Synthesis of Gal determinant epitopes, their glycomimetic variants, and trimeric clusters - Relevance to tumor associated antigens and to discordant xenografts

Article abstract of DOI:10.1016/S0040-4020(01)00195-8

The synthesis of Galα1→3Gal-quinic acid pseudo-trisaccharides and their elaboration into trimeric clusters is described.

Full text of DOI:10.1016/S0040-4020(01)00195-8

TETRAHEDRON
Pergamon
Tetrahedron 57 62001) 3281±3290
Synthesis of Gal determinant epitopes, their glycomimetic
variants, and trimeric clustersÐrelevance to tumor associated
antigens and to discordant xenografts
Stephen Hanessian,a,p Hoan K. Huynh,a Gurijala V. Reddy,a Rudolph O. Duthaler,b
Andreas Katopodis,b Markus B. Streiff,b Willy Kinzyb and Reinhold Oehrleinc
a
 Â
Department of Chemistry, Universite de Montreal, P.O. Box 6128, Succursale Centre-ville, Montreal, Quebec, Canada H3C 3J7
bNovartis Pharma AG, CH-4002, Basel, Switzerland
cCiba Specialty Chemicals, CH-4002, Basel, Switzerland
Received 19 December 2000; accepted 12 February 2001
AbstractÐThe synthesis of Gala1!3Gal-quinic acid pseudo-trisaccharides and their elaboration into trimeric clusters is described. q 2001
Elsevier Science Ltd. All rights reserved.
1. Introduction
transplantation.14 In an ideal scenario, the depletion of
anti-aGal antibodies from an organ recipient's blood before
6if at all) using a suitable formulation of the above
mentioned disaccharide and trisaccharide would then set
the stage for the administration of immunosuppressant
drugs, thus minimizing the risk of humoral immune rejec-
tion. The involvement of Gal epitopes in important cell
surface phenomena with obvious practical therapeutic
consequences has revived an interest in their synthesis.
Carbohydrates play a critical role in cell surface phenomena
during the proliferation of cancer.1 The presence of so-
called tumor associated antigens can in fact be related to
tumor progression.2 The terminal a-Gal residue, particularly
in conjunction with other sugar containing oligosaccharides,
plays a dominant role in recognition and antibody response
in autoimmune processes. For example, Gala1!3Gal 6B
disaccharide) 1 epitopes are more abundantly expressed
on cell surfaces of human breast cancers, compared to
normal cells.3 The overexpression of other tumor associated
antigens such as GalNAc 6Tn)4,5 and Galb1!3GalNAc
6T),3,6 has been linked to certain types of tumors.7 In a recent
paper, we reported on the synthesis of trimeric, clustered Tn
and T motifs on a pentaerythritol scaffold.8,9 The potential
importance of clustered antigens for cooperative binding to
protein receptors such as antibodies has been previously
reviewed.10 Another relevant epitope is associated with
the Gala1!3Galb1!4GlcNAc 6linear B type 2 trisacchar-
ide) which interacts with natural IgG, IgM and IgA anti-
bodies.11 The overexpression of this trisaccharide epitope
on cell surface glycoproteins may illicit an anti-Gal anti-
body mediated autoimmune response.12
In this paper, we report on a synthesis of the 3-aminopropyl
glycoside of the linear B type 2 trisaccharide 1, an analog in
which the GlcNAc unit is replaced by a carbocyclic variant
derived from quinic acid 2, and the deployment of the
pseudo-trisaccharide 2 as a trimeric cluster on a penta-
erythritol scaffold,8,15 3 6Fig. 1).
Several syntheses of the linear B type 2 trisaccharide have
been recorded during the past two decades.16±22 For example
in one case, the Gala1!3Gal unit was prepared ®rst using
halide ion catalyzed a-galactosylation.16
A shorter
sequence19 involving 15 steps relied on d-lactal as a pre-
cursor to the Galb1!4GlcNAc unit. Enzymes, galacto-
sidase21 or a61!3)Gal transferase,22 were used as well for
the construction of the Gala61!3) linkage. In the preceding
paper, we reported on practical syntheses of 6-aminohexyl
glycosides of Gala1!3Gal and Gala3Gal!4GlcNAc.23
Of great interest is the identi®cation of the Gala1!3Gal
and Gala1!3Galb1!4GlcNAc, epitopes on all tissue of
pig.13 Thus, the prospects of this epitope being recognized
by natural anti-aGal antibodies will have major conse-
quences on the success of pig-to-human organ xeno-
The synthesis of the Gala1!3Gal donor 624 was realized
utilizing methodology developed in our laboratory for a-
glycosylations.25 Thus, treatment of the acceptor 516 with
a slight excess of donor 4 in the presence of Cu6OTf)2 as
promoter led to the formation of the desired disaccharide 6
as a single anomer in 75% yield 6Scheme 1). Coupling to a
Keywords: carbohydrate mimetics; pseudo-saccharides; clustering.
Corresponding author. Tel.: 1514-343-6738; fax: 1514-343-5728;
e-mail: hanessia@ere.umontreal.ca
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020601)00195-8

3282
S. Hanessian et al. / Tetrahedron 57 52001) 3281±3290
Figure 1.
pre-formed GlcNAc acceptor 7 using NIS as a promoter26
afforded the protected trisaccharide 8 in modest yield.
Finally, deprotection under standard conditions gave the
desired 3-aminopropyl trisaccharide 1 as an amorphous
solid.
derivative 10 using tin-mediated selective tosylation29 and
displacement of the primary tosylate with sodium azide.
Coupling with the thioethyl disaccharide 6 as a glycosyl
donor proceeded in excellent yield to afford the pseudo-
trisaccharide 11. Systematic removal of the protective
groups gave the intended carbocyclic analog 2 in excellent
overall yield.
We next considered the replacement of the GlcNAc unit in 2
with a carbocyclic spatial mimic derived from quinic acid
6Scheme 2).27 The known intermediate 9,27,28 readily avail-
able in two steps, was transformed into the azidomethyl
The recognition of compound 2 by natural anti-a-Gal
antibodies of pooled human AB-serum was tested by a
Scheme 1. a. Cu6OTf)2, 75%; b. NIS/TfOH, 45%; c. NaOMe/MeOH, 95%; d. H2, Pd6OH)2/C, 85%.

S. Hanessian et al. / Tetrahedron 57 52001) 3281±3290
3283
Scheme 2. a. Bu2SnO, then TsCl, 90%; b. NaN3, 65%; c. NIS/TfOH, 90%; d. 80% aq., AcOH, 88%; e. NaOMe/MeOH, 85%; f. H2, Pd6OH)2/C, 90%.
Scheme 3. a. TBAF, THF, rt, 24 h, 88%; b. allyl bromide, NaH, DMF, rt, 10 h, 78%; c. O3, CH2Cl2, then NaBH4, MeOH, 30 min., 81%; d. TBDPSCl,
Imidazole, DMAP, rt, 12 h, then Pd/C, H2, MeOH, rt, 10 h, 71%; e. Pd/C, H2, MeOH, rt, 10 h, 70%.
Scheme 4. a. AgOTf, CH2Cl2, 08C, 1 h, 70%; b. 1N NaOH, MeOH, rt, 1 h, 94%; c. EDC, HOBT, Et3N, DMF, rt, 24 h, 72%; d. Bu4NF, THF, 79%.

3284
S. Hanessian et al. / Tetrahedron 57 52001) 3281±3290
Scheme 5. a. AgOTf, CH2Cl2, 08C, 3 h, 70%; b. 1 N NaOH, MeOH, rt, 1 h, 90%; c. Trimethylsilylethanol, EDC, HOBT, Et3N, DMF, rt, 24 h, 71%; d. DDQ,
CH2Cl2, H2O, rt, 1 h, 87%; e. NIS, TfOH, CH3CN, 2408C, 1 h, 71%; f. TBAF, THF, rt, 3 h, 90%; g. EDC, HOBT, Et3N, DMF, rt, 24 h, 74%; h. TBAF, THF,
rt, 12 h, 75%; i. MsCl, Pyridine, CH2Cl2, then NaN3, DMF, 608C, 6 h, 81%; j. AcOH-H2O 64:1), 608C, 75%; k. LiOH, eq. MeOH, 608C, 68%; l. Pd6OH)2/C, H2,
aq. dioxane, 67%.
competitive enzyme-linked immunosorbent assay 6ELISA),
with a high-af®nity polyvalent a-galactoside conjugate as
primary coating reagent, and horseradish peroxidase labeled
anti-IgG or anti-IgM antibodies as secondary reagents.
Interestingly, the pseudo-trisaccharide 2 6IC50 19 mM/IgG,
2000 mM/IgM) was found to be qualitatively equal in its
binding properties compared to the trisaccharide 1 6IC50
15 mM/IgG, 1100 mM/IgM).30
The design of an oligomeric cluster that deploys pseudo-di-

S. Hanessian et al. / Tetrahedron 57 52001) 3281±3290
3285
and trisaccharides related to B disaccharide and linear B
2. Experimental
2.1. General information
type 2 trisaccharide was based on previous experience in
our group using pentaerythritol as a scaffold 6Scheme 3).8,31
Pentaerythritol was transformed to the tri-azidoethyl ether
derivative 12 as previously described.15 Reduction of
the azido groups led to the triamine 13. Desilylation of 12
and attachment of the allyl group afforded 14, which was
transformed into 15 using standard methodology. Protection
of the primary alcohol and reduction of the azide groups
gave the triamine 16. To explore an alternative a-galacto-
sylation method, we utilized the crystalline 2-pyridyl-
thiocarbonate 6TOPCAT) donor 1732 in conjunction with
an acceptor derived from quinic acid 18. Using AgOTf as
promoter,33 the pseudo-disaccharide 19 was obtained in
70% yield 6Scheme 4). Hydrolysis to the acid 20 and
coupling with the triamine 13 in the presence of EDC and
HOBT led to the trimeric cluster 21 in 72% yield. Removal
of the TBDPS group with ¯uoride ion afforded the corre-
sponding alcohol, 22.
Solvents were distilled under positive pressure of dry
nitrogen before use and dried by standard methods: THF
and ether, from Na/benzophenone; and CH2Cl2, from
CaCl2. All commercially available reagents were used with-
out further puri®cation. All reactions were performed under
nitrogen atmosphere. NMR 61H, 13C) spectra were recorded
on AMX-300 and ARX-400 spectrometers. Low- and high-
resolution mass spectra were recorded on VG Micromass,
Ael-MS902 or Kratos MS-50 spectrometers using fast atom
bombardment 6FAB) or electrospray techniques. Optical
rotations were recorded on a Perkin±Elmer 241 polarimeter
in a 1 dm cell at ambient temperature. Analytical thin-layer
chromatography was performed on Merck 60F254 pre-coated
silica gel plates. Visualization was performed by ultraviolet
light and/or by staining with ceric ammonium molybdate or
ninhydrine. Flash column chromatography was performed
using 640±60 mm) silica gel at increased pressure.
Having established the conditions for amide coupling with
the triamine 13 and the pseudo-disaccharide 20, we
proceeded to elaborate the corresponding pseudo-
trisaccharide trimer 6Scheme 5). p-Methoxybenzylation of
the quinic ester derivative 18 under mild conditions, with
p-methoxybenzyl 2-pyridylthiocarbonate in the presence of
silver tri¯ate,34 afforded the ether 24, which was further
elaborated to the corresponding trimethylsilylethyl ester
25. Coupling with the acceptor disaccharide 6 in the
presence of NIS as described above led to the expected
pseudo-trisaccharide analog 26 in 75% yield. Hydrolysis
to the acid 27 and coupling with the triamine 16, in the
presence of EDC and HOBT, gave the fully protected trimer
28 in 74% yield. There remained to introduce an amino
group on the pendant hydroxyethyl side-chain, and to
remove the protective groups. Desilylation of 28 followed
by mesylation and displacement with sodium azide gave the
desired azido derivative 29 in excellent overall yield.
Sequential removal of the acetonide groups led to 30,
which upon treatment with sodium methoxide gave the
partially benzylated target compound 31 in good overall
yield. Complete debenzylation with simultaneous reduc-
tion of the azido group in 31 proved to be problematic
at ®rst. Eventually, it was found that catalytic reduction
with 20% Pd6OH)2/C 6Pearlman's catalyst),35 in aqueous
dioxane, and repeating the process again led to the
desired unprotected trimeric cluster 3. The structure of
3 6C70H122N4O49) was ascertained from spectroscopic
studies including two-dimensional 1H±13C correlation
6HSQC).
2.1.1. Ethylthio 2,3,4,6-tetra-O-benzyl-a-d-galacto-
pyranosyl-+1!3)-4-O-acetyl-2,6-di-O-benzoyl-b-d-galacto-
pyranoside, 6. To a mixture of the glycosyl donor 425
61.1 g; 1.69 mmol), 0.4 g 60.843 mmol) of the acceptor
5,24 activated 4 A MS, and 30 mL of dry CH2Cl2 was
Ê
added 1.22 g 63.37 mmol) of oven-dried 61008C) Cu6OTf)2
under argon at room temperature. The mixture was stirred
for 6 h, three drops of pyridine were added, then concen-
trated. The residue was puri®ed by ¯ash chromatography on
silica gel column 6hexanes/EtOAc 1:1) to give 600 mg
675%) of the desired disaccharide 8 as the a-anomer only;
[a]Dˆ1314.9 6c 0.28, CHCl3); HRMS: m/z calcd for
C58H60O13S 6M11) 998.1553; found 998.1573.
2.1.2. 3-Benzyloxycarbonylaminoethyl 2,3,4,6-tetra-O-
benzyl-a-d-galactopyranosyl-+1!3)-4-O-acetyl-2,6-di-
O-benzoyl-b-+1!4)-2-acetamido-2-deoxy-3,6-di-O-benzyl-
b-d-glucopyranoside, 8. To a solution of disaccharide 6
680 mg; 0.079 mmol) and glycosyl acceptor 7 645 mg;
0.076 mmol) in dichloromethane 615 mL) were added
successively NIS 645 mg; 0.197 mmol) and TfOH
61.5 mL; 0.019 mmol) at 2408C. The reaction mixture
was stirred for 1 h, warmed up to room temperature and
®ltered through Celite, then washed with dichloromethane.
The ®ltrate was washed successively with Na2S2O3,
NaHCO3, brine, dried over Na2SO4, the ®ltrate concentrated
and puri®ed by ¯ash chromatography on silica gel 6hexanes/
EtOAc 2:1) to give the desired trisaccharide 8 653 mg;
45%). [a]Dˆ129.6 6c 0.57, CHCl3); HRMS: m/z calcd for
C89H94O21N2 6M11) 1528.6996; found 1528.6349.
In conclusion, we have described methods for the stereo-
selective a-galactosylations in conjunction with the syn-
thesis of biologically relevant oligosaccharides. In
addition, we have shown that the synthesis of pseudo-oligo-
saccharides related to Gala1!3Gal 6B disaccharide) and
Gala1!3Galb1!4GlcNAc 6linear B type 2 trisaccharide)
provides alternative approaches to the design of glyco-
mimetic motifs. Finally, we have constructed a trimeric
clustered `pseudo-hapten' that can be conjugated to appro-
priate carriers for the study of their possible immunogenic
responses. Further studies in the area will be reported in due
course.
2.1.3. 3-Aminoethyl a-d-galactopyranosyl-+1!3)-b-d-
glucopyranosyl-+1!4)b-d-2-acetamido-2-deoxy-d-gluco-
pyranoside, 1. To a solution of trisaccharide 8 650 mg;
0.0324 mmol) in dry methanol 62 mL) was slowly added
freshly prepared sodium methoxide at 08C and the pH of
the solution was kept at 9. The solution was stirred at room
temperature for 5 h, then neutralized with Amberlite IR-120
6H1), ®ltered and concentrated. The residue was puri®ed by
¯ash chromatography on silica gel 6MeOH±CH2Cl2 1:20) to
give 40 mg of the desired product 695%); [a]Dˆ134.8 6c

3286
S. Hanessian et al. / Tetrahedron 57 52001) 3281±3290
2.38, CHCl3); HRMS: m/z calcd for C72H82O18N2 6M11)
1263.4320; found 1263.1230.
76.7, 75.5, 74.8, 73.5, 73.3, 73.1, 73.0, 71.6, 71.3, 70.6,
69.8, 69.5, 64.9, 62.3, 60.5, 60.2, 36.1, 33.4, 27.4, 24.8,
20.4 and 14.1; HRMS: m/z calcd for C66H71N3O17 6M1)
1177.4783; found 1177.4795.
The product 636.0 mg; 0.027 mmol) in methanol 63 mL) was
hydrogenolyzed in the presence of 20% Pd6OH)2/C 620 mg)
under pressure 650 psi) for 12 h at room temperature. The
mixture was ®ltered through Celite, the latter washed with
methanol and water and the ®ltrate was lyophilized to give
the title product 1 614.5 mg; 85%); [a]Dˆ159.8 6c 0.59,
CHCl3); HRMS: m/z calcd for C65H78O16N2 6M11)
1144.3250; found 1144.6330.
2.1.6. Pseudo-trisaccharide 2. A solution of 13 6120 mg,
0.1 mmol) in 80% aqueous AcOH was heated at 608C
for 4 h. The solvents were evaporated to dryness and the
resultant material was puri®ed on silica gel using EtOAc/
hexanes 61:2) as eluent to provide 100 mg of the diol in 88%
yield.
2.1.4. +1R,3R,4S,5R)-1-Azidomethyl-1,3,4,5-tetrahydroxy-
3,4-O-isopropylidene-1-cyclohexane, 10. A solution of 9
60.5 g, 2.29 mmol) in benzene containing Bu2SnO 60.38 g,
1.5 mmol) was azeotropically distilled at 1008C for 6 h. The
mixture was cooled down to 08C and was treated with tosyl
chloride 60.48 g, 2.5 mmol). After being stirred for 30 min
at this temperature, the mixture was evaporated to dryness
and puri®ed by column chromatography using EtOAc/
hexanes 61:1) to give 0.77 g of the corresponding tosylate
in 90% yield.
To a solution of this compound 6100 mg, 0.088 mmol) in
MeOH 65 mL) was slowly added freshly prepared NaOMe
in MeOH until pH of the reaction reached ,10. After
stirring at 408C for 16 h, the mixture was cooled down to
room temperature, and carefully neutralized at 08C with
Amberlite IR 6H1) resin and ®ltered through a bed of Celite
which was washed with a small amount of water several
times. The combined ®ltrates were evaporated at low bath
temperatures and the resulting concentrate was freeze-dried
to obtain the polyol precursor 666 mg) in 85% yield.
1
[a]Dˆ143.6 6c 2.0, CHCl3). H NMR 6400 MHz, CDCl3):
To a solution of the tosylate 60.5 g, 1.63 mmol) in dry DMF
610 mL) was added NaN3 60.16 g, 2.4 mmol), and the
contents were stirred at 908C for 12 h. The reaction mixture
was cooled to room temperature, diluted with EtOAc
650 mL), the solids were ®ltered, and the ®ltrate was washed
with brine 63£10 mL). The organic layer was dried, evapo-
rated, and the resultant residue was puri®ed on silica gel
using EtOAc/hexanes 63:2) to afford the azido compound
10 60.40 g) in 65% yield; [a]Dˆ107.7 6c 1.0, CHCl3). IR
d 7.4±7.16 6m, 20H, aromatic), 4.9±4.6 6m, 7H), 4.54±4.34
6m, 3H), 4.32 6d, 1H, Jˆ7.9 Hz, H-1), 4.32 6m, 1H), 4.20
6bs, 1H), 4.15 9m, 1H), 4.06 6dd, 1H), 4.01 6m, 2H), 3.93
6dd, 1H, Jˆ7.6, 12.3 Hz), 3.78 6bs, 1H), 3.71 6q, 2H, Jˆ9.2,
17.0 Hz), 3.57±3.43 6m, 5H), 3.06 6q, 2H), 2.12.0 6m, 2H),
1.44±1.35 6m, 2H). 13C NMR 6100 MHz, CDCl3): d 138.3,
137.6, 128.4±127.2, 102.7, 95.7, 79.2, 77.8, 75.8, 74.7,
74.5, 74.4, 74.3, 74.1, 73.3, 72.4, 70.3, 69.2, 68.7, 66.3,
62.3, 60.3 and 39.7. HRMS: m/z calcd for C47H57N3O14
6M1) 887.3810; found 887.3740.
1
6CHCl3): 2110 cm21 6N3); H NMR 6400 MHz, CDCl3): d
4.44 6m, 1H, H-5), 4.10 6m, 1H, H-3), 3.92 6t, 1H, Jˆ6.0,
12.0 Hz, H-4), 3.3±2.89 6m, 4H), 2.28±1.97 6m, 2H, CH2),
1.80 6dd, 1H, Jˆ3.9, 15.6 Hz), 1.51, 1.35 [2s, 6H, C6CH3)2].
13C NMR 6100 MHz, CDCl3): d 109.2, 80.3, 74.9, 72.8,
68.7, 60.5, 39.0, 33.7, 28.2 and 25.6. HRMS: m/z calcd
for C10H18N3O4 6M1) 244.1297; found 244.1305.
To a solution of the above precursor 660 mg, 0.068 mmol) in
80% aq. AcOH/EtOH 61:9, 10 ml) was added 10% Pd6OH)2/
C 650 mg) and the suspension was stirred under H2 at 45 psi
in a medium pressure hydrogenator for 16 h. Filtration of
the catalyst over Celite, washing the residue with water,
evaporation of the combined ®ltrates, evaporation and
freeze-drying gave compound 2 630 mg) in 90% yield;
2.1.5. Pseudo-trisaccharide 11. Compound 10 635 mg,
0.14 mmol) and disaccharide 6 6170 mg, 0.17 mmol) in
dry CH2Cl2 620 ml) were stirred at room temperature in
1
[a]Dˆ147.1 6c 1.0, H2O). H NMR 6D2O): d 5.14 6d, 1H,
Jˆ3.1 Hz, H-100), 4.53 6d, 1H, Jˆ7.6 Hz, H-10), 4.23±4.0
6m, 4H), 3.96 6bs, 1H), 3.83 6m, 1H), 3.79±3.62 6m, 9H),
3.29±3.12 6m, 3H), 2.95 6m, 2H), 2.2±1.68 6m, 5H), 1.97 6s,
3H). 13C NMR 6D2O): d 101.8, 96.0, 77.9, 75.7, 73.3, 72.2,
71.5, 69.9, 69.8, 68.8, 65.5, 64.8, 62.1, 61.8, 61.6, 56.5,
51.3, 44.6, 43.9, 37.4 and 10.8. HRMS: m/z calcd for
C19H35N3O14 6M11) 501.2057; found 501.2566.
Ê
the presence of 4 A molecular sieves for 5 h. The solution
was cooled down to 2108C and NIS 644 mg, 0.19 mmol)
followed by TfOH 63 mL, catalytic) were added. The
contents were stirred at this temperature for 10 h under an
inert atmosphere. The mixture was diluted with CH2Cl2
620 mL), ®ltered over Celite, and the ®ltrates were washed
successively with 10% NaHCO3, 10% brine solution and the
organic phase was dried. Evaporation and puri®cation over
silica gel using EtOAc/hexanes 61:3) as eluent gave 150 mg
of the title compound 11 in 90% yield; [a]Dˆ151.0 6c 1.0,
CHCl3); IR 6CHCl3): 2110 cm21 6N3); 1H NMR 6400 MHz,
CDCl3): d 8.08±8.02 6m, 4H), 7.5±7.13 6m, 26H, aromatic),
5.65 6bs, 1H, H-40), 5.52 6t, 1H, H-20), 5.23 6d, 1H,
Jˆ3.3 Hz, H-100), 4.79±4.32 6m, 11H), 4.21 6m, 1H), 4.14
6m, 2H), 3.98±3.91 6m, 3H), 3.61 6dd, 1H), 3.45 6t, 1H),
3.27 6m, 2H), 2.96 6q, 2H), 2.04±1.65 6m, 4H), 1.90 6s, 3H),
1.45, 1.26 [2s, 6H, C6CH3)2]; 13C NMR 6100 MHz, CDCl3):
d 170.3, 165.9, 164.7, 138.5, 138.3, 138.2, 133.2, 133.1,
129.7, 129.7, 129.3, 128.4, 128.3, 128.1, 128.0, 127.8,
127.6, 127.5, 127.4, 127.3, 127.2, 108.8, 100.8, 93.5, 78.7,
2.1.7. Tri-O-+2-azidoethyl)-1-+2-hydroxyethyl)-pentaery-
thritol, 15. Ozone was bubbled through a solution of 148
688 mg; 0.2297 mmol) in 4 mL dichloromethane±methanol
61:1) at 2788C until the solution was saturated 610 min).
Argon was bubbled through the solution for 15 min to
remove the residual ozone and an excess of methyl sul®de
was added. The solution was allowed to warm to room
temperature and concentrated to a syrup that was dissolved
in ethanol 64 mL). Sodium borohydride 635 mg; 0.919
mmol) was added to a cooled solution at 08C. After stirring
for 24 h at room temperature, the solution was acidi®ed with
20% HCl to pH 6, then concentrated to a syrup that was
partitioned between ethyl acetate and water. After usual

S. Hanessian et al. / Tetrahedron 57 52001) 3281±3290
3287
work-up, a syrupy material was puri®ed by silica gel ¯ash
chromatography 6EtOAc±MeOH 20:1) to give the title
product 15 as a syrup 672.1 mg; 81%). 1H NMR
6400 MHz, CDCl3): d 3.69 6t, 2H), 3.62 6t, 6H), 3.54 6t,
2H), 3.49 6s, 8H), 3.32 6t, 6H), 2.30 6s, 1H, OH). 13C
NMR 6100 MHz, CDCl3). d 72.1, 70.4, 69.6, 69.1, 61.3,
50.6, 44.9. HRMS 6FAB), calcd for C13H25O5N9 6M11)
387.1979; found 387.1995.
Amberlite 6IR-1201), ®ltered and concentrated to give
desired product 20 as a syrup 61.05 g; 94%). HRMS: m/z
calcd for C44H50O11 6M11) 754.6290; found 754.5679.
2.1.11. Trimeric pseudo-disaccharide cluster, 21. To a
solution of 20 6749 mg; 0.994 mmol) in DMF 620 mL)
were added successively HOBT 6161 mg; 1.19 mmol),
EDC 6228 mg; 1.19 mmol) and triethylamine 6207 mL;
1.49 mmol) at 08C. The solution was stirred for 1 h, then
the triamine 13 6100 mg; 0.199 mmol) in 3 mL of DMF was
added dropwise to the reaction mixture. The solution was
warmed up to room temperature, the stirring was continued
for 24 h, DMF was removed by evaporation, the resulting
residue was dissolved in EtOAc 6300 mL), then washed
successively with 5% aqueous HCl 650 mL), NaCHO3 sat.
650 mL), water and brine, dried over Na2SO4, and concen-
trated. Puri®cation by ¯ash chromatography on silica gel
6hexane/EtOAc 1:2) gave the desired product 21 6390 mg;
72%): LRMS 6MALDI): calcd for C159H189O34N3Si
6M1Na) 2737; found 2740.
2.1.8. Tri-O-+2-aminoethyl)-1-O-tert-butyldiphenylsilyl-
oxyethyl-pentaerythritol, 16. To a solution of 3a 672 mg;
0.186 mmol) in 4 mL DMF/THF 61:1) were added succes-
sively imidazole 676 mg; 1.12 mmol), a catalytic amount
of DMAP and tert-butyldiphenylchlorosilane 665 mL;
0.372 mmol) at room temperature. The solution was stirred
for 24 h, poured into 10 mL water, and extracted twice with
diethyl ether. The organic phase was washed with brine and
water, and the extracts were dried 6MgSO4), ®ltered,
concentrated, and puri®ed by silica gel ¯ash chroma-
tography to give a syrup. This was dissolved in methanol
63 mL) and hydrogenated in the presence of 10% Pd/C
640 mg) for 12 h at room temperature. The mixture was
®ltered through Celite, washed with methanol, then concen-
trated to give 16 672 mg; 71%). 1H NMR 6400 MHz,
CD3OD): d 7.72±7.70 6m, 4H), 7.46±7.38 6m, 6H), 3.82
6t, Jˆ4.53 Hz, 2H), 3.57±3.31 6m, 22H), 2.77 6s, 6H). 13C
NMR 6100 MHz, CD3OD): d 136.7, 134.8, 130.9, 128.8,
74.1, 73.3, 71.4, 70.9, 64.8, 46.8, 41.9, 27.4, 20.2. HRMS
6FAB), calcd for C29H49O5N3Si 6M11) 548.3441; found
548.3462.
2.1.12. Trimeric pseudo-disaccharide cluster, 22. To a
solution of 21 691 mg; 0.038 mmol) in THF 62 mL) was
added TBAF 6168 mL; 0.167 mmol) at room temperature.
The resulting solution was stirred for 12 h, then poured into
ice±water and extracted with CH2Cl2. Concentration and
puri®cation by ¯ash chromatography on silica gel 6hexane/
EtOAc 1:12) gave 66.0 mg 679%) of the desired product 22
as a syrup in 79% yield; LRMS 6MALDI): calcd for
C143H171O34N3 6M1Na) 2497; found 2500.
2.1.9. Pseudo-disaccharide methyl ester 19. To a mixture
of the glycosyl donor 1725 64.1 g; 6.1 mmol), glycosyl
acceptor 188 61.3 g; 5.28 mmol), and activated powdered
2.1.13. Methyl 5-O-p-methoxybenzyl-3,4-O-isopropyl-
idene quinate, 24. To
a solution of 18 60.88 g;
3.58 mmol) and 23 61.06 g; 4.3 mmol) in CH2Cl2 620 mL)
was added silver tri¯ate 61.6 g; 6.46 mmol) at room
temperature. The mixture was stirred for 3 h and the suspen-
sion was ®ltered through Celite, washed with dichloro-
methane, concentrated and puri®ed by ¯ash chroma-
tography on silica gel 6hexanes/EtOAc 1:1) to give 24
Ê
4 A molecular sieves 6200 mg) in 50 mL of CH2Cl2 was
added silver tri¯ate 62.3 g; 9.1 mmol). The resulting suspen-
sion was stirred for 1 h under nitrogen at 08C, pyridine
6100 mL) was added, and the mixture was ®ltered through
Celite and washed with CH2Cl2. Concentration of the ®ltrate
and puri®cation by ¯ash chromatography on silica gel
6hexanes/EtOAc 1:1) gave the desired glycoside 19
1
60.92 g; 70%). H NMR 6400 MHz, CDCl3): d 7.29 6m,
2H), 6.87 6m, 2H), 4.66 6d, Jˆ11.43 Hz, 1H), 4.58 6d, Jˆ
11.04 Hz, 1H), 4.47 6m, 1H), 4.12 6dd, Jˆ5.42, 6.56 Hz,
1H), 3.92 6m, 1H), 3.78 6s, 3H, OMe), 3.77 6s, 3H, OMe),
3.44 6s, 1H, OH), 2.23 6m, 2H), 2.16 6m, 1H), 1.82 6dd,
Jˆ11.24, 13.35 Hz, 1H), 1.466s, 3H, Me), 1.35 6s, 3H,
Me). 13C NMR 6100 MHz, CDCl3): d 174.9, 159.0, 130.4,
129.3, 113.6, 108.9, 79.2, 74.5, 73.9, 73.8, 71.3, 55.1, 52.8,
37.2, 34.3, 28.2, 25.8. HRMS: m/z calcd for C19H26O7
6M11) 367.4102; found 367.4676.
1
62.85 g; 70%); H NMR 6400 MHz, CDCl3): d 7.45±7.22
6m, 20H, Ph), 5.54 6d, Jˆ3.85 Hz, 1H, H-1, a), 4.97 6d,
Jˆ11.41 Hz, 1H), 4.88 6d, Jˆ11.63 Hz, 1H), 4.81 6d, Jˆ
11.66 Hz, 1H), 4.78 6d, Jˆ11.70 Hz, 1H), 4.74 6d, Jˆ
11.64 Hz, 1H), 4.59 6d, Jˆ11.44 Hz, 1H), 4.51 6d, Jˆ
11.51 Hz, 1H), 4.46 6m, 1H), 4.42 6d, Jˆ11.60 Hz, 1H),
4.32 6m, 1H, H-5), 4.22 6dd, Jˆ5.6, 7.3 Hz, 1H), 4.08 6dd,
Jˆ3.83, 10.1 Hz, 1H, H-2), 3.98 6m, 2H), 3.93 6dd, Jˆ2.77,
10.2 Hz, 1H, H-3), 3.77 6s, 3H, OMe), 3.60 6m, 2H), 3.45 6s,
1H, OH), 2.26 6m, 2H), 2.07 6m, 1H), 1.55 6s, 3H, Me), 1.39
6s, 3H, Me); 13C NMR 6100 MHz, CDCl3); d 174.9, 138.9,
138.7, 138.6, 137.9, 128.3, 128.2, 128.2, 128.1, 127.9,
127.7, 127.6, 127.5, 127.4, 109.0, 97.3, 79.5, 78.8, 76.3,
75.4, 75.1, 74.7, 74.1, 73.9, 73.4, 73.1, 72.9, 72.6, 69.5,
68.9, 68.6, 60.3, 52.8, 38.4, 34.3, 28.2, 27.9, 26.0, 25.7,
20.9, 14.2. HRMS: m/z calcd for C46H56O11 6M11)
785.9420; found 785.5833.
2.1.14. 2-Trimethylsilylethyl 3,4-O-isopropylidene quinate,
25. To a solution of 24 60.62 g; 1.7 mmol) in methanol
610 mL) was added 1N NaOH 62 mL) at 08C. The mixture
was stirred at room temperature for 1 h, then neutralized
with Amberlite IR-120 6H1) 6pH 6), ®ltered and concen-
trated. The syrupy residue was dissolved in EtOAc
6200 mL), then acidi®ed with HCl 610%), dried over
Na2SO4, and concentrated to give the desired acid 60.55 g;
90%); HRMS: m/z calcd for C18H24O7 6M11) 353.3834;
found 353.3522.
2.1.10. Pseudo-disaccharide free acid, 20. To the solution
of 19 61.14 g; 1.48 mmol) in 30 mL of methanol was added
dropwise 5 mL of aq. NaOH 61N) at 08C. The solution was
stirred for 3 h at room temperature, then neutralized with
To a solution of the acid 61.1 g; 3.1 mmol) in DMF 630 mL)
were added successively HOBT 60.63 g; 4.63 mmol), EDC

3288
S. Hanessian et al. / Tetrahedron 57 52001) 3281±3290
61.2 g; 6.2 mmol) and triethylamine 63 mL; 21.6 mmol) at
08C. The solution was stirred for 1 h, then trimethysilyletha-
nol 62.2 mL; 15.5 mmol) was added to the reaction mixture.
The solution was warmed up to room temperature and the
stirring was continued for 24 h. DMF was removed by
evaporation, and the resulting residue was dissolved in
EtOAc 6300 mL), then washed successively with 5%
aqueous HCl 650 mL), NaHCO3 sat. 650 mL), water and
brine, dried over Na2SO4, concentrated and puri®ed by
¯ash chromatography on silica gel 6hexanes/EtOAc 2:1) to
give the desired product 60.99 g; 71%). HRMS: m/z calcd
for C23H36O7Si 6M11) 453.6190; found 453.6276.
2.37 mmol) at 08C. The solution was stirred at room
temperature for 3 h, then diluted with EtOAc 650 mL).
The reaction mixture was washed successively with 10%
HCl 63£20 mL), water, brine, dried over Na2SO4, concen-
trated and puri®ed by ¯ash chromatography on silica gel
6MeOH/CH2Cl2 1:20) to give the desired product 27
1
60.83 g, 90%). H NMR 6400 MHz, CDCl3): d 8.06±8.04
6m, 4H), 7.60±7.08 6m, 26H, Ph), 5.66 6d, Jˆ2.85 Hz, 1H),
5.44 6dd, Jˆ10.02, 8.11 Hz, 1H), 5.17 6d, Jˆ3.13 Hz, 1H),
0
4.81 6d, Jˆ8.07 Hz, 1H, H1 -b), 4.66±4.23 6m, 15H), 4.08
6t, Jˆ5.27 Hz, 1H), 3.99 6m, 1H), 3.88 6m, 2H), 3.62 6dd,
Jˆ10.2, 2.65 Hz, 1H), 3.42 6dd, Jˆ9.49, 3.11 Hz, 1H),
3.35±3.29 6m, 2H), 3.21 6m, 1H), 2.15±1.99 6m, 4H), 1.92
6s, 3H, OAc), 1.53 6t, Jˆ9.00 Hz, 1H), 1.36 6s, 3H, Me),
1.21 6s, 3H, Me). 13C NMR 6100 MHz, CDCl3): d 180.2,
174.6, 163.7, 163.8, 138.9, 138.6, 138.2, 138.1, 133.5,
133.1, 129.5, 129.6, 129.5, 129.4, 128.3, 128.1, 128.0,
127.8, 127.6, 127.5, 127.4, 127.3, 127.1, 108.9, 100.2,
93.4, 78.7, 76.7, 75.3, 74.8, 74.4, 73.7, 73.5, 73.3, 73.1,
71.6, 71.3, 70.7, 69.6, 69.3, 64.8, 64.4, 37.1, 34.3, 28.1,
25.7, 20.4. HRMS: m/z calcd for C66H70O19 6M11)
1168.2676; found 1168.4570.
To a solution of the above product 60.7 g; 1.66 mmol) in
dichloromethane 620 mL) and water 62 mL) was added
DDQ 60.75 g; 3.32 mmol) at 08C. The suspension was stir-
red at room temperature for 2 h, then diluted with EtOAc
650 mL), washed with NaHCO3 6three times) and brine,
dried over Na2SO4, concentrated and puri®ed by ¯ash chro-
matography on silica gel 6hexanes/EtOAc 1:1) to give the
desired product 25 as an oil 60.48 g; 87%). 1H NMR
6400 MHz, CDCl3): d 4.37 6dd, Jˆ9.18, 3.78 Hz, 1H),
4.19 6m, 2H), 4.04 6m, 2H), 3.90 6t, Jˆ6.13 Hz, 1H), 3.40
6s, 2H, OH), 2.17 6m, 2H), 1.96 6m, 1H), 1.75 6t,
Jˆ11.51 Hz, 1H), 1.44 6s, 3H, Me), 1.29 6s, 3H, Me), 0.96
6m, 2H), 0.03 6s, 9H, SiMe3). 13C NMR 6100 MHz, CDCl3):
d 175.2, 108.9, 80.1, 73.5, 67.8, 64.4, 60.2, 38.9, 34.6, 28.0,
25.5, 20.8, 17.2, 13.9, 21.73. HRMS: m/z calcd for
C23H36O7Si 6M11) 333.4686; found 333.4555.
2.1.17. Trimeric cluster, 28. To a solution of 27 6600 mg;
0.558 mmol) in DMF 620 mL) were added successively
HOBT 6150 mg; 1.12 mmol), EDC 6213 mg; 1.12 mmol)
and triethylamine 6155 mL; 1.12 mmol) at 08C. The solution
was stirred for 1 h, then the triamine 18 661 mg;
0.112 mmol) in 3 mL of DMF was added dropwise to the
reaction mixture. The solution was warmed up to room
temperature, the stirring was continued for 24 h, DMF
was removed by evaporation, the resulting residue was
dissolved in EtOAc 6300 mL), then washed successively
with 5% aqueous HCl 650 mL), NaHCO3 650 mL), water
and brine, dried over Na2SO4, and concentrated. Puri®cation
by ¯ash chromatography on silica gel 6hexanes/EtOAc 1:2)
gave the desired product 28 6329 mg; 74%); [a]Dˆ129.6 6c
0.5, CHCl3). 1H NMR 6400 MHz, CDCl3): d 8.05±7.95 6m,
12H), 7.68±7.10 6m, 88H, Ph), 5.64 6d, Jˆ3.08 Hz, 3H),
5.60 6dd, Jˆ10.14, 8.13 Hz, 3H), 5.21 6d, Jˆ3.32 Hz,
3H), 4.75 6d, Jˆ11.47 Hz, 3H), 4.69 6d, Jˆ8.08 Hz, 3H,
2.1.15. Pseudo-trisaccharide 26. To a solution of disac-
charide 6 60.45 g; 1.35 mmol) and 25 61.47 g; 1.48 mmol)
in dichloromethane 615 mL) were added successively NIS
60.5 g; 2.22 mmol) and TfOH 626 mL; 0.27 mmol) at
2408C. The reaction mixture was stirred for 1 h, then
warmed up to room temperature and ®ltered through Celite,
then washed with dichloromethane. The ®ltrate was washed
successively with Na2S2O3, NaHCO3, brine, dried over
Na2SO4, concentrated, and puri®ed by ¯ash chroma-
tography on silica gel 6hexanes/EtOAc 2:1) to give the
desired product 26 61.2 g; 71%). 1H NMR 6400 MHz,
CDCl3): d 8.07±7.98 6m, 4H), 7.59±7.11 6m, 26H, Ph),
5.66 6d, Jˆ3.21 Hz, 1H), 5.57 6dd, Jˆ10.19, 8.07 Hz,
1H), 5.23 6d, Jˆ4.21 Hz, 1H), 4.47 6d, Jˆ11.47 Hz, 1H),
4.67±4.64 6m, 3H), 4.55±4.50 6m, 2H), 4.43 6d,
Jˆ11.77 Hz, 1H), 4.36 6m, 2H), 4.36 6d, Jˆ11.07 Hz,
1H), 4.31 6d, Jˆ11.49 Hz, 1H), 4.18±4.08 6m, 6H), 3.96±
3.93 6m, 2H), 3.85 6t, Jˆ6.34 Hz, 1H), 3.59 6dd, Jˆ10.14,
2.57 Hz, 1H), 3.47 6m, 2H), 3.22 6s, 1H, OH), 3.20 6m, 1H),
2.17±2.04 6m, 3H), 1.93 6s, 3H, OAc), 1.60 6dd, Jˆ13.07,
11.90 Hz, 1H), 1.47 6s, 3H, Me), 1.29 6s, 3H, Me), 0.89 6m,
2H), 0.03 6s, 9H, SiMe3). 13C NMR 6100 MHz, CDCl3): d
174.4, 170.3, 165.9, 164.7, 163.3, 138.6, 138.4, 138.3,
138.2, 133.1, 133.0, 129.7, 129.6, 129.5, 129.5, 128.3,
128.2, 128.0, 127.9, 127.6, 127.5, 127.4, 127.3, 127.3,
108.9, 100.2, 93.4, 78.7, 77.8, 77.3, 77.0, 76.7, 75.3, 74.8,
74.4, 73.7, 73.5, 73.3, 73.1, 71.6, 71.3, 70.7, 69.6, 69.3,
64.8, 64.4, 62.3, 37.1, 34.3, 28.1, 25.7, 20.4, 17.1, 21.65.
HRMS: m/z calcd for C71H82O19Si 6M11) 1268.5033; found
1268.5218.
0
H1 -b), 4.64 6m, 9H), 4.53 6m, 3H), 4.50 6d, Jˆ11.50 Hz,
3H), 4.40±4.26 6m, 15H), 4.20±4.10 6m, 9H), 3.94±3.91
6m, 6H), 3.87 6t, Jˆ6.43 Hz, 3H), 3.76 6t, Jˆ4.43 Hz,
3H), 3.57 6dd, Jˆ15.72, 3.77 Hz, 3H), 2.05±1.95 6m, 6H),
1.89 6s, 9H, 3Me), 1.85 6t, Jˆ11.99 Hz, 3H), 1.42 6s, 9H,
3Me), 1.23 6s, 9H, 3Me), 1.03 6s, 9H, t-Bu). 13C NMR
6100 MHz, CDCl3): d 173.4, 170.4, 165.9, 164.6, 138.6,
138.5, 138.3, 138.2, 135.4, 133.5, 133.2, 132.9, 129.7,
129.6, 129.5, 128.3, 128.2, 128.2, 128.0, 127.8, 127.5,
127.4, 127.2, 109.0, 98.8 6C-10, a), 93.56C-100, b), 78.7,
77.5, 77.3, 76.9, 76.6, 75.4, 75.2, 74.6, 74.3, 73.2,
73.1, 73.0, 71.7, 71.1, 70.5, 69.7, 69.3, 64.8, 62.1,
45.4, 38.7, 35.9, 33.9, 28.1, 26.7, 25.7, 20.5, 19.1. LRMS
6MALDI): calcd for C227H253O59N3Si 6M1Na)1 3992;
found 4015.
2.1.18. Trimeric cluster, 29. To a solution of 28 6151 mg;
0.038 mmol) in THF 64 mL) was added TBAF 6227 mL;
0.227 mmol) at room temperature. The resulting solution
was stirred for 12 h, then poured into ice±water and
extracted with CH2Cl2. Concentration and puri®cation by
¯ash chromatography on silica gel 6hexanes/EtOAc 1:12)
2.1.16. Pseudo-trisaccharide 27. To a solution of 26 61 g;
0.80 mmol) in THF 620 mL) was added TBAF 62.4 mL;

S. Hanessian et al. / Tetrahedron 57 52001) 3281±3290
3289
gave the desired product in 75% yield. 1H NMR 6400 MHz,
CDCl3): d 8.05±7.95 6m, 12H), 7.59±7.06 6m, 78H, Ph),
5.64 6d, Jˆ2.83 Hz, 3H), 5.59 6dd, Jˆ10.15, 8.15 Hz, 3H),
5.20 6d, Jˆ2.27 Hz, 3H), 4.74 6d, Jˆ 11.45 Hz, 3H), 4.70 6d,
Jˆ9.05 Hz, 3H), 4.67±4.61 6m, 9H), 4.54 6m, 3H), 4.51 6d,
Jˆ11.40 Hz, 3H), 4.39 6d, Jˆ11.62 Hz, 3H), 4.32±4.23 6m,
9H), 4.19±4.07 6m, 15H), 3.94 6m, 9H), 3.86 6t, Jˆ6.16 Hz,
3H), 3.62 6m, 3H), 3.56 6dd, Jˆ10.20, 6.26 Hz, 3H), 3.50±
3.14 6m, 39H), 2.28 6dd, Jˆ15.85, 3.75 Hz, 3H), 2.03±1.94
6m, 6H), 1.89 6s, 9H, 3OAc), 1.80 6t, Jˆ11.75 Hz, 3H), 1.44
6s, 9H, 3Me), 1.22 6s, 9H, 3Me). 13C NMR 6100 MHz,
CDCl3): d 173.6, 170.4, 165.9, 164.6, 138.6, 138.5, 138.3,
138.2, 133.1, 132.9, 129.7, 129.6, 129.5, 128.3, 128.1,
127.9, 127.8, 127.5, 127.4, 127.2, 108.9, 98.9 6C-10, b),
93.5 6C-100, a), 78.7, 77.6, 77.3, 76.9, 76.6, 75.5, 75.2,
74.6, 74.3, 73.2, 73.1, 72.3, 71.7, 71.1, 70.5, 69.9, 69.7,
69.3, 64.8, 62.1, 61.3, 38.6, 36.0, 33.9, 29.6, 28.1, 25.7,
20.4. LRMS 6MALDI): calcd for C211H234O58N6 6M1Na)
3757; found 3780.
Jˆ11, 3 Hz, 3H, 3£ H-C6200)), 4.2-4.8 6m, 39H), 4.88 6b,
3H, 3£ NH), 5.18 6d, Jˆ4 Hz, 3£ H-C6100)), 5.53 6dd, Jˆ11,
8 Hz, 3H, 3£ H-C620)), 5.63 6d, Jˆ3 Hz, 3H, 3£ H-C640)),
7.1±7.15 6m, 6H), 7.2±7.55 6m, 69H), 7.55±7.65 6m, 3H),
7.98 6d, Jˆ7 Hz, 6H), 8.12 6d, Jˆ7 Hz, 6H). Partial assign-
ments relate to two-dimensional 1H±13C-correlation
spectroscopy 6HSQC).
To a solution of the above product 670 mg, 0.019 mmol) in
methanol 62 mL), H2O 60.2 mL) and CHCl3 61 mL) was
added LiOH-monohydrate 624 mg, 0.57 mmol) and the
mixture was heated to 608C for 3 h. After addition of
AcOH 638 mL) volatiles were evaporated under reduced
pressure. The residue 6105 mg) was chromatographed on
silica gel 610 g, CHCl3/MeOH/H2O 15:2:0.2) affording
41 mg of partially saponi®ed material. Treatment with
LiOH in CH3OH/H2O/CHCl3 as above at 608C was repeated
for 15 h. After evaporation of solvents the residue was par-
titioned between CHCl3 625 mL) and H2O 610 mL, emul-
sions!). The aqueous phase was extracted with CHCl3
615 mL), and the combined organic phases were washed
with 0.1N NaOH 615 mL). Chromatography of the residue
of the organic phases on silica gel 69 g) eluting with CHCl3/
To the above product 6123 mg; 0.033 mmol) in a mixture of
pyridine 61 mL) and CH2Cl2 61 mL) was added MsCl
67.5 mL; 0.098 mmol) at 08C. The solution was stirred at
room temperature for 2 h, then poured into ice±water
62 mL), washed with CH2Cl2 and concentrated. The result-
ing residue was dissolved in DMF 62 mL) and treated with
NaN3 611 mg; 0.163 mmol) at 608C for 6 h, then diluted
with EtOAc 620 mL). The solution was washed successively
with HCl 65%), water and brine, and dried over Na2SO4.
Concentration and puri®cation by ¯ash chromatography
on silica gel 6hexanes/EtOAc 1:7) gave 100 mg of desired
product 29 in 81% yield; [a]Dˆ1226.7 6c 2.3, CHCl3). IR
1
CH3OH/H2O 615:2:0.2) yielded 38 mg 668%) of 31; H
NMR 6400 MHz, CDCl3): d 1.75±2.2 6m, 12H, 3£ 2H-
C63), 2H-C67)); 3.2±3.85 6m, ,51H); 3.85±3.95 6m, 3H);
3.95±4.1 6m, 12H); 4.25±4.35 6m, 3H, 3£H-C650); 4.35±
4.55 6m, 12H, 3£H-C610), 9£ OCHHC6H5); 4.6±4.8 6m, 9H,
9£OCHHC6H5); 4.8±5.0 6m, 9H, 3£H-C6100), 6£
OCHHC6H5); 5.1±5.4 6b, 3H, 3£OH); 7.15±7.4 6m, 60H,
12£C6H5); 7.4±7.55 6b, 3H, 3£NH). Partial assignments
1
1
relate to two-dimensional H±1H- and H±13C-correlation
spectroscopy 6COSY and HSQC). HRMS: m/z calcd for
C154H192N6O49 6M1Na). 2911.2581; found 2934.1
1
6CHCl3): 2110 cm21 6N3). H NMR 6400 MHz, CDCl3): d
8.05±7.956m, 12H), 7.59±7.10 6m, 78H), 5.646d, Jˆ
2.9 Hz, 3H), 5.59 6dd, Jˆ10.01, 8.14 Hz, 3H), 5.20 6d,
Jˆ3.27 Hz, 3H), 4.75 6d, Jˆ11.50 Hz, 3H), 4.71 6d, Jˆ
9.1 Hz, 3H), 4.68 6m, 9H), 4.55 6m, 3H), 4.526d, Jˆ
11.60 Hz, 3H), 4.40 6d, Jˆ11.39 Hz, 3H), 4.34±4.25 6m,
9H), 4.19±4.09 6m, 12H), 3.99±3.93 6m, 9H), 3.85 6t,
Jˆ6.1 Hz, 3H), 3.58 6dd, Jˆ10.4, 6.3 Hz, 3H), 3.51±3.35
6m, 24H), 3.29±3.16 6m, 12H), 2.26 6dd, Jˆ15.2, 3.68 Hz,
3H), 2.04±1.96 6m, 6H), 1.90 6s, 9H, 3OAc), 1.85 6t,
Jˆ11.33 Hz, 3H), 1.44 6s, 9H, 3Me), 1.25 6s, 9H, 3Me).
13C NMR 6100 MHz, CDCl3): d 173.5, 170.4, 165.9,
164.4, 138.6, 138.5, 138.3, 138.2, 133.1, 132.9, 129.7,
129.6, 129.5, 129.3, 128.3, 128.3, 128.2, 128.0, 127.8,
127.5, 127.4, 127.3, 127.2, 109.0, 98.9 6C-10, b), 93.5
6C-100, a), 78.7, 77.6, 77.3, 76.9, 76.6, 75.5, 75.3, 74.7,
74.3, 73.2, 73.1, 71.7, 71.1, 70.5, 69.7, 69.3, 64.8, 62.1,
50.5, 45.2, 38.7, 36.0, 33.9, 28.1, 25.7, 20.5. LRMS
6MALDI): calcd for C211H234O58N6 6M1Na) 2779; found
2802.
2.1.20. Trimeric cluster, 3. To a solution of 31 629 mg,
9.81 mmol) in water/dioxane 62:3; 4 mL) was added
Pearlman's catalyst30 620% Pd6OH)2 on C, 10 mg) under
argon and the mixture was stirred for 18 h under hydrogen
at room temperature. After addition of more catalyst
610 mg) stirring under hydrogen was continued for 18 h.
Filtration and evaporation of volatiles gave 3 612 mg,
1
67%). H NMR 6400 MHz, D2O): 1.8±2.2 6m, 12H, 3£
2H-C63), 2H-C67)); 3.2±3.45 6m, 8H, 4£CH2-NHR);
3.45±3.8 6m, t34H); 3.85±3.9 6m, 6H); 3.9±4.0 6m, 9H);
4.05±4.15 6m, 6H); 4.15±4.25 6m, 6H); 4.44 6d, Jˆ8 Hz,
3H, 3£H-C610)); 5.05 6d, Jˆ4 Hz, 3H, 3£H-C6100)). Partial
1
assignments relate to two-dimensional H±13C-correlation
spectroscopy 6HSQC).
Acknowledgements
2.1.19. Trimeric cluster, 31. A solution of 29 695.6 mg,
0.0255 mmol) in AcOH/H2O 64:1; 3 mL) was heated to
608C for 2 h. Volatiles were evaporated at reduced pressure,
followed by coevaporation with toluene and chloroform.
Chromatography of the residue 693 mg) on silica gel
615 g) afforded 70 mg 675%) of 30 upon elution with ethyl
We thank NSERCC for generous ®nancial assistance.
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