ ϭ
ϭ
TEMPO and NaOCl as the co-oxidant. It is to be expected 7.48Ϫ7.45 (m, 2 H, ArH), 7.32Ϫ7.30 (m, 3 H, ArH), 5.56 (s, 1 H,
3
CHPh), 4.31 (d, J1,2 ϭ 8.4 Hz, 1 H, 1-H), 4.26 (dd, 1 H, 6-Ha),
3.77 (dd, J ϭ 10.3, 6.1 Hz, 1 H, 6-Hb), 3.61Ϫ3.42 (m, 6 H, 3-H, 4-
H, 5-H, OCH3), 2.70 (dd, J ϭ 8.1, 3.3 Hz, 1 H, 2-H) ppm. 13C
NMR (75 MHz, CD3OD): δ ϭ 128.2Ϫ126.4 (Ar-C), 102.1 (CHPh),
104.0 (C-1), 68.3 (C-6), 72.1 (C-3), 81.6, 66.3 (C-4, C-5), 57.8 (C-
2), 56.3 (OCH3). MALDI-TOF MS: m/z ϭ 282 [M ϩ Na]ϩ.
that similar procedures and protecting groups used for the
preparation of target 18 should be employable for the pre-
paration of other disaccharides that resemble disaccharide
motives found in HS. Currently, we are employing the new
strategy for the assembly of a number of well defined HS
fragments. For this purpose, the anomeric center of the
building blocks is to be protected as a temporary allyl gly-
coside and the C-4Ј position as a p-methoxybenzyl ether.
Methyl 2-Azido-2-deoxy-4,6-O-benzylidene-β-D-glucopyranoside (5):
A freshly prepared solution of TfN3 (1 , 5 ϫ 20 mL) was added
dropwise over a period of 45 min to a solution of compound 4
(3.3 g, 11.7 mmol) and 4-(dimethylamino)pyridine (1.5 g,
12.8 mmol) in methanol (140 mL). After 18 h, TLC analysis
showed complete conversion of the starting material. The solvent
was concentrated in vacuo to a volume of ca. 5 mL, diluted with
ethyl acetate (200 mL), and washed subsequently with sat.
NaHCO3 (1 ϫ 20 mL), water (1 ϫ 20 mL), and brine (1 ϫ 20 mL).
The organic phase was dried (MgSO4) and filtered, and the solvents
were evaporated in vacuo. Silica gel column chromatography (elu-
ent: 1% methanol in dichloromethane) of the residue gave com-
pound 5 as a white solid (2.62 g, 73%). Rf ϭ 0.19 (1% methanol in
dichloromethane). [α]2D0 ϭ Ϫ37.8 (c ϭ 0.34, chloroform). 1H NMR
(300 MHz, CDCl3): δ ϭ 7.48Ϫ7.34 (m, 5 H, ArCH), 5.53 (s, 1 H,
PhCH), 4.37 (dd, J ϭ 10.4, 5.1 Hz, 1 H, 6-Ha), 4.30 (d, J ϭ 7.9 Hz,
1 H, 1-H) 3.93Ϫ3.21 (m, 8 H, 2-H, 3-H, 4-H, 5-H, 6-Hb, OCH3),
2.78 (br. s, 1 H, 3-OH) ppm. 13C NMR (75 MHz, CDCl3): δ ϭ
137.0 (ArCq), 129.6, 128.6, 126.5 (ArCH), 103.8 (CH, benzylidene),
102.2 (C-1), 80.9, 72.4, 68.8, 66.7, 66.4 (C-2, C-3, C-4, C-5, C-6),
57.7 (OCH3) ppm. C14H17N3O5: calcd. C 54.72, H 5.58, N 13.67;
found C 54.55, H 5.45, N 13.65. MALDI-TOF MS: m/z ϭ 307 [M
ϩ Na]ϩ.
Experimental Section
General Methods: All reactions except those in which water was
used as solvent were performed under argon. Reactions were mon-
itored by TLC on 60 F254 Kieselgel (EM Science), and the com-
pounds were detected by UV light and by charring with 5% sulfuric
acid in methanol. Column chromatography was performed on 60
silica gel (EM Science, 70Ϫ230 mesh), size exclusion column chro-
matography was performed on Sephadex LH-20 (methanol/dichlor-
omethane, 1:1, v/v, Pharmacia Biotech AB) or Sephadex G-25
(water elution) Reversed-Phase Columns. Chromatography was
performed on C18 silica gel (Waters Corp.) with a methanol/water
gradient as eluent. Solvents were removed under reduced pressure
at Ͻ 40 °C. All organic solvents were distilled from the appropriate
drying agents prior to use. Acetonitrile, dichloromethane, diethyl
ether, dioxane, N,N-dimethylformamide, pyridine, and toluene were
distilled from CaH2. THF was distilled from sodium directly prior
to use. Methanol was dried by refluxing with magnesium methox-
ide, distilled, and stored under argon. Molecular sieves were
crushed and activated in vacuo at 390 °C for 3 h prior to use.
NMR spectra were recorded with Varian 300 MHz, 500 MHz, and
600 MHz spectrometers equipped with Sun off-line editing work-
stations. Chemical shifts are reported in parts per million (ppm),
with tetramethylsilane as internal standard. Matrix-assisted Laser
Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spec-
trometry was performed with an HP MALDI-TOF spectrometer
with gentisic acid as matrix. Optical rotations were all measured
with a JASCO P-1020 polarimeter and [α]D values are given in units
Methyl 2-Azido-2-deoxy-3-O-levulinoyl-β-D-glucopyranoside (7):
N,NЈ-Dicyclohexylcarbodiimide (3.81 g, 18.4 mmol) and a catalytic
amount of 4-(dimethylamino)pyridine (10 mg) were added at room
temperature to a stirred solution of 5 (2.57 g, 8.4 mmol) and levul-
inic acid (2.10 g, 18.4 mmol) in pyridine (40 mL). A few minutes
later the formation of a precipitate was observed. After 18 h, TLC
analysis showed complete conversion of the starting material. The
precipitate was filtered off, the cake was washed with toluene (100
mL), and the combined organic phases were concentrated in vacuo
to yield methyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-
β--glucopyranoside (6) as a white solid (3.4 g, 8.3 mmol, 99%),
of deg cm2 mgϪ1
.
Methyl 2-Amino-2-deoxy-4,6-O-benzylidene-β-
D-glucopyranoside
(4): Methyl 3,4,6-tri-O-acetyl-2-N-acetamido-2-deoxy-β--glucopy- which was used without further purification. The crude product
ranoside (1, 4.85 g, 13.42 mmol) was dissolved in methanol (50
mL), and freshly prepared sodium methoxide was added until
pH ϭ 11 was reached. After 5 h, the reaction mixture was neutral-
was dissolved in 10% trifluoroacetic acid in dichloromethane (100
mL), and the solution was stirred at room temperature for 3 h. The
reaction mixture was concentrated in vacuo and coevaporated with
ized with Dowex Hϩ, the mixture was filtered, and the filtrate was toluene (5 ϫ 10 mL), and the residue was purified by silica gel
concentrated in vacuo. The residue was dissolved in N,N-dimethyl-
column chromatography (eluent: 1Ϫ10% methanol in dichlorome-
formamide (30 mL), and benzaldehyde dimethyl acetal (3.55 g, thane) to give the diol 7 as a clear oil (2.13 g, 81%). Rf ϭ 0.29 (5%
23.3 mmol) was added. The pH was adjusted to 4 with 10-cam-
phorsulfonic acid. The reaction mixture was heated (60 °C) for 18
h, neutralized with triethylamine, and then concentrated in vacuo
to yield methyl 2-acetamido-2-deoxy-4,6-O-benzylidene-β--gluco-
methanol in dichloromethane). [α]2D0 ϭ Ϫ31.2 (c ϭ 1.29, chloro-
1
form). H NMR (300 MHz, CDCl3): δ ϭ 4.87 (t, J ϭ 9.6 Hz, 1 H,
3-H), 4.30 (d, 3J1,2 ϭ 7.8 Hz, 1 H, 1-H), 3.88 (dd, J ϭ 12.0, 3.9 Hz,
1 H, 6-Ha), 3.87 (dd, J ϭ 11.7, 3.6 Hz, 1 H, 6-Hb), 3.69 (t, J ϭ
2036
Eur. J. Org. Chem. 2002, 2033Ϫ2038

Disaccharide Building Blocks for Modular Synthesis of Heparin Fragments
8.4 Hz, 1 H, 4-H), 3.57 (s, 3 H, OCH3), 3.43Ϫ3.33 (m, 2 H, 2-H,
1 H, 3-H), 4.80Ϫ4.46 (m, 8 H, 1Ј-H, 2Ј-H, PhCH2), 4.19 (d, 3J1,2 ϭ
FULL PAPER
5-H), 3.19 (br. s, 2 H, 4-OH, 6-OH), 2.98Ϫ2.48 (m, 4 H, CH2- 8.0 Hz, 1 H, 1-H), 4.03Ϫ3.23 (m, 2-H, 4-H, 5-H, 6-Ha,b, 3Ј-H, 4Ј-
CH2), 2.18 (s, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3): δ ϭ H, 5Ј-H, 6Ј-Ha,b, OCH3), 2.68Ϫ2.59, 2.29Ϫ2.25 (m, 4 H, CH2CH2),
98.8 (C-1), 71.6 (C-3), 70.5, 59.7 (C-2, C-5), 65.2 (C-4), 57.8 (C-6), 1.97, 1.74, 1.03 (3 s, 15 H, CH3, C(CH3)3] ppm. 13C NMR
53.2 (OCH3), 34.1, 23.9 (CH2CH2), 25.9 (CH3) ppm. C12H19N3O7:
(125 MHz, CDCl3): δ ϭ 172.6 (CϭO, lev), 169.3 (CϭO, acetyl),
calcd. C 45.42, H 6.04, N 13.24; found C 45.55, H 6.10, N 13.33. 137.8Ϫ128.3 (ArCq, ArCH), 102.6 (C-1), 100.8 (C-1Ј), 83.4, 77.7,
MALDI-TOF MS: m/z ϭ 317.3 [M ϩ Na]ϩ.
75.3, 75.4, 75.2, 75.1, 75.0, 74.3, 73.2, 72.9, 72.3, 68.8, 63.4, 60.6,
60.5 (C-3, C-4, C-5, C-6, C-2Ј, C-3Ј, C-4Ј, C-5Ј, C-6Ј, PhCH2), 29.5
(CH3, lev), 34.2, 26.9 (CH2CH2), 26.4 [C(CH3)3], 20.7 (CH3, acetyl)
ppm. C57H67N3O13Si (1030.24): calcd. C 66.45, H 6.55, N 4.08;
found C 66.39, H 6.55, N 4.15. MALDI-TOF MS: m/z ϭ 1031.9
[M ϩ Na]ϩ.
Methyl
noyl-β-
2-Azido-6-O-(tert-butyldiphenylsilyl)-2-deoxy-3-O-levuli-
-glucopyranoside (8): tert-Butylchlorodiphenylsilane (2.5
D
mL, 9.4 mmol) was added dropwise to a solution of compound 7
(2.13 g, 6.7 mmol) and imidazole (1.15 g, 16.8 mmol) in N,N-di-
methylformamide (5 mL). The mixture was stirred for 18 h at room
temperature and then poured into ice-cold water (500 mL) and ex-
tracted with dichloromethane (3 ϫ 100 mL). The combined organic
layers were dried (MgSO4) and filtered, and the filtrate was concen-
trated in vacuo. Silica gel column chromatography (eluent: 2% acet-
one in dichloromethane) of the residue yielded 8 as a clear oil
Methyl 4-O-(2-O-Acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl)-2-
azido-6-O-(tert-butyldiphenylsilyl)-2-deoxy-β- -glucopyranoside (12):
D
A solution of hydrazine acetate (81.4 mg, 0.88 mmol) in methanol
(2 mL) was added to a solution of disaccharide 11 (729 mg,
0.71 mmol) in dichloromethane (18 mL), and the resulting reaction
mixture was stirred until TLC analysis showed complete consump-
tion of starting material. The solvents were evaporated in vacuo,
and the residue was purified by silica gel column chromatography
(1Ϫ2% acetone in dichloromethane) to yield compound 12
(561.4 mg, 84%) as a white solid. Rf ϭ 0.15 (1% acetone in dichloro-
methane). [α]2D0 ϭ ϩ22.0 (c ϭ 1.17, chloroform). 1H NMR
(500 MHz, CDCl3): δ ϭ 7.72Ϫ7.14 (m, 25 H, ArCH), 4.99 (t, J ϭ
8.7 Hz, 1 H, 2Ј-H), 4.81Ϫ4.50 (m, 7 H, 1Ј-H, PhCH2), 4.15 (d,
(2.70 g, 72%). Rf ϭ 0.43 (3% acetone in dichloromethane). [α]2D0
ϭ
Ϫ18.2 (c ϭ 1.0, chloroform). 1H NMR (600 MHz, CDCl3): δ ϭ
7.69Ϫ7.68, 7.42Ϫ7.36 (m, 10 H, ArCH), 4.90Ϫ4.86 (t, J ϭ 9.6 Hz,
3
1 H, 3-H), 4.26 (d, J1,2 ϭ 7.8 Hz, 1 H, 1-H), 3.96Ϫ3.90 (2dd, J ϭ
9.6, 6.3 Hz, 2 H, 6-Ha,b), 3.74Ϫ3.70 (t, J ϭ 9.3 Hz, 1 H, 4-H), 3.55
(s, 3 H, OCH3), 3.42Ϫ3.35 (m, 2 H, 2-H, 5-H), 2.91Ϫ2.57 (m, 4 H,
CH2CH2), 2.17 (s, 3 H, CH3), 1.04 [s, 9 H, C(CH3)3] ppm. 13C
NMR (75 MHz, CDCl3): δ ϭ 207.7 (CϭO, ketone), 173.1 (CϭO,
ester), 135.9Ϫ127.9 (ArCq, ArCH), 102.9 (C-1), 76.4, 75.8, 70.3,
64.0, 63.9 (C-2, C-3, C-4, C-5, C-6), 57.2 (OCH3), 38.6, 28.4
(CH2CH2), 29.9 (CH3), 27.0 [C(CH3)3], 19.5 [C(CH3)3] ppm.
C28H37N3O7Si (555.69): calcd. C 60.52, H 6.71, N 7.56; found C
61.09, H 6.58, N 7.46. MALDI-TOF MS: m/z ϭ 555.6 [M ϩ Na]ϩ.
J1,2 ϭ 8.0 Hz, 1 H, 1-H), 3.87Ϫ3.53 (m, 12 H, 3-H, 4-H, 6-Ha,b
,
3Ј-H, 4Ј-H, 5Ј-H, 6Ј-Ha,b, OCH3), 3.34Ϫ3.28 (m, 2 H, 2-H, 5-H),
1.60 (s, 3 H, CH3, acetyl), 1.03 [s, 9 H, C(CH3)3] ppm. 13C NMR
(75 MHz, CDCl3): δ ϭ 169.4 (CϭO), 138.2Ϫ127.8 (ArCq, ArCH),
102.5 (C-1), 101.4 (C-1Ј), 83.1, 80.2, 78.0, 75.5, 75.4, 75.1, 74.8,
73.9, 73.8, 73.0, 68.6, 65.9, 62.0 (C-2, C-3, C-4, C-5, C-6, C-2Ј, C-
3Ј, C-4Ј, C-5Ј, C-6Ј, PhCH2), 56.9 (OCH3), 27.0 [C(CH3)3], 20.7
(CH3), 19.6 [C(CH3)3] ppm. C52H61N3O11Si (932.14): calcd. C
67.00, H 6.60, N 4.51; found C 67.19, H 6.66, N 4.61. MALDI-
TOF MS: m/z ϭ 932.8 [M ϩ Na]ϩ.
Methyl 4-O-(2-O-Acetyl-3,4,6-tri-O-benzyl-β-
azido-6-O-(tert-butyldiphenylsilyl)-2-deoxy-3-O-levulinoyl-β-
D-glucopyranosyl)-2-
D
-
glucopyranoside (11). Method A: A suspension of donor 9 (39.8 mg,
0.07 mmol), acceptor 8 (61 mg, 0.11 mmol), and molecular sieves
˚
(4 A, 110 mg) in dichloromethane (2 mL) was stirred at room tem-
Methyl 2-Acetamido-4-O-(2-O-acetyl-3,4,6-tri-O-benzyl-β-
pyranosyl)-6-(O-tert-butyldiphenylsilyl)-2-deoxy-β-
D
-gluco-
perature for 3 h. N-Iodosuccinimide (33.3 mg, 0.15 mmol) was ad-
ded, and the reaction mixture was cooled (0 °C). Trifluoromethane-
sulfonic acid (0.6 µL, 0.007 mmol) was added; after 15 min, TLC
analysis showed complete consumption of the donor. The reaction
was quenched by addition of triethylamine, the resulting mixture
was filtered, and the cake was washed with dichloromethane (100
mL). The combined organic phases were subsequently washed with
sat. NaHCO3 (1 ϫ 10 mL) and brine (1 ϫ 10 mL), dried (MgSO4),
and filtered, and the filtrate was concentrated in vacuo. Purification
of the residue by silica gel flash chromatography (eluent: 2% acet-
one in dichloromethane), followed by Sephadex LH-20 size-exclu-
sion column chromatography (eluent: 50% methanol in dichloro-
methane) yielded disaccharide 11 (42.8 mg, 56%) as a white solid.
D
-gluco-
pyranoside (13): Thioacetic S-acid (400 µL, 5.6 mmol) was added
to a solution of disaccharide 12 (548.3 mg, 0.59 mmol) in pyridine
(1 mL). After 18 h, the reaction mixture was concentrated in vacuo,
and the residue was purified by silica gel column chromatography
(eluent: 0Ϫ5% methanol in toluene) to yield disaccharide 13
(500 mg, 89%) as a white foam. Rf ϭ 0.14 (12% methanol in dichlo-
romethane). [α]2D0 ϭ ϩ22.2 (c ϭ 0.89, chloroform). 1H NMR
(500 MHz, CDCl3): δ ϭ 7.73Ϫ7.69, 7.39Ϫ7.23, 7.14Ϫ7.13 (m, 25
3
H, ArCH), 5.49 (d, J ϭ 8.0 Hz, 1 H, NH,), 4.99 (t, J ϭ 8.75 Hz,
1 H, 2Ј-H), 4.80Ϫ4.46 (m, 7 H, 1Ј-H, PhCH2), 3.91Ϫ3.37 (m, 14
H, 2-H, 3-H, 4-H, 5-H, 6-Ha,b, 3Ј-H, 4Ј-H, 5Ј-H, 6Ј-Ha,b, OCH3),
2.00, 1.61 (2 s, 6 H, CH3), 1.02 [s, 9 H, C(CH3)3] ppm. 13C NMR
(75 MHz, CDCl3): δ ϭ 170.7, 169.5 (CϭO), 138.3Ϫ127.9 (ArCq,
ArCH), 101.3, 101.2 (JC,1-H ϭ 163.9 Hz, 164.5 Hz, C-1β, C-1Јβ),
83.1, 80.3, 77.9, 77.4, 75.4, 75.3, 75.1, 74.9, 73.8, 73.0, 71.8, 68.6,
62.2 (C-2, C-3, C-4, C-5, C-6, C-2Ј, C-3Ј, C-4Ј, C-5Ј, C-6Ј, PhCH2),
56.7 (OCH3), 27.0 [C(CH3)3], 24.0 (NHAc), 20.7 (CH3), 19.5
[C(CH3)3] ppm. C54H65NO12Si (948.18): calcd. C 68.40, H 6.91, N
1.48; found C 67.91, H 7.21, N 1.87. MALDI-TOF MS: m/z ϭ
948.0 [M ϩ Na]ϩ.
Method B:
A suspension of trichloroacetimidate 10 (1.18 g,
1.9 mmol), acceptor 8 (788.8 mg, 1.4 mmol), and molecular sieves
˚
(4 A, 200 mg) in dichloromethane was stirred at room temperature
for 30 min. After this had been cooled to Ϫ40 °C, trimethylsilyl
trifluoromethanesulfonate (30 µL, 0.17 mmol) was added. After
15 min, the reaction was quenched by addition of triethylamine,
the mixture was filtered, and the cake was washed with dichlorome-
thane (50 mL). The filtrate was concentrated in vacuo, and the
residue was purified by silica gel column chromatography (eluent:
20% ethyl acetate in hexanes), followed by size exclusion column
chromatography over Sephadex LH-20 to yield disaccharide 11
Methyl
glucopyranosyl)-6-(O-tert-butyldiphenylsilyl)-2-deoxy-3-O-sulfo-α-
D-glucopyranoside Sodium Salt (14): Sulfur trioxideϪtriethylamine
2-Acetamido-4-O-(2-O-acetyl-3,4,6-tri-O-benzyl-l-β-D-
(1.04 g, 72%). Rf ϭ 0.28 (3% acetone in dichloromethane). [α]2D0
ϭ
1
ϩ21.6 (c ϭ 1.54, chloroform). H NMR (500 MHz, CDCl3): δ ϭ complex (250 mg, 1.38 mmol) was added to a heated solution (55
7.73Ϫ7.70, 7.40Ϫ7.23 (m, 25 H, ArCH), 4.97Ϫ4.88 (t, J ϭ 9.6 Hz, °C) of disaccharide 13 in N,N-dimethylformamide (1 mL). After 18
Eur. J. Org. Chem. 2002, 2033Ϫ2038
2037

M. F. Haller, G.-J. Boons
FULL PAPER
3
h, the heating was stopped, sodium acetate (200 mg) in water (20 NMR (500 MHz, D2O): δ ϭ 4.67 (d, J1,2 ϭ 3.5 Hz, 1 H, 1-H),
3
mL) was added, and the reaction mixture was stired for 1 h. The
solvents were evaporated in vacuo and coevaporated with water
4.53 (d, J1Ј,2Ј ϭ 7.5 Hz, 1 H, 1Ј-H), 4.47 (t, J ϭ 10.0 Hz, 1 H, 3-
H), 4.02 (dd, J ϭ 10.0, 3.5 Hz, 1 H, 2-H), 3.90Ϫ3.72 (m, 5 H, 4-
(5 ϫ 10 mL) to remove remaining triethylamine. Purification on H, 5-H, 6-Ha,b, 5Ј-H), 3.48Ϫ3.38 (m, 2 H, 4Ј-H, 3Ј-H), 3.33Ϫ3.28
Sephadex LH-20 (eluent: 50% methanol in dichloromethane) gave
(m, 4 H, 2Ј-H, OCH3), 1.89 (s, 3 H, CH3) ppm. 13C NMR
(125 MHz, D2O): δ ϭ 174.6 (CϭO), 174.1 (CO2Na), 101.0 (C-1),
compound 14 (271 mg, 78%) as a clear glass. [α]2D0 ϭ Ϫ12.6 (c ϭ
0.1, chloroform). 1H NMR (500 MHz, CDCl3): δ ϭ 7.63Ϫ6.97 (m, 97.6 (C-1Ј), 77.4 (C-3), 75.4, 75.0, 72.7, 72.5, 71.0, 70.8, 59.7 (C-4,
3
25 H, ArCH), 5.33 (t, J ϭ 8.7 Hz, 1 H, 2Ј-H), 5.12 (d, JNH,H
ϭ
C-5, C-6, C-2Ј, C-3Ј, C-4Ј, C-5Ј), 55.3 (OCH3), 52.7 (C-2), 22.0
3.5 Hz, 1 H, NH), 4.69Ϫ4.26 (m, 9 H, 1-H, 3-H, 1Ј-H, PhCH2), (NHAc) ppm.
4.04Ϫ3.97 (m, 2 H, 4-H, 4Ј-H), 3.92Ϫ3.47 (m, 6 H, 2-H, 5-H, 6-
Ha,b, 6Ј-Ha,b), 3.39 (t, J ϭ 9.6 Hz, 1 H, 3Ј-H), 3.22Ϫ3.15 (m, 4 H,
[1] [1a]
[1b]
5Ј-H, OCH3), 1.84, 1.57 (2 s, 6 H, CH3), 1.03 [s, 9 H, C(CH3)3]
ppm. 13C NMR (75 MHz, CDCl3): δ ϭ 172.1, 171.5 (CϭO),
139.0Ϫ126.4 (ArCq, ArCH), 102.3 (JC,1-H ϭ 164.8 Hz, C-1Јβ), 97.8
(JC,1-H ϭ 173.5 Hz, C-1α), 83.4, 77.4, 75.8, 75.5, 74.7, 74.3, 74.2,
73.9, 73.4, 71.1, 67.1, 61.2, 55.2, 54.7 (C-2, C-3, C-4, C-5, C-6, C-
2Ј, C-3Ј, C-4Ј, C-5Ј, C-6Ј, PhCH2, OCH3), 26.9 [C(CH3)3], 23.3
(NHAc), 20.7 (CH3), 19.3 [C(CH3)3] ppm.
U. Lindahl, Haemostasis 1999, 38.
S. Tumova, A.
Woods, J. R. Couchman, Int. J. Biochem. Cell Biol. 2000, 32,
269.
[2] [2a]
H. E., Conrad, Heparin-Binding Proteins, Academic Press,
London, 1998. [2b] L. Kjellen, U. Lindahl, Annu. Rev. Biochem.
1991, 60, 443.
1987, 43, 51.
´
[2c]
B. Casu, Adv. Carbohydr. Chem. Biochem.
I. Capila, R. J. Lindhardt, Angew. Chem. Int.
[2d]
Ed. 2002, 41, 390.
[3]
[4]
[5]
[6]
[7]
`
R. R. Vives, D. A. Pye, M. Salmivirta, J. J. Hopwood, U. Lind-
Methyl 2-Acetamido-2-deoxy-3-O-sulfo-4-O-(β-D-glucopyranosyl-
ahl, J. T. Gallagher, Biochem. J. 1999, 339, 767.
M. Haller, G.-J. Boons, J. Chem. Soc., Perkin Trans. 1 2001,
814.
J. H. van Boom, P. M. J. Burgers, Tetrahedron Lett. 1976, 19,
4875.
uronic acid)-α- -glucopyranoside Disodium Salt (18): A suspension
D
of disaccharide 14 (15.5 mg, 14.8 µmol) and Pd/C (10 mg) in EtOH
(5 mL) under H2 was stirred for 5 d until TLC analysis showed
complete conversion into one product. The reaction mixture was
filtered, and the cake was washed thoroughly with 50% MeOH in
chloroform. Evaporation of the solvents in vacuo yielded com-
pound 15 (9.7 mg, 84%), which was used without further purifica-
tion. Crude 15 (9.7 mg, 12.4 µmol) was dissolved in phosphate buf-
fer (500 µL, 0.1 , 0.2 m NaCl, 3 m CaCl2, pH ϭ 7.1), and
Pseudomonas sp. lipase type B (139 U/mg, 10 mg) was added. After
the mixture had been shaken for 3 d at 37 °C, TLC analysis indic-
ated complete conversion of starting material. The enzyme was fil-
tered off and the cake was washed repeatedly with methanol/water
(15 mL, 1:1, v/v). The combined extracts were concentrated in va-
cuo, and residual enzyme was removed by purification on a re-
versed-phase C-18 column (eluent: H2O, then 30% methanol in
H2O). An aqueous NaOCl (13%) solution was adjusted to pH ϭ
8.5 with 4  aq. HCl and added dropwise to a cooled (0 °C) solu-
tion of 16 (9 mg, 98%, 12.2 µmol), TEMPO (0.22 mg, 1.4 µmol),
and NaBr (0.6 mg, 5.5 µmol) in H2O (350 µL). The pH of the
resulting solution was carefully maintained at 8.5 Ϯ 0.5 by addition
of 1  NaOH. Addition of the NaOCl solution and NaOH was
continued until the pH remained constant for a prolonged period
of time upon addition of NaOCl. The reaction mixture was directly
K. C. Nicolaou, N. J. Bockovich, D. R. Carcanague, J. Am.
Chem. Soc. 1993, 115, 8843.
S. Hanessian, P. Lavallee, Can. J. Chem. 1975, 53, 2975.
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[8b]
N. J. Davis, S. L. Flitsch, Tetrahedron Lett. 1993, 34, 1181.
A. E. J. de Nooy, A. C. Besemer, H. van Bekkum, Car-
bohydr. Res. 1995, 269, 89.
[9]
˚
H. B. Boren, G. Ekborg, K. Eklind, P. J. Garegg, A. Pilotti,
C.-G. Swahn, Acta Chem. Scand. 1973, 27, 2639.
R. R. Schmidt, G. Effenberger, Liebigs Ann. Chem. 1987, 825.
A. Moggridge, C. Neuberger, J. Chem. Soc. 1938, 745.
F. Sugawara, H. Nakayama, T. Ogawa, Carbohydr. Res. 1982,
108, C5ϪC9.
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[11]
[12]
[13]
S. Fujita, M. Numata, M. Sugimoto, T. Ogawa, Carbohydr.
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P. B. Alper, S.-C. Hung, C.-H. Wong, Tetrahedron Lett.
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1996, 34, 6029.
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applied to
a reversed-phase chromatography column (eluent
J. C. Jacquinet, Carbohydr. Res. 1990, 199, 153.
0Ϫ30% methanol in H2O) to give compound 17 (7.1 mg, 76%).
HF·pyridine (5 µL; 70% HF in pyridine) was added to a cooled (0
°C) solution of 17 (5 mg, 6.5 µmol) in pyridine (500 µL). The mix-
ture was allowed to warm to room temperature and was stirred for
18 h. The solution was neutralized with solid NaHCO3 (9 mg), di-
luted with methanol (5 mL), and filtered, and the solvents were
evaporated in vacuo. The residue was purified by reversed-phase
column chromatography (eluent: H2O) to give disaccharide 18
[19] [19a]
[19b]
J. R. Turvey, T. P. Williams, J. Chem. Soc. 1963, 2242.
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K. M. Koeller, C.-H. Wong, Nature 2001,
Received December 21, 2001
[O01597]
1
(2.8 mg, 81%) as a white solid. [α]2D0 ϭ Ϫ1.47 (c ϭ 0.75, H2O). H
2038
Eur. J. Org. Chem. 2002, 2033Ϫ2038