Divergent heparin oligosaccharide synthesis with preinstalled sulfate esters

Article abstract of DOI:10.1002/chem.201101108

Traditional chemical synthesis of heparin oligosaccharides first involves assembly of the full length oligosaccharide backbone followed by sulfation. Herein, we report an alternative strategy in which the O-sulfate was introduced onto glycosyl building blocks as a trichloroethyl ester prior to assembly of the full length oligosaccharide. This allowed divergent preparation of both sulfated and non-sulfated building blocks from common advanced intermediates. The O-sulfate esters were found to be stable during glycosylation as well as typical synthetic manipulations encountered during heparin oligosaccharide synthesis. Furthermore, the presence of sulfate esters in both glycosyl donors and acceptors did not adversely affect the glycosylation yields, which enabled us to assemble multiple heparin oligosaccharides with preinstalled 6-O-sulfates. Copyright

Full text of DOI:10.1002/chem.201101108

DOI: 10.1002/chem.201101108
Divergent Heparin Oligosaccharide Synthesis with Preinstalled Sulfate Esters
Gopinath Tiruchinapally, Zhaojun Yin, Mohammad El-Dakdouki, Zhen Wang, and
Xuefei Huang*[a]
Abstract: Traditional chemical synthe-
sis of heparin oligosaccharides first in-
volves assembly of the full length oligo-
saccharide backbone followed by sulfa-
tion. Herein, we report an alternative
strategy in which the O-sulfate was in-
troduced onto glycosyl building blocks
as a trichloroethyl ester prior to assem-
bly of the full length oligosaccharide.
This allowed divergent preparation of
both sulfated and non-sulfated building
blocks from common advanced inter-
mediates. The O-sulfate esters were
found to be stable during glycosylation
as well as typical synthetic manipula-
tions encountered during heparin oligo-
saccharide synthesis. Furthermore, the
presence of sulfate esters in both glyco-
syl donors and acceptors did not ad-
versely affect the glycosylation yields,
which enabled us to assemble multiple
heparin oligosaccharides with preinstal-
led 6-O-sulfates.
Keywords: carbohydrates · glycosy-
lation · heparin oligosaccharides ·
sulfation · synthetic methods
Introduction
it is often very difficult to completely sulfate all the desired
positions. Due to the highly charged nature of the sulfation
products, it is extremely challenging to separate and purify
the partially sulfated oligosaccharides, which can lead to
very low yields (<10%) as encountered in sulfation of a
heparin heptasaccharide and a nonasaccharide.[17] Another
disadvantage is that to access differentially sulfated oligosac-
charides, multiple protected building blocks are required,
which can be very time consuming to prepare, limiting our
abilities to generate diverse heparin structures.
As an alternative to the post-glycoassembly sulfation
strategy, we became interested in investigating the usage of
pre-sulfated building blocks for the assembly of full length
oligosaccharides. To accomplish this, sulfates will be prein-
stalled onto glycosyl building blocks as sulfate esters, which
reduce the need for selectively removable protective groups
in oligosaccharide synthesis. Furthermore, to expedite build-
ing block preparation, a divergent strategy is designed in
which both the non-sulfated and sulfated glycosyl building
blocks can be obtained from a common intermediate.
Heparin and heparan sulfate belong to the glycosaminogly-
can family of carbohydrates. They consist of disaccharide re-
peating units of glucosamine a-1,4 linked to a pyranosoic
acid, which can be either glucuronic or iduronic acid. The 2-
O of the pyranosoic acid, 3-O, 6-O as well as the 2-N posi-
tions of the glucosamine can be sulfated, leading to a large
diversity of possible sequences.[1,2] Heparin and heparan sul-
fates play important roles in many biological events, such as
pathogen infection, blood coagulation, and tumor metasta-
sis.[1–5] Their biological functions can be critically dependent
on the sulfation patterns and backbone sequences.[6] This
has led to intense current interests in preparing heparin and
heparan sulfate oligosaccharides with defined sulfation and
backbone structures to decipher their structure–activity rela-
tionships.
Since the ground breaking synthesis of the anti-coagulant
heparin pentasaccharide,[6,7] significant advancements have
been achieved in heparin oligosaccharide assembly.[8–19] Typi-
cally, to prepare different sulfation patterns, building blocks
with strategically placed protective groups are utilized. Fol-
lowing assembly of the full length oligosaccharides, these
protective groups are selectively removed and the newly li-
berated hydroxyl and amino groups are sulfated. With the
need to introduce a large number of sulfates simultaneously,
Several sulfate esters[20] including phenyl,[21–23] ethyl,[24] tri-
fluoroethyl,[25–27] neopentyl,[28] isobutyl,[28] and trichloroethyl
(TCE) sulfates[29–32] have been explored in carbohydrate
chemistry. The majority of these studies were focused on the
preparation and deprotection of sulfate esters with several
reports investigating monosaccharide glycosylations.[25,31,32]
Since the outcome of glycosylation in complex oligosacchar-
ide synthesis can be quite different from the simpler model
systems,[33–37] we set out to test the feasibility of using sul-
fate-ester-containing building blocks in the synthesis of com-
plex oligosaccharides such as heparin and establish the com-
patibility of sulfate esters with the synthetic manipulations
encountered in heparin oligosaccharide assembly.
[a] G. Tiruchinapally, Z. Yin, M. El-Dakdouki, Z. Wang, Prof. X. Huang
Department of Chemistry
Michigan State University, East Lansing
MI 48824 (USA)
Fax : (+1)517-353-1793
E-mail: xuefei@chemistry.msu.edu
Supporting information for this article (including detailed experimen-
tal procedures and selected NMR spectra) is available on the WWW
under http://dx.doi.org/10.1002/chem.201101108.
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FULL PAPER
Results and Discussion
ylcarbodiimide (EDC) and N,N-dimethylaminopyridine
(DMAP)-mediated esterification with levulinic acid generat-
ed the sulfated donor disaccharide 2. It was important to
use EDC for esterification, as a significant amount of the
sulfate ester was displaced by chloride when the more
common hydrochloride salt of EDC was employed.
Next, we tested the sulfation of diol 10, which was ob-
tained from 1 under the Zemplꢁn deacylation condition.
The primary hydroxyl group in 10 could be selectively sul-
fated with the salt 8 leading to
Among the several sulfate esters available,[20–28] we decided
to investigate the utility of TCE sulfates, which could be in-
troduced and deprotected under mild conditions in good
yields.[29,32] To access diverse heparin oligosaccharide struc-
tures by a divergent route, we designed an advanced disac-
charide 1 to serve as a key intermediate, which can be readi-
ly transformed into multiple building blocks (Scheme 1a).
the monosulfate ester 11 in
95% yield under microwave ir-
radiation (Scheme 2). Addition
of excess sulfation reagent 8 or
prolonged microwave irradia-
tion to sulfate the secondary
hydroxyl group led to decom-
position of the disaccharide,
which was presumably due to
participation of the anomeric
thioether group displacing the
neighboring 2-O sulfate ester.
A similar phenomenon was ob-
served during sulfation of the 2-
OH of a monosaccharide thio-
glycoside.[29]
With the monosulfate ester
11 in hand, the effects of 2-O
acyl groups on glycosylation
were explored. The free 2-OH
was first protected as a chloro-
AHCTUNGTRENGNaUN cetate (ClAc) ester (disacchar-
ide 12), because ClAc can be
selectively removed without af-
fecting other types of acyl pro-
Scheme 1.
Disaccharide 1 was formed ste-
reospecifically
through
the
treatment of glucoside 6[38] with
the azide bearing glucosamine
donor 5[9] promoted by pTol-
SOTf,[39] which was formed in
situ through the reaction of
pTolSCl with AgOTf. The
newly formed a linkage was
confirmed by NMR analysis
(3JH1’,H2’ =4.2 Hz,
173.9 Hz). Selective removal of
1JC1’,H1’
=
Scheme 2.
the acetate in 1 by magnesium
methoxide led to compound 7 (Scheme 1b), which was sul-
fated with the trichloroethyl sulfuryl dimethylimidazolium
triflate salt 8[29] over four hours (80% yield). The sulfation
reaction time was significantly shortened to 20 min when
the reaction was assisted with microwave irradiation, pro-
ducing 9 in an excellent 94% yield. Removal of p-methoxy-
benzyl ether (PMB) by 2,3-dichloro-5,6-dicyanobenzoqui-
none (DDQ) followed by 1-(3-dimethylaminopropyl)-3-eth-
tective groups.[40] Although the donor 12 was cleanly activat-
ed with pTolSCl/AgOTf, addition of acceptor 3 to the preac-
tivated 12 did not yield the desired tetrasaccharide. This
could be due to donor deactivation by the electron-with-
drawing sulfate ester, since we have demonstrated previous-
ly that electron withdrawing groups on the glycon ring could
significantly reduce the glycosylation power of donors even
after the donors were completely activated.[41] To test this
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X. Huang et al.
possibility, the non-sulfated acceptor 4 was subjected to gly-
cosylation with donor 12, which again failed to produce the
desired oligosaccharide products. Furthermore, the reaction
of donor 13 with acceptor 14 gave a very low yield (<5%)
of the desired product. These results suggested that 2-O
ClAc was not a suitable protective group for this glycosyla-
tion.
(Scheme 3b). To test the sulfated donor reactivity, the reac-
tion between sulfated disaccharide donor 2 with disacchar-
ide 4 was performed, which produced tetrasaccharide 18 in
82% yield with a sulfate ester at the non-reducing end
(Scheme 3d). Further chain extension was performed by
treating donor 2 with tetrasaccharide 19, to generate a hexa-
saccharide with two TCE sulfate esters (Scheme 3e). Inter-
estingly, upon completion of the glycosylation and warming
up the reaction mixture to 08C, the TBS moiety in the hexa-
saccharide was cleanly removed, presumably by the acid
generated during the reaction, leading to hexasaccharide 20
without the need of an additional deprotection step. All the
oligosaccharide products were fully characterized by NMR
spectroscopy and mass spectrometry. These results suggest
that the TCE sulfate ester does not significantly affect the
reactivities of both the glycosyl donor and the acceptor. The
usage of sulfate esters enabled us to access the differentially
sulfated oligosaccharide sequences starting from a single di-
The benzoyl (Bz) group was explored next as the 2-O pro-
tecting group. In contrast to the 2-O ClAc donor, the reac-
tion between the Bz-containing donor 2 and alcohol 14 pro-
ceeded smoothly producing disaccharide 15 in 80% yield
(Scheme 3a). To elongate the saccharide chain, the tert-bu-
tyldimethylsilyl (TBS) moiety in 15 needed to be removed.
This was accomplished by treating 15 with trimethylsilyl tri-
fluoromethansulfonate (TMSOTf) at À208C, whereas the
AHCTUNGTRENNUNG
rin, the generality of the sulfate ester method was tested in
the preparation of iduronic-acid-containing heparin oligo-
saccharides. To accomplish this, the key idosyl disaccharide
building block 21 was assembled by reacting the glucosa-
mine donor 5 with idosyl acceptor 22[9] (Scheme 4a). The se-
lective removal of the acetate group with magnesium meth-
oxide followed by sulfation and protective group adjustment
created the disaccharide donor 23. The idosyl containing di-
usage of HF·pyridine or tetra-n-butylACTHNUTRGNEaNUG mmonium fluoride
(TBAF) for this reaction led to partial displacement of the
sulfate ester. The presence of the TCE sulfate in acceptor 3
did not disarm it towards glycosylation, since the disacchar-
ide donor 16 successfully glycosylated 3 in 80% yield
Scheme 3.
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Divergent Heparin Oligosaccharide Synthesis
FULL PAPER
Scheme 4.
A
leading to tetrasaccharide 29 in 72% yield. Removal of the
TBS moiety was achieved by reacting 29 with TMSOTf and
the next task was to convert the azido groups in tetrasac-
charide 30 to amines. Although the Staudinger reduction of
azides using trimethylphosphine has been successfully ap-
plied in heparin oligosaccharide synthesis,[9,12,44] the analo-
gous reaction on tetrasaccharide 30 did not lead to the de-
sired amines. Instead, the tetrasaccharide backbone was
cleaved at the glucosamine and glucuronic acid junctions.
This was presumably due to the intramolecular deprotona-
tion of C5 proton of the glucuronic acid unit assisted by the
aza-ylide intermediate formed in the Staudinger reduction
followed by elimination of the glucosamine unit (Scheme 6).
The contrasting outcome of our reaction can be attributed
hol 14 with disaccharide 24 followed by the subsequent re-
moval of the TBS group (Scheme 4b). The reaction of the
sulfated donor 23 with the acceptor 26 produced the idosyl
tetrasaccharide 27 smoothly demonstrating TCE-sulfate-
containing idosyl building blocks are compatible with glyco-
assembly (Scheme 4c).
The deprotection of tetrasaccharide 17 began with the re-
moval of the two Lev moieties with hydrazine acetate with-
out affecting the TCE sulfate ester (Scheme 5). Conversion
of the newly liberated primary hydroxyl groups to carboxylic
esters was accomplished by 2,2,6,6-tetramethyl piperidine 1-
oxyl (TEMPO)/NaOCl-mediated oxidation followed by Pin-
nick oxidation[42] and treatment with phenyl diazomethane[43]
Scheme 5.
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X. Huang et al.
stall the O-sulfates at desired
positions prior to assembly of
the full length oligosaccharides.
This in turn allowed the genera-
tion of heparin sequences with
different sulfation patterns
from common disaccharide in-
termediates without resorting
to
differential
protective
groups. Further studies are on-
going in exploring the utility of
this method for the assembly of
larger heparin oligosaccharides.
Scheme 6. Proposed mechanism for trimethylphosphine-mediated backbone cleavage of the oligosaccharide
through a six-membered-ring transition state.
to the substrate structure, because the successful Staudinger
reductions from the literature were all performed on hepa-
rin oligosaccharides with carboxylates rather than carboxyl
Experimental Section
esters as in 30.[9,12,44] The pKa values of the C5 protons in
the carboxylate-bearing molecules would be higher due to
the anionic nature of the structures, thus less prone to de-
protonation by the aza-ylide. The difficulty in the Staudinger
reduction was overcome by a zinc/ammonium formate re-
duction of tetrasaccharide 30, which not only cleanly re-
duced the two azides but also simultaneously deprotected
the TCE sulfate ester[32] generating tetrasaccharide 31. The
free amine groups were then sulfated with SO3·pyridine fol-
lowed by saponification and catalytic hydrogenation leading
to heparin oligosaccharide 34 with full N-sulfation and a
6-O sulfation in the glucosamine unit closer to the reducing
end. Besides sulfation, the amines in 31 were acetylated
with acetic anhydride. Saponification and hydrogenation of
the amide 33 created tetrasaccharide 35 as an acetamide-
containing heparin tetrasaccharide. In an analogous manner,
the tetrasaccharides 18 and 27 were deprotected and modi-
fied leading to four more heparin tetrasaccharides (36–39).
The availability of these compounds with systematically
varied structures can be useful for probing the biological
roles of N-sulfation, 6-O sulfation and different backbone
structures in heparin oligosaccharides.
General experimental procedures: All reactions were carried out under
nitrogen with anhydrous solvents in flame-dried glassware, unless other-
wise noted. All glycosylation reactions were performed in the presence
of molecular sieves, which were flame-dried immediately before use in
the reaction under high vacuum. Glycosylation solvents were dried using
a solvent purification system and used directly without further drying.
The chemicals used were reagent grade as supplied, except where noted.
Analytical thin-layer chromatography was performed using silica gel 60
F254 glass plates. Compound spots were visualized by UV light (254 nm)
and by staining with a yellow solution containing CeACHTNURTGENNUG(NH4)2ACHTUGNTERN(NUGN NO3)6 (0.5 g)
and (NH4)6Mo7O244H2O (24.0 g) in 6% H2SO4 (500 mL). Flash column
chromatography was performed on silica gel 60 (230–400 mesh). NMR
spectra were referenced using Me4Si (0 ppm), residual CHCl3 (1H NMR
d=7.26 ppm, 13C NMR d=77.0 ppm), CD3OD (1H NMR d=3.30 ppm,
13C NMR d=49.00 ppm), CD3SOCD3 (1H NMR d=2.49 ppm, 13C NMR
d=39.5 ppm), D2O (1H NMR d=4.67 ppm). Peak and coupling constant
1
1
1
assignments are based on 1H NMR,
H H gCOSY and (or) H
13C
À
À
gHMQC experiments. All optical rotations were measured at 258C using
the sodium D line.
Characterization of anomeric stereochemistry: The stereochemistry of
the newly formed glycosidic linkages in the heparin-like tetrasaccharide
3
and intermediates are determined by J(H1,H2) through 1H NMR and/or
1JC1,H1 through gHMQC 2D NMR (without 1H decoupling). Smaller cou-
3
pling constants of J(H1,H2) (around 4 Hz) indicate a linkages and larger
3
coupling constants J(H1,H2) (7.2 Hz or larger) indicate b linkages. This can
1
1
be further confirmed by J(C1,H1) (ꢀ170 Hz) for a linkages and J(C1,H1)
AHCTUNGTRENNUNG
General procedure for preactivation-
based glycosylation: A solution of
donor (60 mmol) and freshly activat-
ed molecular sieve MS 4 ꢂ (200 mg)
in CH2Cl2 (2 mL) was stirred at
room temperature for 30 min, and
cooled to À788C, which was fol-
lowed by addition of AgOTf (47 mg,
180 mmol) dissolved in Et2O (1 mL)
without touching the wall of the
flask. After 5 min, orange-colored
pTolSCl (9.5 mL, 60 mmol) was added
to the solution through a microsyr-
Conclusion
inge. Since the reaction temperature was lower than the freezing point of
pTolSCl, it was added directly into the reaction mixture to prevent it
from freezing on the flask wall. The characteristic yellow color of
pTolSCl in the reaction solution dissipated rapidly within a few seconds
indicating the depletion of pTolSCl. After the donor was completely con-
sumed according to TLC analysis (about 5 min at À788C), a solution of
acceptor (60 mmol) in CH2Cl2 (0.2 mL) was slowly added dropwise
through a syringe (one equivalent of TTBP was added with acceptor if
We have demonstrated that TCE sulfate esters are compati-
ble with common synthetic manipulations encountered in
heparin oligosaccharide assembly. The presence of O-sulfate
esters in the glycosyl donor or the acceptor does not signifi-
cantly impact the glycosylation yields, enabling us to prein-
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Divergent Heparin Oligosaccharide Synthesis
FULL PAPER
the donor or acceptor contains PMB protective group). The reaction mix-
ture was warmed to À208C while stirring for over 2 h. Then, the mixture
was diluted with CH2Cl2 (20 mL) and filtered over Celite. The Celite was
further washed with CH2Cl2 until no organic compounds were observed
in the filtrate by TLC. All CH2Cl2 fractions were combined and washed
twice with a saturated aqueous solution of NaHCO3 (20 mL) and twice
with water (10 mL). The organic layer was collected and dried over
Na2SO4. After removal of the solvent, the desired oligosaccharide was
purified from the reaction mixture by silica gel flash chromatography.
0.014 mmol, 0.3 equiv per hydroxyl group) and a saturated aqueous solu-
tion of NaHCO3 (125 mL) were added to
a solution of alcohol
(0.045 mmol) in CH2Cl2 (1 mL) and H2O (170 mL) in an ice–water bath.
The resulted mixture was treated with an aqueous NaOCl solution
(150 mL, chlorine content not less than 5%. Fresh NaOCl solution should
be used (as the reaction became sluggish with old NaOCl) and continu-
ously stirred for 1 h as the temperature increased from 08C to room tem-
perature. It was crucial to closely monitor the progress of the reaction,
once the starting material was completely consumed, the reaction should
be quenched with glacial AcOH (150 mL) to pH 6–7. After adjusting the
reaction pH to near neutral, tBuOH (0.7 mL), 2-methyl 2-butene in THF
(2m, 1.4 mL), and a solution of NaClO2 (50 mg, 0.44 mm) and NaH2PO4
(40 mg, 0.34 mm) in H2O (0.2 mL) were added. The reaction mixture was
kept at room temperature for 1–2 h, diluted with saturated aqueous
NaH2PO4 solution (5 mL), and extracted with EtOAc (3ꢃ10 mL). The
organic layers were then combined and dried over MgSO4. After removal
of the solvent, the resulting residue was dissolved in CH2Cl2 (5 mL), fol-
lowed by addition of phenyldiazomethane in diethyl ether until the color
of the reaction remained red. The mixture was stirred at room tempera-
ture for 3 h and then was diluted with CH2Cl2 (50 mL). The organic
phase was washed with saturated NaHCO3 and then dried over Na2SO4.
The solvent was concentrated in vacuo and the compound was purified
by silica gel column chromatography.
General procedure for deprotection of PMB: The PMB-protected com-
pound (1.0 equiv) was dissolved in a mixture of CH2Cl2/H2O (for 0.5 g of
compound, 9 mL/1 mL) and the solution was cooled to 08C. DDQ
(1.5 equiv) was added to the reaction mixture and stirred at room tem-
perature for 4–6 h. The reaction was quenched with saturated NaHCO3
solution and diluted with CH2Cl2 (50 mL) and extracted with water. The
organic phase was washed with H2O until the solution became colorless.
The solvent was concentrated in vacuo and the compound was purified
by silica gel column chromatography.
General procedure for protection of 6-OH with Lev (for substrates with-
À
out the OSO3TCE group): The compound containing 6-OH (1 equiv)
was dissolved in CH2Cl2 (for 0.5 g of compound, 5 mL), followed by addi-
tion of levulinic acid (1.4 equiv), EDC·HCl (1.6 equiv) and DMAP
(1.0 equiv). The mixture was stirred at room temperature overnight and
was then diluted with CH2Cl2 (100 mL) and extracted with water. The or-
ganic phase was washed with saturated NaHCO3 solution, water and then
dried over Na2SO4. The solvent was concentrated in vacuo and the com-
pound was purified by silica gel column chromatography.
General procedure for saponification: The compound (for 100 mg of
compound, 1 equiv) in THF (2.5 mL) was cooled to À58C and LiOH
(1m, 12 equiv per COOBn) was added followed by H2O2 (150 equiv per
COOBn, 30%). The mixture was stirred at room temperature for 16 h
and then MeOH (6 mL) and LiOH (3m, 10 equiv per COOBn) were
added to the solution. The mixture was stirred for another 24 h, which
was acidified with acetic acid to pH 7–7.5 at 08C and concentrated to
dryness on a rotavapor. The resulting residue was purified by a short size
exclusion column (LH-20, 1:1, CH2Cl2/MeOH).
General procedure for protection of 6-OH with Lev (for substrates with
À
a
OSO3TCE group): The compound containing 6-OH (1 equiv) was dis-
solved in CH2Cl2 (for 0.5 g of compound, 5 mL), followed by addition of
levulinic acid (1.4 equiv), EDC (1.6 equiv) and DMAP (0.1 equiv). The
mixture was stirred at À158C to À108C for 6–8 h, then diluted with
CH2Cl2 (100 mL) and extracted with water. The organic phase was
washed with saturated NaHCO3 solution and then dried over Na2SO4.
The solvent was concentrated in vacuo and the compound was purified
by silica gel column chromatography.
General procedure for azide reduction to amine & deprotection of tri-
chloroethoxysulfate ester: To a solution of the azide-containing com-
pound (for 50 mg of compound, 1 equiv) in MeOH (1 mL) was added
ammonium formate solution (1m) in MeOH (2 mL) and Zn power (8–10
equiv). The mixture was stirred at room temperature overnight. The salts
were filtered and washed with MeOH. The mixture was concentrated to
dryness on a rotavapor and the resulting residue was purified through a
size exclusion column (LH-20) (1:1, CH2Cl2/MeOH).
General procedure for the deprotection of TBS (for substrates without a
À
OSO3TCE group): The TBS-protected compound was dissolved in pyri-
dine in a plastic vial (for 100 mg of compound, 1.5 mL). The mixture was
cooled to 08C, followed by addition of HF in pyridine (0.75 mL, 65–70%
in pyridine). The mixture was stirred at room temperature overnight. The
solvents were evaporated under vacuum and then the residue was diluted
in CH2Cl2 (100 mL) and then washed with saturated NaHCO3 solution.
The organic layer was dried over Na2SO4. The solvent was concentrated
in vacuo and the compound was purified by silica gel column chromatog-
raphy.
General procedure for selective N-sulfation: The mixture of NH2-con-
taining compound (for 20 mg of compound, 1 equiv), methanol (1 mL),
pyridine (0.3 mL), Et3N (0.2 mL), 1m Na2CO3 solution (0.15 mL), and
SO3·Py (5 equiv per NH2) was stirred at room temperature. The mixture
was stirred for 12 h and acidified with acetic acid to pH 8.0. In order to
remove salts, the mixture was filtered through a plug of cotton, washed
with methanol and concentrated to dryness on a rotavapor. The residue
was diluted with CH2Cl2/MeOH (1 mL/1 mL) and the resulting solution
was layered on the top of Sephadex LH-20 chromatography column that
was eluted with CH2Cl2/MeOH (1:1, v/v). The appropriate fractions were
evaporated to dryness under vacuo and the residue was used for next
step without further purification.
General procedure for deprotection of TBS (for substrates with
a
À
OSO3TCE group): The TBS-protected compound was dissolved in
CH2Cl2 (for 100 mg of compound, 2.5 mL). The mixture was cooled to
À208C, followed by addition of TMSOTf (4–6 equiv). The mixture was
stirred at À208C for 6–8 h, then the residue was diluted in CH2Cl2
(100 mL) and washed with saturated NaHCO3 solution. The organic
layer was dried over Na2SO4. The solvent was concentrated in vacuo and
the compound was purified by silica gel column chromatography.
General procedure for N-acetylation: The mixture of OH-, NH2-contain-
ing compound (for 12 mg of compound, 1 equiv), Ac2O (10 equiv per
NH2), Et3N (0.15 mL), and pyridine (1 mL) was stirred at room tempera-
ture for overnight. The solvent was evaporated to dryness at room tem-
perature on a rotavapor. The residue was dissolved with CH2Cl2/MeOH
(1 mL/1 mL) and the resulting solution was layered on the top of Sepha-
dex LH-20 chromatography column that was eluted with CH2Cl2/MeOH
(1:1, v/v). The appropriate fractions were evaporated to dryness under
vacuo and the residue was used for next step without further purification.
General procedure for deprotection of Lev: The Lev-protected com-
pound (1 equiv) was dissolved in pyridine (for 150 mg of compound,
2.4 mL) and acetic acid (1.6 mL). The mixture was cooled to 08C, fol-
lowed by addition hydrazine monohydrate (5 equiv for each Lev). The
mixture was stirred at 08C for 6 h and then quenched by acetone
(0.28 mL). The mixture was stirred at room temperature for another 1 h
and the acetone was evaporated under vacuum. The residue was diluted
with EtOAc (50 mL) and extracted with water. The organic phase was
washed with saturated NaHCO3 solution, HCl solution (10%), water,
and then dried over Na2SO4. The solvent was concentrated in vacuo and
the compound was purified by silica gel column chromatography.
General procedure for global debenzylation: The mixture of the Bn-con-
taining compound (for 12 mg of compound, 1 equiv), MeOH/H2O (4 mL/
2 mL), and Pd(OH)2 (50 mg) was stirred under H2 at room temperature
for 24 h, which was filtered through a plug of cotton to remove Pd. The
filtrate was concentrated to dryness under vacuum and then diluted with
H2O (15 mL). The aqueous phase was further washed with CH2Cl2
(5 mLꢃ3), EtOAc (5 mLꢃ3), and then the water was evaporated under
General procedure for 6-OH oxidation to carboxylic acid and benzyl
ester formation: An aqueous solution of NaBr (1m, 25 mL), an aqueous
solution of tetrabutylammonium bromide (1m, 50 mL), TEMPO (2.2 mg,
Chem. Eur. J. 2011, 17, 10106 – 10112
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Published online: July 22, 2011
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