Solid-phase synthesis of a branched hexasaccharide using a highly efficient synthetic strategy

Article abstract of DOI:10.1021/jo016018p

The solid-phase synthesis of branched lacto-N-neohexaose derivative 1 occurring in human milk is described. The new building block of lactose 3 bearing the orthogonal temporary hydroxy protecting groups 9-fluorenylmethyloxycarbonyl (Fmoc) and levulinoyl (Lev) has been prepared. Its use, together with that of lactosamine donor 4, glucosamine donor 5, and O-galactosyl trichloroacetimidate 6, has enabled the preparation of hexasaccharide 22 following two different approaches in excellent overall yield (43%, 90% per step over eight steps). An additional key feature of this work is the successful use of newly prepared ester-type linker 2, having a benzylic spacer connected to the anomeric oxygen. This linker presents the advantage of producing a benzylic anomeric moiety after cleavage from the polymer support, which could be easily removed to obtain the unprotected oligosaccharide 1.

Full text of DOI:10.1021/jo016018p

8540
J . Org. Chem. 2001, 66, 8540-8548
Solid -P h a se Syn th esis of a Br a n ch ed Hexa sa cch a r id e Usin g a
High ly Efficien t Syn th etic Str a tegy
Fabien Roussel, Mohamed Takhi, and Richard R. Schmidt*
Fachbereich Chemie, Universita¨t Konstanz, Fach M 725, D-78457 Konstanz, Germany
richard.schmidt@uni-konstanz.de
Received August 9, 2001
The solid-phase synthesis of branched lacto-N-neohexaose derivative 1 occurring in human milk is
described. The new building block of lactose 3 bearing the orthogonal temporary hydroxy protecting
groups 9-fluorenylmethyloxycarbonyl (Fmoc) and levulinoyl (Lev) has been prepared. Its use,
together with that of lactosamine donor 4, glucosamine donor 5, and O-galactosyl trichloroace-
timidate 6, has enabled the preparation of hexasaccharide 22 following two different approaches
in excellent overall yield (43%, 90% per step over eight steps). An additional key feature of this
work is the successful use of newly prepared ester-type linker 2, having a benzylic spacer connected
to the anomeric oxygen. This linker presents the advantage of producing a benzylic anomeric moiety
after cleavage from the polymer support, which could be easily removed to obtain the unprotected
oligosaccharide 1.
In tr od u ction
n-pentenyl glycosides12 have also been explored for solid-
phase oligosaccharide synthesis.
Oligosaccharides are known to be important in a
multitude of biological processes; therefore, they have
attracted a lot of interest in recent years.1 However,
contrary to oligopeptides2 and oligonucleotides3 which are
routinely constructed on automated synthesizers employ-
ing polymer-supported synthesis, no general synthetic
strategy has yet emerged for solid-phase oligosaccharide
synthesis.4 The construction of complex oligosaccharides
in solution is quite laborious. Though their synthesis on
a solid phase remains a challenging task, this method
would present several advantages over solution-phase
techniques. Notably, an excess of reagents can be used
to drive reactions to completion, and syntheses can be
accomplished faster with simpler purification procedures.
One important key issue for the construction of oligosac-
charides on a solid phase is the use of a high-yielding
and stereoselective glycosylation strategy. Several meth-
odologies available for the solution-phase construction of
oligosaccharides were applied to the solid-phase synthesis
of these oligomers. Besides the successful application of
O-glycosyl trichloroacetimidates5 widely developed in our
group,6 thioglycosides,7 glycosyl sulfoxides,8 glycosyl fluo-
rides,9 1,2-anhydrosugars,10 glycosyl phosphates,11 and
Another fundamental key issue for the construction of
oligosaccharides on a solid phase is the protecting group
strategy. Recently, we have reported the first preparation
of O-glycosyl trichloroacetimidate possessing Fmoc-
protected (Fmoc ) 9-fluorenylmethyloxycarbonyl) hy-
droxy groups13 and their high efficiency for the solid-
phase construction of oligosaccharides.6a,14 The mild basic
conditions required for the Fmoc removal have proven
to be compatible with the use of Fmoc-containing building
blocks in conjunction with a novel ester-type linker.13 The
usefulness of this new solid-phase system was demon-
strated by the successful synthesis of a set of tri- and
tetrasaccharides.6a
In an endeavor to develop this promising methodology,
we report here on a highly efficient strategy for the solid-
phase synthesis of human milk branched lacto-N-neo-
hexaose derivative 1 (Scheme 1).15 An important require-
ment for the synthesis of the desired compound was the
design of a galactose moiety (b) possessing orthogonal
protecting groups in the 3b- and 6b-O-positions. For this
purpose, a novel key building block of lactose 3 bearing
levulinoyl (Lev) and Fmoc groups16 in the 6b- and 3b-O-
positions, respectively, was prepared allowing for selec-
* To whom correspondence should be addressed.
(1) Varki, A. Glycobiology 1993, 3, 97.
(2) Bodzanszky, M. Peptide Chemistry: A Practical Textbook, 2nd
ed.; Springer-Verlag: Berlin, 1993.
(3) J ung, G.; Beck-Sickinger, A. G. Angew. Chem., Int. Ed. Engl.
1992, 31, 367.
(4) (a) Haase, W. C.; Seeberger, P. H. Curr. Org. Chem. 2000, 4,
481. (b) Haase, W. C.; Seeberger, P. H. Chem. Rev. 2000, 100, 4349.
For a first investigation of solid-phase oligosaccharide synthesis with
an automated synthesizer, see: (c) Plante, O. J .; Palmacci, E. R.;
Seeberger, P. H. Science 2001, 291, 1523.
(7) (a) Zhu, T.; Boons, G.-J . Chem.sEur. J . 2001, 7, 2382. (b) Zhu,
T.; Boons, G.-J . J . Am. Chem. Soc. 2000, 122, 10222. (c) Nicolaou, K.
C.; Watanabe, N.; Li, J .; Pastor, J .; Winssinger, N. Angew. Chem., Int.
Ed. Engl. 1998, 37, 1559. (d) Nicolaou, K. C.; Winssinger, N.; Pastor,
J .; DeRoose, F. J . Am. Chem. Soc. 1997, 119, 449.
(8) Liang, R.; Yan, L.; Loebach, J .; Ge, M.; Uozumi, Y.; Sekanina,
K.; Horan, N.; Gildersleeve, J .; Smith, A.; Biswas, K.; Kahne, D. E.
Science 1996, 274, 1520.
(9) Ito, Y.; Ogawa, T. J . Am. Chem. Soc. 1997, 119, 5562.
(10) Danishefsky, S. J .; McClure, K. F.; Randolph, J . T.; Ruggeri,
R. B. Science 1993, 260, 1307.
(11) (a) Hewitt, M. C.; Seeberger, P. H. J . Org. Chem. 2001, 66, 4233.
(b) Andrade, R. B.; Plante, O. J .; Melean, L. G.; Seeberger, P. H. Org.
Lett. 1999, 1074.
(12) Rodebaugh, R.; J oshi, S.; Fraser-Reid, B.; Geysen, M. H. J . Org.
Chem. 1997, 62, 5660.
(13) Roussel, F.; Knerr, L.; Grathwohl, M.; Schmidt, R. R. Org. Lett.
2000, 2, 3043.
(14) For Fmoc-containing thioglycoside donors see ref 7.
(15) Kobata, A.; Ginsburg, V. Biochem. Biophys. 1972, 150, 273.
(5) Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212. (b)
Schmidt, R. R., Kinzy, W. Adv. Carbohydr. Chem. Biochem. 1994, 50,
21.
(6) (a) Roussel, F.; Knerr, L.; Schmidt, R. R. Eur. J . Org. Chem. 2001,
2067. (b) Wu, X.; Grathwohl, M.; Schmidt, R. R. Org. Lett. 2001, 3,
747. (c) Knerr, L.; Schmidt, R. R. Eur. J . Org. Chem. 2000, 2803. (d)
Knerr, L.; Schmidt, R. R. Synlett 1999, 1802. (e) Rademann, J .; Geyer,
A.; Schmidt, R. R. Angew. Chem. 1998, 110, 1309; Angew. Chem., Int.
Ed. Engl. 1998, 37, 1241. (f) Rademann, J .; Schmidt, R. R. J . Org.
Chem. 1997, 62, 3650. (g) Heckel, A.; Mross, E.; J ung, K. H.;
Rademann, J .; Schmidt, R. R. Synlett 1998, 171.
10.1021/jo016018p CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/15/2001

Solid-Phase Synthesis of a Branched Hexasaccharide
J . Org. Chem., Vol. 66, No. 25, 2001 8541
Sch em e 1. Bu ild in g Block s for th e Solid -P h a se Syn th esis of Br a n ch ed La cto-N-n eoh exa ose Der iva tive 1
tive elongation at both positions. In addition to lactosyl
Sch em e 2. P r ep a r a tion of Lin k er 2
donor 3, three other building blocks, namely, lactosamine
4,6a,c glucosamine 5,17 and galactose 6,18 were used. The
recently introduced dimethylmaleoyl (DMM) group19 was
selected as an amino-protecting group of both glucos-
amine moieties of 4 and 5.
The previous ester-type linker that we have developed
has proven to be stable to all reaction conditions all along
the synthesis and allowed the release of the oligosaccha-
ride from the polymeric support in high yield.6a,13 How-
ever, the oligosaccharides obtained after cleavage from
the solid support possessed an anomeric hydroxyoctyl
aglycon moiety which could not be removed. To circum-
vent this problem, a novel ester-type linker (2) having a
benzylic spacer connected to the anomeric oxygen was
designed and successfully used, allowing the deprotection
of the anomeric position in the solution phase and
therefore target compound 1 to be obtained.
Resu lts a n d Discu ssion
P r ep a r a t ion of Novel E st er -Typ e Lin k er 2. The
tritylated alcohol 7 was obtained from readily available
3-(4-bromophenyl)propan-1-ol20 (8) (Scheme 2). The al-
cohol 8 was protected as tert-butyldiphenylsilyl (TBDPS)
ether in practically quantitative yield under conventional
conditions. The silylated compound 9 thus obtained was
then subjected to hydroxymethylation using tert-butyl-
lithium (t-BuLi) as base and DMF as electrophile, fol-
lowed by a one-pot reduction with sodium borohydride
(NaBH4). The resultant crude alcohol was tritylated
under standard conditions to furnish 10 in 82% yield.
Subsequent desilylation was performed by treatment
with tetrabutylammonium fluoride (TBAF) in tetrahy-
drofuran, affording the corresponding alcohol 7 in 89%
yield.
The attachment of tritylated alcohol 7 onto a polysty-
(16) While this work was in progress, Boons et al. reported the
rene support was carried out under two different condi-
preparation of a thioglycoside bearing Fmoc and Lev groups: Zhu, T.;
tions. In the first route, carboxypolystyrene resin com-
mercially available from Advanced ChemTech (100-200
mesh, 1% cross-linked, 2 mmol/g) was esterified by
reaction with 0.19 equiv of 7 using N,N-diisopropylcar-
bodiimide (DIC) and a catalytic amount of Steglich’s
reagent (4-(dimethylamino)pyridine, DMAP) as activa-
tors. The unreacted carboxyl sites were then capped by
Boons, G.-J . Tetrahedron: Asymmetry 2000, 11, 199.
(17) Aly, M. R. E.; Castro-Palomino, J . C.; Ibrahim, E. I.; El-Ashry,
E. H.; Schmidt, R. R. Eur. J . Org. Chem. 1998, 2305, 5.
(18) Glover, S. A.; Rowbottom, C. A.; Scott, A. P.; Schoonraad, J . L.
Tetrahedron 1990, 20, 7247.
(19) Kuyama, H.; Nukada, T.; Nakahara Y.; Ogawa T. Tetrahedron
Lett. 1993, 34, 2171.
(20) Alais, J .; Maranduba, A.; Veyrie`res A. Tetrahedron Lett. 1983,
24, 2383.

8542 J . Org. Chem., Vol. 66, No. 25, 2001
Roussel et al.
Sch em e 3. Syn th esis of La ctosyl Don or 3
Sch em e 4. Syn th esis of Glu cosa m in e Don or 4
quantitative yield. The C-3b allyl ether was removed
using a two-step, one-pot sequence. An isomerization step
was first accomplished by treatment with Wilkinson
catalyst and 1,8-diaza[5.4.0]bicycloundec-7-ene (DBU),
and then hydrolysis of the enol ether was performed
using a mixture of HCl and acetone to provide 16 in good
yield. The Fmoc group was subsequently introduced by
reaction with FmocCl and catalytic amounts of DMAP,
giving the corresponding fully protected derivative in
excellent yield. The anomeric p-methoxyphenyl group
was oxidatively cleaved by treatment with ceric am-
monium nitrate (CAN), and then, the trichloroacetimi-
date function was introduced under the previously de-
scribed conditions6a using trichloroacetonitrile and sodium
hydride to furnish 3 in good yield.
The glucosamine donor 5 was prepared starting from
glucosamine (Scheme 4). The Fmoc group was installed
under standard conditions in the 4-O-position of 17,
which was obtained in five steps starting from D-glu-
cosamine,19,24 to give the corresponding fully protected
glucosamine derivative. The anomeric thexyldimethylsi-
lyl (TDS) was then removed by treatment with an excess
of HF‚pyridine complex. The resulting hemiacetal 18 was
converted into trichloroacetimidate 5 under the condi-
tions described above.
Solid -P h a se Syn th esis of La cto-N-n eoh exa ose De-
r iva tive 1. With the required building blocks at our
disposal, the multistep synthesis of branched hexasac-
charide 1 was initiated. Linker-loaded polystyrene resin
2 was glycosylated with 3 equiv of new O-lactosyl
trichloroacetimidate 3 under trimethylsilyl triflate (TM-
SOTf) activation (0.24 equiv) to furnish the corresponding
support-bound disaccharide 19 (Scheme 5). The glycosy-
lation here and in the next steps was carried out twice
at -40 °C to ensure a complete reaction. The batch of
resin 19 was then split into two pools to prepare the
target hexasaccharide via two different routes, thus
emphasizing the orthogonality between the Fmoc and Lev
protecting groups.
Concerning the first protocol (Scheme 5), the Fmoc
group was selectively removed in the presence of the Lev
group by treatment of 19 with triethylamine in CH2Cl2.
No loss of the Lev group was observed during this
reaction. Next, an elongation of the immobilized disac-
charide was performed by reaction with 4 equiv of
lactosamine donor 4 upon activation with TMSOTf (0.28
equiv), leading to resin-bound tetrasaccharide 20. The
Lev group was subsequently removed by reaction with 2
equiv of hydrazinium acetate (N2H4‚HOAc), releasing the
C-6b primary hydroxyl group. To exhibit further the
reaction with trimethylsilyldiazomethane (Me3SiCHN2)
and MeOH to furnish 11. Trityl groups were cleaved by
treatment with trifluoroacetic acid (TFA), allowing the
calculation of the loading by colorimetric assay (0.32
mmol/g), thus affording linker-loaded resin 2. According
to a second route, acid chloride support 12, derived from
a commercial carboxypolystyrene resin, was reacted with
alcohol 7 (0.24 equiv) in pyridine followed by capping with
anhydrous methanol. After detritylation, the resin-bound
linker 2 was obtained (loading 0.41 mmol/g).
Syn th esis of th e Bu ild in g Block s of La ctose a n d
Glu cosa m in e. 4-Methoxyphenyl â-lactoside (13)21 served
as starting material for the synthesis of lactosyl donor 3
(Scheme 3). According to the conditions developed by
Veyrie`res et al.,22 an assisted regioselective allylation of
the 3b-O-position was carried out. Given the poor solubil-
ity of 13 in methanol, a low yield was obtained for this
reaction. However, a second run enabled us to improve
the yield up to 50%. Under standard conditions a ben-
zylidene acetal function was regioselectively introduced
into the 4b,6b-O-positions followed by a perbenzylation,
furnishing 14. Subsequent removal of the benzylidene
group was accomplished using p-TsOH and ethanethiol
as the nucleophile,23 and the released primary hydroxyl
group was regioselectively silylated in excellent yield.
Next, a benzylation of the axial hydroxyl group was
performed under conventional conditions to yield 15.
Selective removal of the TBDPS group by treatment with
TBAF was followed by reaction with levulinic acid
(LevOH) and DCC in the presence of catalytic amounts
of DMAP to yield the corresponding ester in practically
(21) Williams, D. R.; Sith, S. Y. J . Am. Chem. Soc. 1984, 106, 2949.
(22) Chiesa, V.; Schmidt, R. R. Eur. J . Org. Chem. 2000, 3541.
(23) Takamura, T.; Chiba, T.; Tejima, S. Chem. Pharm. Bull. 1981,
29, 2270.
(24) Chiesa, V.; Schmidt, R. R. Eur. J . Org. Chem. 2000, 3541.

Solid-Phase Synthesis of a Branched Hexasaccharide
J . Org. Chem., Vol. 66, No. 25, 2001 8543
Sch em e 5. F ir st Rou te of th e Solid -P h a se Syn th esis of Br a n ch ed Hexa sa cch a r id e 22
Sch em e 6. Secon d Rou te of th e Solid -P h a se
Syn th esis of Br a n ch ed Hexa sa cch a r id e 22 a n d
efficiency of Fmoc-containing O-glycosyl trichloroacetimi-
dates for solid-phase oligosaccharide synthesis, the sec-
ond lactosamine moiety (e, f) was introduced by using
Rem ova l of th e P r otectin g Gr ou p s
instead of lactosamine donor 4 at first glucosamine donor
5 and then galactosyl donor 6. Thus, it will be demon-
strated that our future aim to prepare a small library of
branched oligosaccharides starting from resin-bound
lactose 19 is feasible. Glycosylation with 5 (4 equiv) and
TMSOTf (0.28 equiv) as catalyst and then removal of the
Fmoc group furnished resin-bound pentasaccharide 21
as acceptor. New elongation with O-galactosyl trichloro-
acetimidate 618 (3 equiv) as donor under TMSOTf activa-
tion (0.45 equiv) furnished the corresponding resin-bound
hexasaccharide. Cleavage from the polymer support
under alkaline conditions (MeONa) was executed twice
to give an anomeric mixture (â/R, 1:1) of hexasaccharide
22 in 43% overall yield from 2 (90% per step, eight steps).
A second route to the synthesis of 22 was performed
in which the Lev group of 19 was first selectively removed
in the presence of the Fmoc group using the conditions
described above (Scheme 6). This time, the elongation
with 4 started at the 6b-O-position, thus allowing also
the isolation of the corresponding immobilized tetrasac-
charide. After removal of the Fmoc group, furnishing 23,
the branching of the oligosaccharide was continued at the
3b-O-position of 23, applying exactly the sequence used
during the first route. Thus, the desired compound 22
was obtained in almost the same overall yield (42%) as
described above. Thus, the usefulness of lactose building
block 3 and the efficiency of the selected protective group
pattern were demonstrated in the synthesis of 22 with
two different approaches.
With derivative 22 in hand, the removal of protecting
groups was then undertaken. At first, the two DMM
groups were removed following the conditions previously
described,19 and the released amino groups were con-
verted into N-acetamido functions by treatment with
acetic anhydride. Finally, the benzylic groups were
smoothly cleaved by catalytic hydrogenation over pal-
ladium to produce, after acetylation, the linker-free
peracetylated lacto-N-neohexaose 1. The spectroscopic
data of 1 were in agreement with the values reported in
the literature.25
Con clu sion s
We have described a highly efficient synthesis of
branched peracetylated lacto-N-neohexaose 1 on a solid
phase. For this purpose, the efficient combination of Fmoc
and Lev as temporary orthogonal hydroxy protecting
(25) Takamura, T.; Chiba, T.; Tejima, S. Chem. Pharm. Bull. 1981,
29, 2270.

8544 J . Org. Chem., Vol. 66, No. 25, 2001
Roussel et al.
dissolved in pyridine (25 mL) were added trityl chloride (1.2
equiv, 1.58 g, 6.88 mmol) and a catalytic amount of DMAP.
After the resulting solution was stirred for 6 h at room
temperature, the pyridine was removed azeotropically with
toluene in vacuo, and the product was purified by flash
chromatography (petroleum ether/EtOAc, 9:1) to afford 10
(3.04 g, 4.69 mmol) as a yellow oil in 82% yield: TLC
(petroleum ether/EtOAc, 9:1) Rf ) 0.55; 1H NMR (CDCl3) δ
7.67-7.14 (m, 29H), 4.13 (s, 2H), 3.69 (t, J ) 6.4 Hz, 2H), 2.71
(t, J ) 7.2 Hz, 2H), 1.87 (m, 2H), 1.06 (s, 9H); 13C NMR (CDCl3)
δ 144.1, 141.0, 136.4, 135.5, 134.0, 129.5, 128.7, 128.3, 127.8,
127.5, 127.0, 126.9, 86.8, 65.5, 63.0, 34.1, 31.7, 26.8, 19.2. Anal.
Calcd for C45H46O2Si (646.94): C, 83.54; H, 7.16. Found: C,
83.43; H, 7.18.
3-(4-O-Tr iph en ylm eth yloxym eth ylph en yl)pr opan ol (7).
To a solution of 10 (2.8 g, 4.32 mmol) in THF (40 mL) was
added a 1.0 M solution of TBAF in THF (6.91 mL, 6.91 mmol)
at 0 °C. The solution was allowed to warm to room temperature
and stirred for 1 h. The solvent was evaporated in vacuo, the
residue diluted in CH2Cl2, and the organic phase washed twice
with water. After the organic phase was dried over MgSO4 and
concentration in vacuo, the crude mixture was purified by flash
chromatography (petroleum ether/EtOAc, 4:1) to produce 7
(1.57 g, 3.84 mmol) as a colorless oil in 89% yield: TLC
(petroleum ether/EtOAc, 3:2) Rf ) 0.45; 1H NMR (CDCl3) δ
7.58-7.24 (m, 19H), 4.20 (s, 2H), 3.70 (t, J ) 6.4 Hz, 2H), 2.75
(t, J ) 7.5 Hz, 2H), 1.92 (m, 2H), 1.62 (br s, 1H); 13C NMR
(CDCl3) δ 144.0, 140.6, 136.5, 128.8, 128.6, 128.5, 128.4, 128.2,
127.9, 127.8, 127.7, 127.6, 127.0, 126.9, 86.8, 65.5, 62.1, 34.1,
31.7. Anal. Calcd for C29H28O2 (408.53): C, 85.25; H, 6.90.
Found: C, 85.20; H, 6.92.
P r ep a r a tion of Tr ityla ted Lin k er -Loa d ed Resin 11.
P r oced u r e A. To carboxypolystyrene resin commercially
available from Advanced ChemTech (loading 2 mmol/g, 689
mg, 1.37 mmol) suspended in CH2Cl2 (4.5 mL) were added 7
(0.19 equiv, 109 mg, 0.26 mmol), DIC (5 equiv/7, 206 µL, 1.33
mmol), and DMAP (0.2 equiv/7, 6.5 mg, 0.05 mmol) under
argon. The stirring was continued for 40 h. The solvent was
filtered, and the resin was washed, successively, with THF (4
× 6 mL) and CH2Cl2 (4 × 6 mL). The resin was then dried
under high vacuum.
P r oced u r e B. To acid chloride resin 12 (loading 2 mmol/g,
670 mg, 1.27 mmol) suspended in pyridine (6 mL) was added
7 (0.24 equiv, 125 mg, 0.30 mmol) under argon. Stirring was
continued for 24 h. An excess of MeOH (2 mL) was added to
the mixture, and the suspension was agitated for 48 h. The
resin was then filtered off, washed with CH2Cl2 (4 × 7 mL)
and THF (4 × 7 mL), and dried under high vacuum.
Dry resin was swollen in THF (5 mL), and the resulting
suspension was shaken under argon for 10 min. MeOH (500
µL) and a 2.0 M solution of Me3SiCHN2 in hexane (3.5 equiv,
2.06 mL, 4.12 mmol) were added to the mixture. The yellow
suspension was agitated for 7 h and resin 11 filtered off,
washed with CH2Cl2 (4 × 7 mL) and THF (4 × 7 mL), and
dried under high vacuum.
groups through the use of newly prepared O-lactosyl
trichloroacetimidate 3 was demonstrated. The efficiency
of this temporary protecting group system was shown by
the preparation of the desired human milk hexasaccha-
ride derivative in two different approaches in excellent
overall yield. A novel ester-type linker, which has proven
to be inert to all reaction conditions all along the
construction of the oligosaccharide, was successfully used.
This linker offers the great advantage of releasing, after
cleavage from the polymer support, a benzylic aglycon
moiety which was easily removed by hydrogenolysis.
Thus, the preparation of fully unprotected oligosaccha-
rides can be carried out. We believe that our combination
of linker system, protective group pattern, and glycosyl
donors provides one of the most simple and efficient
methodologies used until now for solid-phase oligosac-
charide synthesis.
Exp er im en ta l Section
The solvents were purified and dried in the usual way. All
reactions were performed with dry solvents and under argon
unless otherwise stated. TLC was performed on plastic plates,
silica gel 60 F254. Detection was achieved by treatment with a
solution of 20 g of ammonium molybdate and 0.4 g of cerium-
(IV) sulfate in 400 mL of 10% H2SO4 or with 15% H2SO4, and
heating at 150 °C. Flash chromatography was carried out on
silica gel (Baker 30-60 mm). Adsorption of reaction crudes
was performed using silica gel (Baker 60-200 mm). Petroleum
ether was used in the boiling range 35-70 °C; toluene, CH2-
Cl2, MeOH, and EtOAc were distilled. Optical rotations were
determined at 21 °C with a Perkin-Elmer 241/MC polarimeter
(1 dm cell). NMR spectra were recorded with Bruker 600 DRX
instruments by using tetramethylsilane as internal standard.
MS spectra were recorded with a MALDI-kompakt (Kratos)
instrument in the positive mode using 2,5-dihydroxybenzoic
acid in THF as matrix. Microanalyses were performed in the
unit of Microanalysis at the Fachbereich Chemie, Universita¨t
Konstanz.
3-(4-Br om op h e n yl)p r op yloxy-t er t -b u t yld ip h e n ylsi-
la n e (9). To a solution of commercially available alcohol 8 (3.22
g, 14.97 mmol) in CH2Cl2 (50 mL) were added imidazole (1.6
equiv, 1.62 g, 23.96 mmol) and TBDPSCl (1.2 equiv, 4.92 g,
17.97 mmol) under an argon atmosphere, and the mixture was
stirred at room temperature for 4 h. The reaction mixture was
diluted with CH2Cl2, washed successively with water and
brine, and dried over MgSO4. The solvent was removed in
vacuo, and the residue was purified by flash chromatography
(petroleum ether/EtOAc, 9:1) to furnish 9 (6.52 g, 14.3 mmol)
as a yellow viscous oil in 96% yield: TLC (petroleum ether/
EtOAc, 9:1) Rf ) 0.65; 1H NMR (CDCl3) δ 7.67-7.03 (m, 14H),
3.68 (t, J ) 6.1 Hz, 2H), 2.69 (t, J ) 7.5 Hz, 2H), 1.84 (m, 2H),
1.08 (s, 9H); 13C NMR (CDCl3) δ 141.1, 135.5, 133.8, 131.2,
130.2, 129.5, 127.6, 119.3, 62.7, 33.9, 31.4, 26.8, 19.2. Anal.
Calcd for C25H29BrOSi (453.49): C, 66.21; H, 6.44. Found: C,
66.11; H, 6.46.
3-(4-Tr iph en ylm eth yloxym eth ylph en yl)pr opyloxy-ter t-
bu tyld ip h en ylsila n e (10). To a solution of 9 (2.6 g, 5.72
mmol) in THF was added t-BuLi (1 equiv, 3.81 mL, 1.5 M
solution in pentane, 5.72 mmol) dropwise at -78 °C. After 15
min of stirring, DMF (1.2 equiv, 0.52 mL, 6.86 mmol) was
added, and the reaction mixture was further stirred for 30 min
and quenched by addition of a saturated solution of NH4Cl.
The solution was extracted with diethyl ether (3×), and the
combined organic layers were washed with water and brine
and dried over MgSO4. The solvent was removed in vacuo, the
crude product was dissolved in MeOH, NaBH4 (1.5 equiv, 0.3
g, 8.82 mmol) was added, and the solution was stirred at room
temperature for 15 min. The reaction mixture was concen-
trated in vacuo, diluted with EtOAc, and subsequently washed
with water and brine. The organic layer was dried over MgSO4
and concentrated in vacuo. To a solution of the crude alcohol
Lin k er -Loa d ed Resin 2. Dry resin 11 was swollen in CH2-
Cl2 (0.40 mL/10 mg of resin), and the resulting suspension was
agitated for 10 min under argon. MeOH (10% by volume) and
TFA (5% of the total volume) were added, and shaking was
continued for 15 min. The resin was filtered off, washed with
CH2Cl2/MeOH (9:1) (4 × 7 mL) and THF/MeOH (9:1) (4 × 7
mL), and dried under high vacuum.
P r oced u r e for Ca lcu la tion of th e Loa d in g. Resin 11 was
swollen in CH2Cl2 (0.40 mL/10 mg of resin), and the resulting
suspension was shaken for 10 min under argon. TFA (5% of
the total volume) was added, and agitation was continued for
10 min. The resin was then filtered off and washed with a CH2-
Cl2/TFA solution (95:5, v/v). UV measurement was then carried
out on this solution (calculation of the concentration employing
a standard straight line prepared with the UV values of TrOH
in a solution of 5% TFA in CH2Cl2), giving the loading (0.32-
0.41 mmol/g).
p-Meth oxyp h en yl O-(3-O-Allyl-2-O-ben zyl-4,6-O-ben -
zylid en e-â-d -ga la ctop yr a n osyl)-(1f4)-2,3,6-tr i-O-ben zyl-
â-d -glu cop yr a n osid e (14). To a suspension of p-methoxyphe-

Solid-Phase Synthesis of a Branched Hexasaccharide
J . Org. Chem., Vol. 66, No. 25, 2001 8545
nyl lactoside (13)21 (13.94 g, 31.09 mmol) in MeOH (450 mL)
was added dibutyltin oxide (1.1 equiv, 8.51 g, 34.19 mmol)
under an argon atmosphere. The mixture was strirred for 24
h at 70 °C, MeOH was evaporated, and the residue was dried
under high vacuum for 30 min. The same procedure was
repeated to obtain further reaction. To a suspension of the
crude mixture in toluene were added allyl bromide (10 equiv,
26.30 mL, 310.9 mmol) and n-tetrabutylammonium iodide (1
equiv, 11.48 g, 31.09 mmol). After being stirred at 70 °C for
40 h, the crude mixture was concentrated in vacuo and purified
by flash chromatography (CH2Cl2/MeOH, 98:2 f 9:1) to afford
p-methoxyphenyl O-(3-O-allyl-â-D-galactopyranosyl)-(1f4)-â-
D-glucopyranoside (7.59 g, 15.54 mmol) as a white foam in 50%
yield: TLC (CH2Cl2/MeOH, 88:12) Rf ) 0.45; [R]D ) -12.0 (c
1, MeOH); 1H NMR (CD3OD) δ 7.03 (d, J ) 9 Hz, 2H), 6.82 (d,
J ) 9 Hz, 2H), 6.01-5.95 (m, 1H), 5.32 (dd, J ) 17.2, 1.3 Hz,
1H), 5.15 (d, J ) 10.4 Hz, 1H), 4.40 (d, J ) 7.9 Hz, 1H), 4.22
(dd, J ) 5.6, 12.7 Hz, 1H), 4.13 (dd, J ) 5.7, 12.6 Hz, 1H),
4.00-3.99 (m, 1H), 3.92-3.85 (m, 2H), 3.73-3.69 (m, 4H),
3.67-3.59 (m, 3H), 3.57-3.52 (m, 2H), 3.49-3.47 (m, 1H), 3.32
(dd, J ) 3.1, 9.8 Hz, 1H); 13C NMR (CD3OD) δ 156.7, 153.0,
136.4, 119.2, 117.4, 115.4, 105.0, 103.2, 82.1, 80.3, 76.9, 76.5,
76.3, 74.6, 71.7, 71.6, 67.0, 62.5, 61.7, 56.0; MALDI-MS m/z
511.4 [MNa+]. Anal. Calcd for C22H32O12 (488.48): C, 54.09;
H, 6.60. Found: C, 53.98; H, 6.58.
A mixture of p-methoxyphenyl O-(3-O-allyl-â-D-galactopy-
ranosyl)-(1f4)-â-D-glucopyranoside (7.0 g, 14.33 mmol), ben-
zaldehyde dimethyl acetal (1.1 equiv, 2.36 mL, 15.76 mmol),
and p-TsOH (0.1 equiv, 0.27 g, 1.43 mmol) in dry DMF (35
mL) was stirred at room temperature for 19 h. After neutral-
ization with NEt3, the solvents were evaporated in vacuo. The
resulting crude was purified by flash chromatography (CH2-
Cl2/MeOH, 95:5) to afford p-methoxyphenyl O-(3-O-allyl-4,6-
O-benzylidene-â-D-galactopyranosyl)-(1f4)-â-D-glucopyrano-
side (6.77 g, 11.75 mmol) as a white foam in 82% yield: TLC
(CH2Cl2/MeOH, 92.5:7.5) Rf ) 0.39; [R]D ) - 13.0 (c 1,
pyridine); 1H NMR (DMSO-d6) δ 7.40-7.34 (m, 5H), 6.98 (d, J
) 9 Hz, 2H), 6.84 (d, J ) 9 Hz, 2H), 5.93-5.87 (m, 1H), 5.60
(s, 1H), 5.42 (d, J ) 5.3 Hz, 1H), 5.40 (d, J ) 4.8 Hz, 1H), 5.30
(dd, J ) 17.3, 1.4 Hz, 1H), 5.12 (d, J ) 10.4 Hz, 1H), 4.80 (d,
J ) 7.8 Hz, 1H), 4.75 (s, 1H), 4.57 (t, J ) 5.9 Hz, 1H), 4.46 (d,
J ) 7.8 Hz, 1H), 4.32 (d, J ) 3.0 Hz, 1H), 4.16-4.09 (m, 3H),
4.03 (d, 1H), 3.75-3.73 (m, 1H), 3.71-3.65 (m, 5H), 3.63 (s,
1H), 3.58-3.46 (m, 6H), 3.41 (dd, J ) 3.2, 9.7 Hz, 1H), 3.27
(m, 1H); 13C NMR (DMSO-d6) δ 154.3, 151.3, 138.4, 135.6,
128.6, 127.9, 126.0, 117.5, 116.3, 114.3, 102.7, 101.0, 99.6, 78.7,
78.4, 74.8, 74.5, 73.1, 72.5, 69.7, 68.8, 68.5, 66.3, 66.1, 59.9,
55.3; MALDI-MS m/z 599.3 [MNa+]. Anal. Calcd for C29H36O12
(576.22): C, 60.41; H, 6.29. Found: C, 60.29; H, 6.31.
55.6; MALDI-MS m/z 960.0 [MNa+]. Anal. Calcd for C57H60O12
(937.08): C, 73.06; H, 6.45. Found: C, 73.13; H, 6.47.
p-Meth oxyp h en yl O-(3-O-Allyl-2,4-d i-O-ben zyl-6-O-ter t-
bu tyld ip h en ylsilyl-â-D-ga la ctop yr a n osyl)-(1f4)-2,3,6-tr i-
O-ben zyl-â-D-glu cop yr a n osid e (15). A mixture of 14 (3.30
g, 3.52 mmol), EtSH (6 equiv, 1.56 mL, 21.1 mmol), and
p-TsOH (0.2 equiv, 0.13 g, 0.70 mmol) in CH2Cl2 (23 mL) was
stirred at room temperature under an argon atmosphere for
19 h. The reaction was then quenched by the addition of excess
Et3N. After evaporation of the solvents in vacuo, the residue
was purified by flash chromatography (toluene/EtOAc, 7:3) to
give p-methoxyphenyl O-(3-O-allyl-2-O-benzyl-â-D-galactopy-
ranosyl)-(1f4)-2,3,6-tri-O-benzyl-â-D-glucopyranoside (2.86 g,
3.37 mmol) as a white foam in 96% yield: TLC (toluene/EtOAc,
1
7:3) Rf ) 0.28; [R]D ) -5.2 (c 1, CHCl3); H NMR (CDCl3) δ
7.44 (m, 2H), 7.36-7.26 (m, 15H), 7.04 (d, J ) 9 Hz, 2H), 6.81
(d, J ) 9 Hz, 2H), 5.96-5.90 (m, 1H), 5.31 (dd, J ) 7.3, 1.3
Hz, 1H), 5.21 (dd, J ) 10.4, 0.9 Hz, 1H), 5.03-5.02 (m, 2H),
4.89-4.81 (m, 3H), 4.77 (s, 2H), 4.52 (d, J ) 12 Hz, 1H), 4.42-
4.41 (m, 2H), 4.21-4.14 (m, 2H), 3.97 (m, 1H), 3.92-3.91 (m,
1H), 3.83-3.75 (m, 5H), 3.71-3.64 (m, 3H), 3.53-3.51 (m, 1H),
3.30 (dd, J ) 9.3, 3.3 Hz, 1H), 3.24-3.22 (m, 1H), 2.61 (m,
1H); 13C NMR (CDCl3) δ 155.2, 151.5, 138.9, 138.5, 138.3,
138.2, 134.5, 128.3, 128.2, 128.1, 127.8, 127.6, 127.5, 127.4,
118.5, 117.3, 114.5, 102.8, 102.7, 82.7, 81.5, 81.0, 79.1, 77.1,
75.5, 75.3, 75.1, 73.9, 73.1, 71.3, 68.2, 67.2, 62.3, 55.6; MALDI-
MS m/z 871.8 [MNa+]. Anal. Calcd for C50H56O12 (848.97): C,
70.74; H, 6.65. Found: C, 70.69; H, 6.66.
To a solution of p-methoxyphenyl O-(3-O-allyl-2-O-benzyl-
â-D-galactopyranosyl)-(1f4)-2,3,6-tri-O-benzyl-â-D-glucopyra-
noside (2.41 g, 2.84 mmol) in DMF (13 mL) were added
TBDPSCl (1.15 equiv, 851 µL, 3.26 mmol) and Et3N (1.15
equiv, 456 µL, 3.26 mmol). The mixture was stirred at room
temperature for 13 h under an inert atmosphere, the volatile
compounds were evaporated in vacuo, and the residue was
diluted with Et2O. The organic phase was washed twice with
water, dried over MgSO4, and concentrated in vacuo. The
residue was purified by flash chromatography (toluene/EtOAc,
95:5 f 9:1) to afford p-methoxyphenyl O-(3-O-allyl-2-O-benzyl-
6-O-tert-butyldiphenylsilyl-â-D-galactopyranosyl)-(1f4)-2,3,6-
tri-O-benzyl-â-D-glucopyranoside (2.96 g, 2.72 mmol) as a
yellow gum in 96% yield. TLC (toluene/EtOAc, 9:1) Rf ) 0.41;
[R]D ) -7.8 (c 1, CHCl3); 1H NMR (CDCl3) δ 7.66 (m, 2H), 7.61
(m, 2H), 7.39-7.37 (m, 6H), 7.36-7.24 (m, 17H), 7.04-6.99
(m, 5H), 6.77 (d, J ) 9 Hz, 2H), 5.96-5.90 (m, 1H), 5.30 (d, J
) 17.6 Hz, 1H), 5.20 (d, J ) 10.5 Hz, 1H), 4.94 (d, J ) 10.7
Hz, 1H), 4.90 (d, J ) 10.7 Hz, 1H), 4.82 (d, J ) 7.6 Hz, 1H),
4.77-4.68 (m, 4H), 4.51 (d, J ) 11.9 Hz, 1H), 4.40-4.37 (m,
2H), 4.19-4.16 (m, 2H), 4.09 (s, 1H), 3.97-3.94 (m, 1H), 3.85
(dd, J ) 9.2, 8.8 Hz, 1H), 3.77-3.75 (m, 5H), 3.71-3.69 (m,
1H), 3.61 (dd, J ) 7.8 Hz, 1H), 3.59-3.51 (m, 2H), 3.45-3.43
(m, 1H), 3.28 (dd, J ) 9.4, 3.3 Hz, 1H), 3.26-3.24 (m, 1H),
1.06-1.03 (m, 9H); 13C NMR (CDCl3) δ 155.1, 151.6, 138.6,
138.5, 135.6, 135.5, 134.8, 133.2, 129.7, 128.2, 128.0, 128.1,
127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.2, 118.4, 117.1,
114.4, 102.7, 102.5, 82.7, 81.5, 81.1, 79.5, 76.4, 75.4, 75.3, 75.2,
75.1, 73.8, 73.1, 71.2, 68.2, 67.0, 65.6, 61.5, 55.6, 26.8, 19.1;
MALDI-MS m/z 1110.3 [MNa+]. Anal. Calcd for C66H74O12Si
(1087.37): C, 72.90; H, 6.86. Found: C, 72.96; H, 6.88.
p-Methoxyphenyl O-(3-O-allyl-2-O-benzyl-6-O-tert-butyl-
diphenylsilyl-â-D-galactopyranosyl)-(1f4)-2,3,6-tri-O-benzyl-
â-D-glucopyranoside (2.90 g, 2.61 mmol) was benzylated ac-
cording to the procedure used for the preparation of 14. The
crude mixture was purified by flash chromatography (toluene/
EtOAc, 98:2 f 96:4) to furnish 15 (2.92 g, 2.48 mmol) as a
white foam in 95% yield: TLC (toluene/EtOAc, 95:5) Rf ) 0.42;
Dry NaH at 95% in oil (2.5 equiv/OH, 963 mg, 38.1 mmol)
was added to a solution of p-methoxyphenyl O-(3-O-allyl-4,6-
O-benzylidene-â-D-galactopyranosyl)-(1f4)-â-D-glucopyrano-
side (2.2 g, 3.81 mmol) and benzyl bromide (2.2 equiv/OH, 4
mL, 33.5 mmol) in DMF (26 mL) at 0 °C. The solution was
allowed to warm to room temperature and stirred under argon
for 14 h. The reaction was quenched by careful addition of
MeOH, and the solvents were evaporated in vacuo. The residue
was diluted in diethyl ether, and the organic phase was washed
with water and brine, dried over MgSO4, and concentrated in
vacuo. The crude residue was purified by flash chromatography
(toluene/EtOAc, 9:1) to give 14 (3.35 g, 3.58 mmol) as a gum
1
in 94% yield: TLC (toluene/EtOAc, 9:1) Rf ) 0.28; H NMR
(CDCl3) δ 7.47-7.43 (m, 4H), 7.37-7.26 (m, 18H), 7.21-7.18
(m, 3H), 7.03 (d, J ) 9 Hz, 2H), 6.80 (d, J ) 9 Hz, 2H), 5.99-
5.93 (m, 1H), 5.53 (s, 1H), 5.34 (dd, J ) 17.3, 1.3 Hz, 1H), 5.24-
5.19 (m, 2H), 5.02 (d, J ) 10.9 Hz, 1H), 4.89-4.81 (m, 5H),
4.75 (d, J ) 11.2 Hz, 1H), 4.53-4.51 (m, 2H), 4.38 (d, J ) 11.9
Hz, 1H), 4.26-4.18 (m, 4H), 4.16-4.15 (m, 1H), 4.05-4.02 (m,
1H), 3.93-3.91 (m, 1H), 3.86 (dd, J ) 10.9, 4.7 Hz, 1H), 3.82-
3.71 (m, 7H), 3.50-3.49 (m, 1H), 3.40 (dd, J ) 3.5, 9.7 Hz,
1H), 3.10 (m, 1H); 13C NMR (CDCl3) δ 155.2, 151.6, 138.9,
138.8, 138.8, 138.4, 138.0, 135.1, 128.8, 128.5, 128.3, 128.2,
128.1, 128.0, 127.7, 127.6, 127.5, 127.4, 127.3, 127.2, 126.5,
118.4, 117.0, 114.4, 103.0, 102.8, 101.3, 83.0, 81.6, 79.7, 78.8,
77.9, 75.8, 75.3, 75.2, 75.1, 73.9, 73.0, 71.1, 68.9, 68.3, 66.4,
1
[R]D ) -15.0 (c 1, CHCl3); H NMR (CDCl3) δ 7.57 (m, 2H),
7.54 (m, 2H), 7.36-7.35 (m, 2H), 7.33-7.22 (m, 26H), 7.03-
7.02 (m, 1H), 7.00-6.99 (m, 2H), 6.95-6.93 (m, 2H), 6.77 (d,
J ) 9 Hz, 2H), 6.00-5.93 (m, 1H), 5.36 (dd, J ) 17.2, 1.5 Hz,
1H), 5.19 (d, J ) 10.5 Hz, 1H), 5.05 (d, J ) 11.3 Hz, 1H), 4.97
(d, J ) 10.5 Hz, 1H), 4.93 (d, J ) 10.9 Hz, 1H), 4.81 (d, J )
7.5 Hz, 1H), 4.79-4.72 (m, 3H), 4.62-4.59 (m, 2H), 4.50 (d, J
) 11.9 Hz, 1H), 4.39-4.37 (m, 2H), 4.24-4.23 (m, 2H), 4.05
(s, 1H), 3.90 (dd, J ) 9.2 Hz, 1H), 3.85 (dd, J ) 9.3 Hz, 1H),

8546 J . Org. Chem., Vol. 66, No. 25, 2001
Roussel et al.
3.78-3.74 (m, 5H), 3.72-3.65 (m, 2H), 3.61-3.54 (m, 2H),
3.44-3.42 (m, 1H), 3.34 (dd, J ) 9.7, 2.9 Hz, 1H), 3.29-3.27
(m, 1H), 1.06-1.03 (m, 9H); 13C NMR (CDCl3) δ 155.1, 151.6,
139.2, 138.6, 135.5, 135.1, 133.2, 129.7, 128.2, 128.1, 128.0,
127.9, 127.7, 127.6, 127.5, 127.4, 127.2, 127.1, 127.0, 118.4,
116.3, 114.4, 102.7, 82.9, 82.5, 81.5, 80.0, 76.6, 75.6, 75.4, 75.2,
75.1, 74.5, 74.2, 73.4, 73.1, 71.6, 68.2, 61.2, 55.6, 26.9, 19.1;
MALDI-MS m/z 1200.8 [MNa+]. Anal. Calcd for C73H80O12Si
(1177.49): C, 74.46; H, 6.85. Found: C, 74.42; H, 6.84.
p-Meth oxyp h en yl O-(2,4-Di-O-ben zyl-6-O-levu lin oyl-â-
D-ga la ctop yr a n osyl)-(1f4)-2,3,6-tr i-O-ben zyl-â-D-glu cop y-
r a n osid e (16). 15 (2.80 g, 2.37 mmol) was desilylated accord-
ing to the procedure used for the preparation of 10 to give,
after purification by flash chromatography (toluene/EtOAc,
8:2), p-methoxyphenyl O-(3-O-allyl-2,4-di-O-benzyl-â-D-galac-
topyranosyl)-(1f4)-2,3,6-tri-O-benzyl-â-D-glucopyranoside (2.03
g, 2.16 mmol) as a white foam in 91% yield: TLC (toluene/
and the residue was diluted in Et2O. The organic layer was
washed successively with saturated aqueous NaHCO3 solution
and water and dried with MgSO4, and the solvents were
removed in vacuo. The residue was adsorbed onto silica gel in
toluene and purified by flash chromatography (toluene/EtOAc,
83:17 f 73:27) to afford 16 (2.57 g, 2.58 mmol) as a white
amorphous solid in 84% yield: TLC (toluene/EtOAc, 75:25) Rf
) 0.25; [R]D ) -3.2 (c 1, CHCl3); 1H NMR (CDCl3) δ 7.35-
7.22 (m, 25H), 7.03 (d, J ) 9 Hz, 1H), 6.80 (d, J ) 9 Hz, 1H),
5.05-4.99 (m, 2H), 4.88 (d, J ) 7.2 Hz, 1H), 4.85-4.82 (m,
3H), 4.77 (d, J ) 10.7 Hz, 1H), 4.67-4.63 (m, 2H), 4.59 (d, J
) 11.9 Hz, 1H), 4.47-4.44 (m, 2H), 4.14-4.10 (m, 2H), 4.06-
4.02 (m, 1H), 3.84-3.77 (m, 6H), 3.70-3.66 (m, 2H), 3.55-
3.51 (m, 3H), 3.48-3.46 (m, 1H), 2.69-2.67 (m, 2H), 2.49-
2.47 (m, 2H), 2.23 (d, 1H), 2.16 (s, 3H); 13C NMR (CDCl3) δ
206.3, 172.1, 155.2, 151.5, 138.8, 138.4, 138.3, 138.2, 138.1,
128.5, 128.3, 128.1, 128.0, 127.9, 127.6, 127.5, 127.3, 118.4,
114.4, 102.7, 102.5, 82.5, 81.5, 80.1, 76.6, 75.4, 75.3, 75.2, 75.1,
75.0, 74.1, 73.2, 72.0, 68.2, 62.5, 55.6, 37.8, 29.8, 27.7; MALDI-
MS m/z 1020.0 [MNa+]. Anal. Calcd for C59H64O14 (997.13): C,
71.07; H, 6.47. Found: C, 71.09; H, 6.46.
1
EtOAc, 75:25) Rf ) 0.27; [R]D ) -3.3 (c 1, CHCl3); H NMR
(CDCl3) δ 7.36-7.20 (m, 25H), 7.01 (d, J ) 9 Hz, 2H), 6.77 (d,
J ) 9 Hz, 2H), 5.97-5.91 (m, 1H), 5.34 (dd, J ) 17.2, 1.4 Hz,
1H), 5.19 (d, J ) 10.5 Hz, 1H), 5.04 (d, J ) 10.7 Hz, 1H), 4.99
(d, J ) 10.9 Hz, 1H), 4.95 (d, J ) 11.7 Hz, 1H), 4.86-4.75 (m,
5H), 4.56 (d, J ) 11.7 Hz, 1H), 4.47 (d, J ) 11.9 Hz, 1H), 4.39-
4.37 (m, 2H), 4.19 (s, 1H), 3.90 (dd, J ) 9.2, 9.0 Hz, 1H), 3.82-
3.80 (m, 1H), 3.78-3.71 (m, 5H), 3.69-3.68 (m, 1H), 3.65-
3.62 (m, 2H), 3.53-3.50 (m, 2H), 3.33-3.30 (m, 2H), 3.22-
3.20 (m, 1H); 13C NMR (CDCl3) δ 155.2, 151.5, 138.8, 138.7,
138.5, 138.3, 134.8, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8,
127.7, 127.6, 127.4, 127.3, 118.4, 116.6, 114.4, 103.0, 102.7,
82.7, 82.5, 81.5, 79.7, 77.2, 75.5, 75.2, 75.1, 74.8, 74.3, 73.4,
73.1, 71.8, 68.3, 61.8, 55.6; MALDI-MS m/z 962.3 [MNa+].
Anal. Calcd for C57H62O12 (939.09): C, 72.90; H, 6.65. Found:
C, 72.86; H, 6.64.
O-[(2,4-Di-O-ben zyl-3-O-(9-flu or en ylm eth oxycar bon yl)-
6-O-levu lin oyl-â-D-ga la ct op yr a n osyl)-(1f4)-2,3,6-t r i-O-
ben zyl-â-D-glu cop yr a n osyl] Tr ich lor oa cetim id a te (3). To
a solution of 16 (2.27 g, 2.28 mmol) in pyridine (15 mL) were
added FmocCl (4 equiv, 2.36 g, 9.12 mmol) and DMAP (0.1
equiv, 27.9 mg, 0.22 mmol) at room temperature under argon.
The yellow solution was stirred for 8 h (complete disappear-
ance of the starting material by TLC), and the solvent was
then evaporated and coevaporated three times with toluene
in vacuo. The crude residue was adsorbed onto silica gel in
toluene and purified by flash chromatography (toluene/EtOAc,
93:7 f 9:1) to provide p-methoxyphenyl O-(2,4-di-O-benzyl-3-
O-(9-fluorenylmethoxycarbonyl)-6-O-levulinoyl-â-D-galactopy-
ranosyl)-(1f4)-2,3,6-tri-O-benzyl-â-D-glucopyranoside (2.61 g,
2.14 mmol) as a white foam in 94% yield: TLC (toluene/EtOAc,
To a solution of p-methoxyphenyl O-(3-O-allyl-2,4-di-O-
benzyl-â-D-galactopyranosyl)-(1f4)-2,3,6-tri-O-benzyl-â-D-glu-
copyranoside (3.08 g, 3.28 mmol) in CH2Cl2 (40 mL) were added
levulinic acid (10 equiv, 3.81 g, 32.8 mmol), DCC (5 equiv, 3.38
g, 16.4 mmol), and DMAP (0.1 equiv, 0.04 g, 0.32 mmol) at
room temperature under argon. After the resulting solution
was stirred for 35 min, the precipitate was removed by
filtration over Celite and washed with CH2Cl2 and the solution
concentrated in vacuo. The residue was purified by flash
chromatography (toluene/EtOAc, 85:15) to give p-methoxyphe-
nyl O-(3-O-allyl-2,4-di-O-benzyl-6-O-levulinoyl-â-D-galactopy-
ranosyl)-(1f4)-2,3,6-tri-O-benzyl-â-D-glucopyranoside (3.29 g,
3.18 mmol) as a white amorphous solid in 97% yield: TLC
1
7:3) Rf ) 0.56; [R]D ) +1.8 (c 1, CHCl3); H NMR (CDCl3) δ
7.76-7.72 (m, 2H), 7.60-7.59 (m, 2H), 7.35-7.19 (m, 25H),
7.02 (d, J ) 9 Hz, 1H), 6.80 (d, J ) 9 Hz, 1H), 4.99 (m, 2H),
4.85 (d, J ) 7.6 Hz, 1H), 4.83 (d, J ) 11.9 Hz, 1H), 4.77-4.68
(m, 5H), 4.54 (d, J ) 11.9 Hz, 1H), 4.50-4.46 (m, 2H), 4.44-
4.40 (m, 3H), 4.24 (dd, J ) 7.3 Hz, 1H), 4.09-4.07 (m, 2H),
4.03-4.00 (m, 1H), 3.87 (m, 1H), 3.81-3.77 (m, 6H), 3.67-
3.63 (m, 2H), 3.47-3.43 (m, 2H), 2.72-2.65 (m, 2H), 2.50-
2.46 (m, 2H), 2.16 (m, 3H); 13C NMR (CDCl3) δ 206.3, 171.9,
155.2, 154.6, 151.5, 143.3, 143.0, 141.2, 138.7, 138.4, 138.1,
137.8, 128.3, 128.2, 128.0, 127.9, 127.7, 127.6, 127.5, 127.3,
127.1, 125.0, 120.0, 118.4, 114.4, 102.7, 102.3, 82.6, 81.5, 79.5,
77.6, 76.8, 75.3, 75.1, 73.7, 73.1, 71.4, 69.9, 68.1, 62.0, 55.6,
46.7, 37.8, 29.8, 27.7; MALDI-MS m/z 1242.5 [MNa+]. Anal.
Calcd for C74H74O16 (1219.37): C, 72.89; H, 6.12. Found: C,
72.86; H, 6.14.
1
(toluene/EtOAc, 8:2) Rf ) 0.42; [R]D ) -13.2 (c 1, CHCl3); H
NMR (CDCl3) δ 7.35-7.26 (m, 22H), 7.21-7.20 (m, 3H), 7.03
(d, J ) 9 Hz, 1H), 6.80 (d, J ) 9 Hz, 1H), 5.99-5.93 (m, 1H),
5.35 (dd, J ) 17.2, 1.7 Hz, 1H), 5.21 (dd, J ) 10.4, 1.2 Hz,
1H), 5.04 (d, J ) 10.7 Hz, 1H), 5.00-4.98 (m, 2H), 4.87 (d, J
) 7.2 Hz, 1H), 4.83-4.79 (m, 2H), 4.76-4.74 (m, 2H), 4.60 (d,
J ) 11.4 Hz, 1H), 4.52 (d, J ) 12 Hz, 1H), 4.45 (d, J ) 7.7 Hz,
1H), 4.41 (d, J ) 12 Hz, 1H), 4.24-4.21 (m, 2H), 3.98-3.97
(m, 1H), 3.82-3.78 (m, 5H), 3.72 (dd, J ) 9.7, 7.7 Hz, 1H),
3.68-3.63 (m, 2H), 3.51-3.49 (m, 1H), 3.40-3.38 (m, 1H), 3.33
(dd, J ) 6.6 Hz, 1H), 2.69-2.67 (m, 2H), 2.48-2.42 (m, 2H),
2.16 (s, 3H); 13C NMR (CDCl3) δ 206.4, 155.1, 151.5, 138.9,
138.7, 138.6, 138.4, 138.3, 134.8, 128.3, 128.2, 128.0, 127.9,
127.8, 127.6, 127.4, 127.2, 118.4, 116.5, 114.4, 102.7, 102.6,
82.8, 82.3, 81.5, 79.7, 76.7, 75.3, 75.2, 75.1, 74.4, 73.0, 71.7,
68.3, 62.7, 55.6, 37.8, 29.8, 27.7; MALDI-MS m/z 1060.3
[MNa+]. Anal. Calcd for C62H68O14 (1037.19): C, 71.80; H, 6.61.
Found: C, 71.74; H, 6.62.
To a solution of p-methoxyphenyl O-(2,4-di-O-benzyl-3-O-
(9-fluorenylmethoxycarbonyl)-6-O-levulinoyl-â-D-galactopyra-
nosyl)-(1f4)-2,3,6-tri-O-benzyl-â-D-glucopyranoside (2.97 g,
2.43 mmol) in CH3CN/H2O/toluene (4:1:1, 50 mL) was added
ceric ammonium nitrate (2 equiv, 2.66 g, 4.86 mmol). The
orange solution was stirred at room temperature for 90 min
and quenched by addition of solid NaHCO3, and the solvents
were removed in vacuo. The crude residue was then diluted
in Et2O and the organic phase washed three times with water,
dried over MgSO4, and concentrated in vacuo. A purification
by flash chromatography (toluene/EtOAc, 82:18 f 75:25) was
carried out to give an anomeric mixture (R/â, 1:1) of O-(2,4-
di-O-benzyl-3-O-(9-fluorenylmethoxycarbonyl)-6-O-levulinoyl-
â-D-galactopyranosyl)-(1f4)-2,3,6-tri-O-benzyl-R/â-D-glucopy-
ranose (2.05 g, 1.84 mmol) as a white foam in 76% yield: TLC
A mixture of p-methoxyphenyl O-(3-O-allyl-2,4-di-O-benzyl-
6-O-levulinoyl-â-D-galactopyranosyl)-(1f4)-2,3,6-tri-O-benzyl-
â-D-glucopyranoside (3.20 g, 3.08 mmol), Wilkinson’s catalyst
(0.1 equiv, 0.28 g, 0.30 mmol), and DBU (0.05 equiv, 0.02 mL,
0.15 mmol) in EtOH/toluene/water (8:3:1, 16 mL) was stirred
at 100 °C for 12 h. The solvents were evaporated, and the
residue was diluted in Et2O. The organic phase was washed
with water and brine, and the solvents were removed in vacuo.
The residue was dissolved in an acetone/HCl (1 M) mixture
(9:1, 26 mL), and the reaction was stirred at room temperature
for 20 min. The solvents were subsequently removed in vacuo,
1
(toluene/EtOAc, 7:3) Rf ) 0.27 and 0.20; H NMR (CDCl3) δ
7.76-7.72 (m, 1H), 7.60-7.59 (m, 1H), 7.39-7.19 (m, 12.5H),
5.19 (d, J ) 3.8 Hz, 0.5H), 5.02-4.97 (m, 2H), 4.88 (d, J )
11.0 Hz, 0.5H), 4.79-4.55 (m, 12H), 4.47-4.40 (m, 5H), 4.37-
4.34 (m, 2H), 4.26-4.23 (m, 1H), 4.11-4.04 (m, 3H), 3.98-
3.95 (m, 2H), 3.87-3.81 (m, 2.5H), 3.78-3.74 (m, 2.5H), 3.67-
3.65 (m, 1H), 3.59-3.54 (m, 1.5H), 3.40-3.33 (m, 1.5H), 3.22
(s, 0.5H), 3.07 (s, 0.5H), 2.70-2.67 (m, 2H), 2.50-2.46 (m, 2H),

Solid-Phase Synthesis of a Branched Hexasaccharide
J . Org. Chem., Vol. 66, No. 25, 2001 8547
2.16 (m, 3H); 13C NMR (CDCl3) δ 206.2, 171.9, 154.6, 143.0,
141.3, 141.2, 138.7, 138.1, 137.8, 128.4, 128.3, 128.2, 128.0,
127.9, 127.6, 127.5, 127.2, 127.1, 125.0, 120.0, 102.2, 97.3, 91.5,
82.5, 82.4, 79.5, 79.1, 77.5, 76.5, 75.1, 74.9, 74.8, 73.6, 73.2,
71.4, 70.4, 69.9, 68.0, 67.9, 62.1, 46.7, 37.8, 29.8, 27.7; MALDI-
MS m/z 1136.4 [MNa+]. Anal. Calcd for C67H68O15 (1113.25):
C, 72.29; H, 6.16. Found: C, 72.25; H, 6.15.
NaHCO3 cold aqueous solution, water, and brine. The organic
layer was dried over MgSO4 and concentrated in vacuo. The
residue was purified by flash chromatography (petroleum
ether/EtOAc, 4:1) to produce 18 (1.49 g, 2.16 mmol) as a
colorless oil in 86% yield: TLC (petroleum ether/EtOAc, 3:2)
1
Rf ) 0.45; [R]D ) +43.6 (c 1, CHCl3); H NMR (CDCl3) δ 7.74
(t, J ) 8.1 Hz, 2H), 7.58 (d, J ) 7.4 Hz, 1H), 7.55 (d, J ) 7.4
Hz, 1H), 7.42-7.24 (m, 8H), 7.20-7.13 (m, 4H), 7.11-7.09 (m,
2H), 5.22 (t, J ) 7.9 Hz, 1H), 4.94 (t, J ) 9.5 Hz, 1H), 4.63 (d,
J ) 12.4 Hz, 1H), 4.52-4.45 (m, 2H), 4.42-4.33 (m, 3H), 4.31-
4.28 (m, 1H), 4.23 (d, J ) 7.3 Hz, 1H), 4.15-4.08 (m, 1H), 3.97
(dd, J ) 8.0, 10.8 Hz, 1H), 3.82-3.79 (m, 1H), 3.64-3.61 (m,
2H), 1.78 (br s, 6H); 13C NMR (CDCl3) δ 154.1, 143.1, 142.9,
141.1, 137.9, 137.5, 136.8, 128.1, 128.0, 127.7, 127.6, 127.5,
127.2, 127.0, 124.9, 124.8, 119.9, 119.7, 92.7, 76.5, 74.3, 74.0,
73.9, 73.3, 72.6, 69.7, 69.1, 68.7, 68.2, 56.5, 55.1, 46.5, 8.4;
MALDI-MS m/z 712.9 [MNa+]. Anal. Calcd for C41H59NO9
(689.75): C, 71.39; H, 5.70; N, 2.03. Found: C, 71.43; H, 5.69;
N, 2.02.
O-(2,4-Di-O-benzyl-3-O-(9-fluorenylmethoxycarbonyl)-6-O-
levulinoyl-â-D-galactopyranosyl)-(1f4)-2,3,6-tri-O-benzyl-R/â-
D-glucopyranose (1.32 g, 1.18 mmol) was dissolved in CH2Cl2/
CCl3CN (1:1, 15 mL) under argon. The minimum amount of
NaH (95% in oil) required for the completion of the reaction
(TLC monitoring) was added to the solution. After 15 min of
stirring, the solution was adsorbed onto silica gel, and the
residue was purified by flash chromatography (petroleum
ether/EtOAc, 7:3 f 65:35) to produce an anomeric mixture of
3 (R/â, 1:1) (1.30 g, 1.03 mmol) as a white foam in 88% yield.
Data for the â anomer: TLC (toluene/EtOAc, 7:3) Rf ) 0.61;
[R]D ) +5.0 (c 1, CHCl3); 1H NMR (CDCl3) δ 8.66 (s, 1H), 7.75-
7.73 (m, 2H), 7.60-7.58 (m, 2H), 7.39-7.37 (m, 2H), 7.31-
7.16 (m, 27H), 5.77 (d, J ) 7.6 Hz, 1H), 4.99 (d, J ) 10.8 Hz,
1H), 4.87 (d, J ) 10.7 Hz, 1H), 4.78-4.64 (m, 7H), 4.59 (d, J
) 12.2 Hz, 1H), 4.49 (d, J ) 7.7 Hz, 1H), 4.46-4.41 (m, 5H),
4.25 (dd, J ) 7.2 Hz, 1H), 4.12 (dd, J ) 9.0 Hz, 1H), 4.06-
4.05 (m, 2H), 3.85-3.82 (m, 2H), 3.77 (dd, J ) 10.2, 7.7 Hz,
1H), 3.70-3.67 (m, 3H), 3.53-3.52 (m, 1H), 3.42-3.40 (m, 1H),
2.70-2.67 (m, 2H), 2.50-2.46 (m, 2H), 2.16 (s, 3H); 13C NMR
(CDCl3) δ (selected data) 102.4, 98.6, 82.8, 80.4, 79.8, 77.6, 76.2,
O-[3,4-Di-O-ben zyl-2-d eoxy-2-d im eth ylm a leim id o-4-O-
(9-fluorenylmethoxycarbonyl)-â-D-glucopyranosyl]Trichlo-
r oa cetim id a te (5). Product 5 was prepared following the
procedure described for the synthesis of 3. 5 was obtained,
after purification by flash chromatography (petroleum ether/
EtOAc, 75:25), as a white foam in 88% yield: TLC (petroleum
ether/EtOAc, 6:4) Rf ) 0.70; [R]D ) +52.7 (c 1, CHCl3); 1H NMR
(CDCl3) δ 8.57 (s, 1H), 7.73-7.70 (m, 2H), 7.55-7.51 (m, 2H),
7.36-7.13 (m, 12H), 7.07-7.05 (m, 2H), 6.22 (d, J ) 8.8 Hz,
1H), 5.02 (dd, J ) 10.0, 8.8 Hz, 1H), 4.65-4.61 (m, 2H), 4.53-
4.51 (m, 2H), 4.43 (dd, J ) 10.8, 8.8 Hz, 1H), 4.35 (dd, J )
10.5, 7.4 Hz, 1H), 4.32-4.26 (m, 3H), 4.11 (dd, J ) 7.0 Hz,
1H), 3.94-3.92 (m, 1H), 3.70-3.65 (m, 2H), 1.78-1.75 (m, 6H);
13C NMR (CDCl3) δ 160.7, 154.2, 143.2, 143.0, 141.3, 137.0,
128.2, 127.7, 127.6, 127.4, 127.1, 125.0, 124.9, 120.0, 93.9, 77.0,
76.3, 74.2, 73.9, 73.4, 70.0, 68.8, 54.1, 46.7, 8.5. Anal. Calcd
for C43H39Cl3N2O9 (834.14): C, 61.92; H, 4.71; N, 3.36. Found:
C, 61.89; H, 4.72; N, 3.37.
Gen er a l P r oced u r e for th e Solid -P h a se Glycosyla tion
(P r oced u r e A). Dry acceptor-loaded resins were directly
swollen under argon in a CH2Cl2 solution (1-1.5 mL/0.1 g of
resin) containing the appropriate donor (3 equiv). The resulting
suspension was cooled to -40 °C, after 10 min under agitation
a freshly prepared 0.25 M TMSOTf solution in CH2Cl2 was
added, and shaking was continued for 1 h. The mixture was
allowed to warm to room temperature, and the resin was then
filtered off, washed with CH2Cl2 (4 × 5 mL) and THF (4 × 5
mL), and dried under high vacuum (1 h, room temperature,
0.05 mbar). This procedure was repeated prior to starting the
next step. Reaction completion for glycosylations and depro-
tections was monitored by TLC and MALDI-TOF analysis
(TLC and MALDI-TOF analysis indicated disappearance of the
corresponding starting material) of the crude cleavage product
(MeONa, CH2Cl2/MeOH) from a very small resin sample (2
mg).
76.1, 74.0, 71.8, 70.2, 67.6, 62.4, 46.9. Anal. Calcd for C69H68
-
Cl3NO15 (1257.63): C, 65.90; H, 5.45; N, 1.11. Found: C, 65.88;
H, 5.44; N, 1.10. Data for the R anomer: TLC (toluene/EtOAc,
7:3) Rf ) 0.46; [R]D ) +25.3 (c 0.76, CHCl3); 1H NMR (CDCl3)
δ 8.50 (s, 1H), 7.69-7.66 (m, 2H), 7.53-7.51 (m, 2H), 7.26-
7.08 (m, 29H), 6.36 (d, J ) 3.5 Hz, 1H), 4.88 (d, J ) 10.7 Hz,
1H), 4.67-4.62 (m, 8H), 4.53 (dd, J ) 10.1, 3.1 Hz, 1H), 4.48
(d, J ) 12.0 Hz, 1H), 4.40-4.35 (m, 4H), 4.28-4.24 (m, 2H),
4.18-4.16 (m, 1H), 4.01-3.98 (m, 4H), 3.85-3.81 (m, 2H),
3.78-3.76 (m, 3H), 3.69 (dd, J ) 10.1, 7.8 Hz, 1H), 3.63 (dd, J
) 9.6, 3.5 Hz, 1H), 3.44-3.42 (m, 1H), 3.31-3.29 (m, 1H),
2.62-2.60 (m, 2H), 2.42-2.39 (m, 2H), 2.08 (m, 3H); 13C NMR
(CDCl3) δ 206.2, 161.1, 154.6, 143.3, 141.3, 141.2, 138.9, 138.1,
137.9, 137.7, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8,
127.7, 127.5, 127.1, 125.0, 120.1, 102.3, 94.4, 79.6, 79.3, 78.3,
77.4, 75.8, 75.3, 75.1, 73.7, 73.2, 73.1, 71.3, 69.9, 67.2, 62.0,
46.7, 37.8, 29.8, 27.7. Anal. Calcd for
C69H68Cl3NO15
(1257.63): C, 65.90; H, 5.45; N, 1.11. Found: C, 65.91; H, 5.47;
N, 1.10.
O-3,4-Di-O-ben zyl-2-d eoxy-2-d im eth ylm a leim id o-4-O-
(9-flu or en ylm et h oxyca r b on yl)-â-D-glu cop yr a n ose (18).
The Fmoc group was introduced to compound 1719,24 according
to the procedure employed for the protection of 16, thus
affording, after purification by flash chromatography (petro-
leum ether/EtOAc, 4:1), thexyldimethylsilyl 3,4-di-O-benzyl-
4-O-(9-fluorenylmethoxycarbonyl)-2-deoxy-2-dimethylmaleimido-
â-D-glucopyranoside as a yellow viscous oil in 83% yield: TLC
(petroleum ether/EtOAc, 3:2) Rf ) 0.65; [R]D ) +23.4 (c 1,
CHCl3); 1H NMR (CDCl3) δ 7.74 (t, J ) 8.2 Hz, 2H), 7.68 (d, J
) 7.5 Hz, 1H), 7.38-7.20 (m, 9H), 7.16-7.15 (m, 3H), 7.14-
7.09 (m, 2H), 5.21 (d, J ) 8.1 Hz, 1H), 4.93 (t, J ) 9.6 Hz,
1H), 4.63 (d, J ) 12.3 Hz, 1H), 4.59-4.54 (m, 2H), 4.38-4.30
(m, 4H), 4.15 (t, J ) 7.2 Hz, 1H), 3.97 (dd, J ) 8.1 Hz, 1H),
3.80-3.76 (m, 1H), 3.67-3.65 (m, 2H), 1.81 (br s, 6H), 1.50-
1.46 (m, 1H), 0.75-0.69 (m, 12H), 0.13 (s, 3H), 0.01 (s, 3H);
13C NMR (CDCl3) δ 154.3, 143.2, 143.0, 141.2, 138.1, 138.0,
136.8, 128.2, 128.1, 127.8, 127.4, 127.3, 127.2, 127.1, 125.0,
124.9, 119.9, 93.3, 73.7, 73.4, 72.9, 69.8, 69.7, 57.1, 46.6, 33.9,
24.4, 19.8, 19.6, 18.3, 8.4, -1.8, -3.9; MALDI-MS m/z 845.3
[MNa+]. Anal. Calcd for C49H51NO9Si (832.07): C, 70.73; H,
6.90; N, 1.68. Found: C, 70.75; H, 6.91; N, 1.69.
Gen er a l P r oced u r e for th e F m oc Rem ova l (P r oced u r e
B). Dry resin-bound protected oligosaccharide was swollen
under argon in a CH2Cl2 solution (1 mL/0.1 g of resin). After
10 min under agitation, NEt3 (20% in volume) was added to
the suspension, and shaking was continued for 4 h. The resin
was then filtered off, washed with CH2Cl2 (4 × 5 mL) and THF
(4 × 5 mL), and dried under high vacuum.
Gen er a l P r oced u r e for Clea va ge (P r oced u r e C). Dry
resin was swollen in CH2Cl2 (1-1.5 mL/0.1 g of resin), and
the resulting suspension was shaken under argon for 10 min.
A solution of 5 equiv of MeONa in MeOH (10% of the complete
volume) was added, and the resulting mixture was agitated
for 6 h under an inert atmosphere. After this period, TLC
analysis confirmed satisfactory cleavage. The resin was rinsed
off with CH2Cl2/MeOH (9:1, 3 × 5 mL). This procedure was
repeated to ensure complete cleavage. Amberlite IR120 (H+
form) was added to the combined filtrate and washings to
neutralize the medium and then filtered off. The solution was
concentrated in vacuo and the residue purified by flash
chromatography. In the case of an analytical cleavage, the
crude residue was directly analyzed by TLC and MALDI-TOF.
To a solution of thexyldimethylsilyl 3,4-di-O-benzyl-4-O-(9-
fluorenylmethoxycarbonyl)-2-deoxy-2-dimethylmaleimido-â-D-
glucopyranoside (2.1 g, 2.52 mmol) in THF (15 mL) was added
HF·pyridine (3 mL) under an argon atmosphere. After being
stirred at room temperature for 2 h, the reaction mixture was
diluted with ethyl acetate and washed succesively with 10%

8548 J . Org. Chem., Vol. 66, No. 25, 2001
Roussel et al.
Resin 19 was prepared according to procedure A employing
0.24 equiv of TMSOTf.
Resin 20. The Fmoc group of 19 was removed using
procedure C. An elongation with lactosamine donor 4 (4 equiv)
was then performed according to procedure B using 0.28 equiv
of TMSOTf.
Resin 21. Resin-bound tetrasaccharide 20 was swollen
under argon in a CH2Cl2 solution (1.5 mL/0.1 g of resin). After
10 min under shaking, MeOH (10% by volume) and NH2NH2-
HOAc (2 equiv) were added to the suspension, and the
agitation was continued for 16 h. The resin was then filtered
off, washed with CH2Cl2/MeOH (9:1, 4 × 5 mL) and THF/
MeOH (9:1, 4 × 5 mL), and dried under high vacuum. The
released hydroxyl group was then glycosylated with 5 (4 equiv)
employing procedure A using 0.28 equiv of TMSOTf. The Fmoc
group was cleaved according to procedure B to afford resin-
bound pentasaccharide 21.
4-(3-H yd r oxyp r op yl)b en zyl (â-D-Ga la ct op yr a n osyl)-
(1f4)-(3,6-d i-O-ben zyl-2-d eoxy-2-d im eth ylm a leim id o-â-
D-glu cop yr a n osyl)-(1f3)-[(â-D-ga la ctop yr a n osyl)-(1f4)-
(3,6-d i-O -b e n zy l-2-d e o x y -2-d im e t h y lm a le im id o -â-D-
g l u c o p y r a n o s y l ) -( 1 f6 ) ] -( 2 , 4 -d i -O -b e n z y l -â-D -
g a la c t o p y r a n o s y l)-(1 f4)-2,3,6-t r i -O -b e n z y l-r,â-D -
glu cop yr a n osid e (22). First route: 22 was obtained from 21
using procedure A and donor 6 (0.45 equiv of TMSOTf)
followed by a cleavage from the polymer support (procedure
C). Second route: (1) glycosylation of 23 with 5 (4 equiv) carried
out (procedure A) using 0.32 equiv of TMSOTf; (2) removal of
the Fmoc group (procedure B); (3) glycosylation employing
procedure A and donor 6 (0.45 equiv of TMSOTf); (4) cleavage
(procedure C). The residue was purified by flash chromatog-
raphy (CH2Cl2/MeOH, 93: 7 f 88:12) to provide 22 (42-43%
overall yield from 2) as an oil (R/â, 1:1): TLC (CH2Cl2/MeOH,
89:11) Rf ) 0.21; 1H NMR (CDCl3) δ 7.21-7.01 (m, 49H), 5.09-
5.04 (m, 1H), 5.02-4.97 (m, 1H), 4.92-4.68 (m, 5.5H), 4.67-
4.56 (m, 2H), 4.54-4.11 (m, 19.5H), 4.09-3.87 (m, 7H), 3.81-
3.68 (m, 10.5H), 3.63-2.91 (m, 32H), 2.61-2.56 (m, 2H), 1.79-
1.72 (m, 2H), 1.66-1.53 (m, 6H); 13C NMR (CDCl3) δ 171.4,
138.9, 138.6, 138.4, 137.5, 136.7, 134.9, 128.4, 128.3, 128.2,
128.1, 128.0, 127.9, 127.8, 127.6, 127.5, 127.3, 127.2, 127.1,
126.6, 103.0, 102.4, 102.2, 102.0, 99.7, 97.4, 95.3, 91.6, 82.9,
81.6, 78.4, 78.1, 77.2, 74.8, 74.4, 74.2, 74.0, 73.8, 73.5, 73.3,
72.8, 72.1, 72.0, 70.8, 69.3, 68.5, 62.2, 62.1, 62.0, 56.1, 55.5,
34.1, 33.9, 31.8, 31.7, 31.6, 29.6, 29.3, 8.6, 8.5; MALDI-MS m/z
2187.4 [M + Na+]. Anal. C121H138N2O34 (2164.38).
of a 0.03 N HCl solution, and stirring was continued for 48 h
with a constant monitoring of the pH with a pH meter. The
solution was neutralized by addition of a solution of NaOH
followed by addition of ethanolamine (2 equiv, 0.77 µL, 0.0012
mmol) and concentration in vacuo. The crude residue was then
diluted in pyridine (2 mL), and Ac2O (1 mL) was added to the
solution. After the resulting solution was stirred for 8 h, the
solvent was evaporated and coevaporated twice with toluene
in vacuo. The crude residue was purified by flash chromatog-
raphy (petroleum ether/EtOAc, 1:1 f 3:7) to furnish 4-(3-O-
acetylpropyl)benzyl (2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-
(1f4)-(2-acetamido-3,6-di-O-benzyl-2-deoxy-â-D-glucopyranosyl)-
(1f3)-[(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-(1f4)-(2-
acetamido-3,6-di-O-benzyl-2-deoxy-â-D-glucopyranosyl)-(1f6)]-
(2,4-di-O-benzyl-â-D-galactopyranosyl)-(1f4)-2,3,6-tri-O-benzyl-
R,â-D-glucopyranoside (0.008 g, 0.003 mmol) in 57% yield as
an oil (R/â, 1:1): TLC (EtOAc/petroleum ether, 7:3) Rf ) 0.17;
1H NMR (CDCl3) δ 7.38-7.22 (m, 49H), 6.45 (d, J ) 7.6 Hz,
0.5H), 5.98 (dd, J ) 9.5, 5.4 Hz, 0.5H), 5.78 (dd, J ) 9.5, 5.7
Hz, 1H), 5.53-5.49 (m, 1H), 5.39-5.37 (m, 0.5H), 5.28-5.19
(m, 2.5H), 5.12-4.97 (m, 4H), 4.90-4.29 (m, 26H), 4.25-4.19
(m, 1.5H), 4.16 (dd, J ) 5.3 Hz, 1H), 4.09-3.95 (m, 5H), 3.93-
3.88 (m, 4H), 3.86-3.29 (m, 23.5H), 3.25-3.22 (m, 1H), 2.65-
2.62 (m, 2H), 2.17-1.78 (m, 35H); 13C NMR (CDCl3) δ (selected
data) 170.1, 170.0, 138.8, 138.4, 138.3, 138.2, 138.1, 128.6,
128.5, 128.4, 128.2, 128.0, 127.9, 127.8, 127.7, 127.5, 126.8,
124.7, 123.1, 121.8, 107.5, 102.9, 102.7, 102.6, 102.2, 101.3,
99.9, 99.8, 95.6, 77.2, 76.9, 74.9, 74.8, 73.4, 70.7, 70.5, 69.4,
66.7, 63.7, 63.3, 60.6, 38.8, 37.4, 31.8, 30.1, 29.6, 29.3, 22.6,
21.3, 20.9, 20.8, 20.6; MALDI-MS m/z 2431.5 [M + Na+]. Anal.
C
131H152N2O41 (2408.99).
The aforementioned intermediate (0.008 g, 0.003 mmol) was
dissolved in a dioxane/MeOH/AcOH mixture (1:1:1, 0.9 mL)
and hydrogenated in the presence of Pd-C (10%, 0.003 g).
After 48 h the mixture was filtered through Celite and washed
with MeOH/water (1:1), and the filtrate was evaporated in
vacuo. The crude residue was then diluted in pyridine (1 mL),
and Ac2O (0.5 mL) was added to the solution. After the
resulting solution was stirred for 6 h, the solvents were
evaporated and coevaporated twice with toluene in vacuo. The
residue was purified by flash chromatography (CHCl3/acetone,
1:1) to give 125 (0.005 g, 0.002 mmol) in 82% yield as an
amorphous solid, which had spectroscopic data in accordance
with those reported in the literature:25 MALDI-MS m/z 1852.8
[M + Na+]. Anal. C76H104N2O49 (1829.62).
Resin 23. The Lev group of 19 was removed according to
the protocol applied to resin 20. Next, a glycosylation with 4
(4.5 equiv) was carried out (procedure A) employing 0.31 equiv
of TMSOTf. The Fmoc group was removed using procedure B.
Acetyl (2,3,4,6-Tetr a -O-a cetyl-â-D-ga la ctop yr a n osyl)-
(1f4)-(2-a ceta m id o-3,6-d i-O-a cetyl-2-d eoxy-â-D-glu cop y-
r a n osyl)-(1f3)-[(2,3,4,6-t et r a -O-a cet yl-â-D-ga la ct op yr a -
n osyl)-(1f4)-(2-a cet a m id o-3,6-d i-O-a cet yl-2-d eoxy-â-D-
g l u c o p y r a n o s y l ) -( 1 f6 ) ] -( 2 , 4 -d i -O -a c e t y l -â-D -
g a l a c t o p y r a n o s y l )-(1 f4 )-2 ,3 ,6 -t r i -O -a c e t y l -r,â-D -
glu cop yr a n osid e (1). A mixture of 22 (0.014 g, 0.006 mmol)
and sodium hydroxide (25 equiv, 0.006 g, 0.16 mmol) in a
dioxane/water mixture (1:1, 1 mL) was stirred at room tem-
perature. After 14 h, the pH was adjusted to 4.5 by addition
Ack n ow led gm en t. We thank the European Com-
munity (Grant No. FAIR-CT97-3142), the Bundesmin-
isterium fu¨r Bildung und Forschung (Grant No. 0311
229), the Deutsche Forschungsgemeinschaft, and the
Fonds der Chemischen Industrie for financial support
of this work.
Su p p or tin g In for m a tion Ava ila ble: Spectral data for all
new compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
J O016018P