Sequential one-pot glycosidations catalytically promoted: Unprecedented strategy in oligosaccharide synthesis for the straightforward assemblage of the antitumor PI-88 pentasaccharide

Article abstract of DOI:10.1021/jo8003953

(Chemical Equation Presented) The pentasaccharide sequence of the most active components of the antitumor drug PI-88, currently in phase II clinical trial, has been rapidly assembled in high overall yield and in only three steps starting from three monosa

Full text of DOI:10.1021/jo8003953

Sequential One-Pot Glycosidations Catalytically Promoted:
Unprecedented Strategy in Oligosaccharide Synthesis for the
Straightforward Assemblage of the Antitumor PI-88
Pentasaccharide
Silvia Valerio, Antonello Pastore, Matteo Adinolfi, and Alfonso Iadonisi*
Dipartimento di Chimica Organica e Biochimica, UniVersita` degli Studi di Napoli “Federico II”,
Via Cynthia 4, Napoli I-80126, Italy
iadonisi@unina.it
ReceiVed February 18, 2008
The pentasaccharide sequence of the most active components of the antitumor drug PI-88, currently in
phase II clinical trial, has been rapidly assembled in high overall yield and in only three steps starting
from three monosaccharide building blocks. The procedure takes advantage of the first reported strategy
of sequential one-pot glycosidations conducted exclusively under catalytic activation. In addition, the
procedure relies only on shelf-stable and mild promoters such as Yb(OTf)3 and Bi(OTf)3.
Introduction
of oligosaccharides has reached an important stage of maturity
with the advent of one-pot strategies that enable the construction
of oligosaccharide fragments by means of sequential multiple
glycosidations.3 In this regard, several approaches have been
usefully exploited. An option is represented by the use of the
orthogonal strategy, which exploits for each coupling the
selective activation of one leaving group over another, the two
groups being chemically different.4 Another opportunity is
offered by a chemoselective approach, which takes advantage
of the feasible reactivity tuning of donors bearing similar
anomeric groups by exploiting the arming-disarming5 effect
Over the past years the therapeutic impact of oligosaccharides
has been widely recognized by the scientific community as
demonstrated by the increasing number of biomedical applica-
tions.1 Consequently, the ever-growing need of pure and
homogeneous oligosaccharide probes has elicited formidable
progress in the field of oligosaccharide synthesis so that even
some examples of automated syntheses via a solid-phase
approach have been reported.2 Concurrently, solution synthesis
(1) (a) Wong, C.-H. Carbohydrate-Based Drug DiscoVery; Wiley-VCH:
Weinheim, 2003. (b) Sinay, P.; Ernst; B.; Hart, G. W. Carbohydrates in Chemistry
and Biology; Wiley-VCH: Weinheim, 2000. (c) Seeberger, P. H.; Werz, D. B.
Nature 2007, 446, 1046–1051.
(3) For recent reviews, see: (a) Wang, Y.; Ye, X.-S.; Zhang, L.-H. Org.
Biomol. Chem 2007, 5, 2189–2200. (b) Codee, J. D. C.; Litjens, R. E. J. N.;
Van den Bos, L. J.; Overkleeft, H. S.; Van der Marel, G. A. Chem. Soc. ReV.
2005, 34, 769–782.
(2) Plante, O. J.; Seeberger, P. H.; Palmacci, E. R. Science 2001, 291, 1523–
1527.
4496 J. Org. Chem. 2008, 73, 4496–4503
10.1021/jo8003953 CCC: $40.75 2008 American Chemical Society
Published on Web 05/15/2008

Assemblage of the Antitumor PI-88 Pentasaccharide
of the functional groups present on the saccharide scaffold. This
latter concept has been broadly elaborated by Wong and co-
workers, who developed a computer-assisted protocol for
choosing the best set of glycosyl donors to assemble a given
target sequence.6,7 Very recently, another ingenious one-pot
approach has been proposed. It is based on a preactivation stage
for every coupling in which a thioglycoside donor is converted
to a highly reactive glycosylating species at low temperature
upon exposure to a suitable stochiometric promoter. Subsequent
addition of a thioglycoside acceptor yields a glycosidation
product that can be preactivated in situ for a further elongation
with a further acceptor.8 This latter strategy is advantageous
because it is independent of the relative reactivity of the
employed donors. A common drawback of all one-pot sequential
glycosidation procedures so far described is the required
activation of glycosyl donors with stoichiometric or excess
amounts of costly and/or sensitive reagents such as NIS, triflic
anhydride, silver triflate, or sterically encumbured pyridines.3
Recently, we have communicated the first examples of
catalytic procedures for the sequential construction of two
glycosidic bonds in a one-pot approach.9 Such protocols were
inspired by the recognition of the well-differentiated activation
conditions of similarly protected glycosyl-trichloroacetimidates10
and (N-phenyl)trifluoroacetimidates11 under the agency of
catalytic ytterbium(III) triflate.12,13 More recently, we have also
disclosed that bismuth(III) triflate is a novel activator of glycosyl
trihaloacetimidates endowed of exceptional reactivity so that
FIGURE 1. Components of PI-88.
reactions can be performed under relevantly milder conditions
in terms of temperature and catalyst loading than when using
Yb(OTf)3.14 The capability of Bi(OTf)3 of promoting high-
yielding glycosidations in short times appeared a good fit with
our attempts to develop ever more efficient catalytic multigly-
cosidation processes to achieve biologically useful oligosac-
charides.
In this paper, we wish to report on the sequential one-pot
glycosidations performed exclusively under catalytic activation
that led us to a very straightforward and flexible approach for
preparing the pentasaccharide component of the antitumor agent
PI-88 (Figure 1). This drug is currently in phase II clinical trial15
and is constituted by a mixture of randomly sulfated tri-, tetra-,
and penta-mannans bearing a phosphate functionality at the
primary site of the nonreducing terminal residue. This phosphate
can be replaced by a sulfate group without appreciable loss of
biological activity.16,17 The drug is obtained by the yeast Pichia
(Hansenula) holstii NRRL Y-2448 as a mixture of phosphory-
lated mannans that are subsequently randomly sulfated via a
chemical approach.18
The antitumor properties of PI-88 are believed to be connected
with its ability to act as inhibitor of both heparanases and
angiogenic growth factors.19 The pentasaccharide fraction is the
most abundant and is considered the most active component of
the drug. To date, only two examples of chemical syntheses of
this pentasaccharide have been reported, including a very recent
paper by Ferro and co-workers.17,20 Both of these schemes are
providing good overall yields, but a large number of synthetic
steps renders them rather laborious. In this paper we wish to
report the feasible assemblage of the penta-mannose sequence
1 (Scheme 1) by an alternative strategy based on only three
steps (two one-pot multiglycosidations and a single intermedi-
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Chem. Soc. 1994, 116, 7919–7920. (b) Grice, P.; Ley, S. V.; Pietuszka, J.;
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C.-H. J. Am. Chem. Soc. 1999, 121, 734–753. For appliations to biologically
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K. T.; Wong, C.-H. Angew. Chem., Int. Ed. 2002, 41, 4087–4090. (c) Lee, J.-
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Ravida`, A.; Schiattarella, M. Synlett 2004, 275–278. (b) Jayaprakash, K. N.;
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Angew. Chem., Int. Ed. 1994, 33, 2292–2294. (b) Douglas, N. L.; Ley, S. V.;
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see: Valerio, S.; Iadonisi, A.; Adinolfi, M.; Ravida`, A. J. Org. Chem. 2007, 72,
6097–6106.
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Valerio et al.
SCHEME 1. Retrosynthetic Scheme of the PI-88 Pentasaccharide
SCHEME 2
SCHEME 3
ate deprotection step) and entailing only three monosaccharide
building blocks for the whole task.
ing 4-O-methoxy phenyl glycoside 8 under the agency of
BF3 ·OEt2 and then 2-O-deacetylated to yield the desired
building block 3.
Results and Discussion
The proposed strategy (Scheme 1) relies on the feasible in situ
generation of disaccharide 2 as the precursor of both the BC and
DE fragments, whereas readily obtainable acceptor 3 represents
the precursor of the reducing terminus A. Disaccharide 2 was
expected to be accessible by the key chemoselective coupling of
trichoroacetimidate donor 4 and the partially protected trifluoro-
acetimidate 5 acting as an acceptor at this stage.
Acceptor 3 was efficiently obtained starting from penta-O-
acetyl-D-mannose (Scheme 2), which was initially converted
into orthoester 6 through a rapid sequence of three reactions
and without any chromatographical purification of intermediates
(50-55% overall yield).21 Compound 6 was submitted to
sequential acid-mediated cyclic orthoester opening and acety-
lation to yield almost quantitatively the diacetylated intermediate
7. This latter was in turn directly converted into the correspond-
Compounds 4 and 5 were prepared through synthetic path-
ways sharing a large number of steps (Scheme 3). Methyl
mannoside 9 was regioselectively 3-O-allylated via a stan-
nylidene intermediate,22 and the resulting triol 10 was then
perbenzylated under standard conditions. Derivative 11 was
hydrolyzed under acidic conditions to yield hemiacetal 12, which
was the last common intermediate of the routes leading to 4
and 5. The former was prepared in high yield (ca. 90%) with
only an additional standard 1-O-trichloroacetimidation step.
Alternatively, 12 was deallylated with PdCl2 in methanol in 72%
yield, and the resulting diol 13 was then selectively converted
into trifluoroacetimidate 5 (R:ꢀ ca. 4:1) adopting Cs2CO313d as
the base.
As shown in the retrosynthetic analysis (Scheme 1), the
successful assemblage of the target pentasaccharide is critically
(21) Adinolfi, M.; Iadonisi, A.; Schiattarella, M.; Ravida`, A. Tetrahedron
Lett. 2003, 44, 7863–7866.
(22) Yang, G. B.; Kong, F.-Z.; Zhou, S. H. Carbohydr. Res. 1991, 211, 179–
182.
4498 J. Org. Chem. Vol. 73, No. 12, 2008

Assemblage of the Antitumor PI-88 Pentasaccharide
SCHEME 4. Chemoselective Coupling of 14 and 5
SCHEME 5
Bi(OTf)3, although a remarkably higher temperature (-10 versus
-60 °C) was required for reactions to proceed within compa-
rable times (Table 1, entry 3).
The donor ability of disaccharide 15r under Bi(OTf)3
activation was then examined in the coupling with acceptor 3
to quickly provide in excellent yield the trisaccharide 17
(Scheme 5), which corresponds to the ABC fragment of the
target pentasaccharide and is itself biologically interesting, being
related to an antiallergenic sequence.23
TABLE 1a
Having established the optimized conditions for generating
and activating the requisite mannose disaccharide trifluoroace-
timidate, their application was targeted toward the assemblage
of the desired pentasaccharide via sequences of one-pot double
glycosidations.
isolated yield
entry promoter (equiv)
T (°C)
time (min)
of 15rb
1
2
3
Bi(OTf)3 (0.10)
Bi(OTf)3 (0.05)
Yb(OTf)3 (0.03)
-60 to -55
-70 to -60
-10
30
60
30
42
47
58
a General conditions: 14 (1.4-1.7 equiv), 5 (1 equiv), PhCH3/Et2O
4/1, Yb(OTf)3 or Bi(OTf)3 (16-20 mg/mL in dioxane), 4Å AW MS.
b Calculated with respect to the overall amount (R and ꢀ anomers) of 5.
The extensible precursor 19 of the ABC fragment was
prepared by initial coupling of 5 and the 3-O-allylated trichlo-
roacetimidate 4, the subsequent addition of acceptor 3 to the
mixture, and the adjustment of the experimental conditions for
activating the trifluroacetimidate leaving group (Scheme 6). The
one-pot synthesis was examined by using either Bi(OTf)3 or
Yb(OTf)3 for the initial coupling. In the former case (Scheme
6, Method A), the first glycosidation step occurred at a very
low temperature (from -70 to -60 °C), and the second coupling
was accomplished without any further amount of promoter by
the simple addition of acceptor 3 and the spontaneous warming
of the mixture. Alternatively (Scheme 6, Method B), Yb(OTf)3
promoted the first coupling between 4 and 5 at -10 °C for 30
min, and then, after cooling to -60 °C, acceptor 3 and Bi(OTf)3
(0.03 equiv) were sequentially added, and the mixture was
allowed to warm spontaneously. As expected from the results
of preliminary experiments shown in Table 1, use of a different
promoter for each glycosidation (Route B) provided an improved
yield, the results being in any way quite satisfying by the means
of both methods (yields in the range of 43-60%). It is worthy
of note that in both cases a very low amount of promoter(s)
was needed for accomplishing the sequential double glycosi-
dation and the whole process took less than 3 h, with a sensible
acceleration in comparison with the previously reported catalytic
procedure based on the sole Yb(OTf)3.8 Trisaccharide 19 was
smoothly deallylated to yield 20 (Scheme 6), the requisite
acceptor for the final one-pot sequence leading to pentasaccha-
dependent on the coupling between the fully protected trichlo-
roacetimidate 4 and the partially protected trifluoroacetimidate
5. Indeed, this reaction should provide in high yield and
stereocontrol the desired glycosidic bond while leaving intact
the activatable trifluoroacetimidate functionality to allow the
subsequent glycosidation. To optimize this delicate step, ac-
ceptor 5 and the model trichloroacetimidate 14, easier to obtain
than 4,10 were coupled under a variety conditions aimed at
achieving exclusive activation of the trichloroacetimidate com-
ponent (Scheme 4). The reactions were quenched at low
temperature with pyridine to minimize any deterioration of the
trifluoroacetimidate functionality. After a preliminary screening,
a toluene/diethyl ether mixture was found as the best serving
reaction solvent. Reactions conducted under Bi(OTf)3 activation
at very low temperature produced the desired disaccharide
trifluoroacetimidate 15r in a satisfying isolated yield (Table 1,
entries 1 and 2) together with minor amounts of the anomer
15ꢀ (equally useful for our purposes in the planned one-pot
application). This latter could not be isolated pure because it
coeluted with the byproduct 16 derived from rearrangement of
the trichloroacetimidate 14. To the best of our knowledge, these
are the first examples of isolated glycosylation products in which
an anomeric (N-phenyl)trifluoroacetimidate group is designed
to act as a temporary protecting group. Interestingly, the milder
promoter Yb(OTf)3 furnished 15r in a higher yield than
(23) Carpenter, C.; Nepogodiev, S. A. Eur. J. Org. Chem. 2005, 3286–3296.
J. Org. Chem. Vol. 73, No. 12, 2008 4499

Valerio et al.
SCHEME 6a
SCHEME 7
a Method A: 4 (1.3-1.5 equiv), 5 (1 equiv), PhCH3/Et2O 4:1, Bi(OTf)3
(0.05 equiv) in dioxane, -70 to -60 °C, 45 min; then 3 (1-1.3 equiv),
-60 to 0 °C, 90 min, 43-46%. Method B: 4 (1.2 equiv), 5 (1 equiv), PhCH3/
Et2O 4:1, Yb(OTf)3 (0.03 equiv) in dioxane, -10 °C, 30 min; then 3 (0.8
equiv), Bi(OTf)3 (0.025 equiv) in dioxane, -60 to 10 °C, 90 min, 60%.
temporary anomeric protecting group in sequential multigly-
cosidation steps. Feasible access to a biological important target
establishes a conceptually novel strategy where the catalytic
activation of all sequential glycosidation steps makes it possible
to overcome important drawbacks of the current strategies such
as the use of costly and/or sensitive stoichiometric promoters
and the production of higher amounts of side products. Elabora-
tion of the developed protocols toward other biologically useful
oligosaccharide sequences are being evaluated in our laboratory
and will be reported in due course.
ride 21. Following the procedure that best served for 19 (Method
B, Scheme 6), 18 was transiently formed from 4 and 5 under
the agency of Yb(OTf)3 and then coupled with 20 in the
presence of Bi(OTf)3 to give 21 in a gratifying yield of 56%
(Scheme 7).
Despite the structural changes in the final acceptor (20 rather
than 3), the yield of this sequence was somewhat similar to
that leading to trisaccharide 19. Pentasaccharide 21 was deal-
lylated and debenzylated under standard conditions to yield the
free pentasaccharide 1, which can be submitted to the procedure
of random sulfation. On the other hand, the presence in 21 of
differentiated protecting groups (allyl, benzyls, and an anomeric
4-methoxy-phenyl group) renders the obtained pentasaccharide
amenable to further structural elaborations.
Experimental Section
1,2-Di-O-acetyl-3,4,6-tri-O-benzyl-r,ꢀ-D-mannopyranoside (7).
Orthoester 6 (3.895 g, 7.9 mmol) was dissolved at room temperature
in 80% aqueous AcOH (20 mL). After 30 min, the mixture was
diluted with DCM and the organic phase washed with water, and
then with aqueous Na2CO3. The aqueous phases were then
re-extracted with DCM and the collected organic phases dried and
concentrated under vacuum. The residue was dissolved in pyridine
(5 mL) and acetic anhydride (2.5 mL). After 2.5 h the mixture was
treated with MeOH and diluted with DCM. The organic phase was
washed with water, dried and concentrated to yield syrupy 7 in a
satisfying purity (3.960 g, R/ꢀ ca. 4, yield 96%) to be directly
Conclusions
In conclusion, in this paper we have shown the notable
applicative potential of one-pot glycosidation of oligosaccharides
conducted under exclusively catalytic conditions of activation,
the pentasaccharide component of the drug PI-88 being rapidly
accessed through a reduced number of synthetic steps and
resorting to only three easily obtained D-mannose building
blocks. Notably, inclusion of Bi(OTf)3 in the activation system
allows the one-pot glycosidation sequences to be accomplished
in short times and high yields. Additionally, very low amounts
of shelf-stable promoter(s) (about 0.10 equiv) are sufficient for
the assemblage of the overall sequence, which entails the
construction of four glycosidic bonds. It is also worthy of note
that some model coupling experiments have shown that the
trifluoroacetimidate anomeric group can play the role of a
1
submitted to the following step. H NMR (CDCl3, 300 MHz): δ
7.50-7.00 (aromatic protons), 6.15 (1H, d, J1,2 ) 2.1 Hz, H-1),
5.39 (1H, bd, H-2), 4.90-4.50 (6H, 3 × AB, 3 × benzyl CH2),
4.00 (1H, bd, J3,4 ) 9.3 Hz, H-3), 3.95-3.75 (3H, H-5, H-6a, H-6b),
3.72 (1H, t, J4,5 ) 9.3 Hz, H-4), 2.17, 2.08 (6H, 2 × s, 2 ×
sCOCH3). 13C NMR (CDCl3, 50 MHz): δ 170.8, 170.0, 138.1
(×2), 137.6, 128.9-127.5, 91.3, 75.3, 73.7, 73.6, 73.5, 71.9, 68.5,
67.5, 20.9. Anal. Calcd for C31H34O8: C, 69.65, H, 6.41. Found: C,
69.93; H, 6.52.
4500 J. Org. Chem. Vol. 73, No. 12, 2008

Assemblage of the Antitumor PI-88 Pentasaccharide
p-Methoxyphenyl 3,4,6-Tri-O-benzyl-r-D-mannopyranoside
(3). To a solution of compound 7 (1.234 g, 2.31 mmol) and
p-methoxyphenol (436 mg, 3.51 mmol) in dry DCM (7 mL) were
added freshly activated 4Å molecular sieves and BF3 ·OEt2 (75 µL,
0.59 mmol) at 0 °C under argon. After removing the ice bath, the
mixture was stirred at room temperature for 3 h and then pyridine
(6 drops) was added to quench the reaction. The mixture was
concentrated in vacuo and the residue submitted to silica gel flash
chromatography to yield 8 slightly contaminated with unreacted
p-methoxyphenol. To this impure material dissolved in methanol
(4 mL) was added dropwise a solution of MeONa (2 M in MeOH)
until completion of the reaction (TLC). The mixture was then
diluted with DCM and washed with 0.1 M aqueous NaOH and
water. Aqueous phases were re-extracted with DCM and the
collected organic phases were dried and concentrated. The residue
was applied to a short silica gel column eluted with petroleum ether/
with DCM, and the organic phase was washed with water. The
aqueous phase was re-extracted with DCM. Collected organic
phases were washed with aqueous sodium carbonate and concen-
trated. The residue was purified by silica gel flash chromatography
(eluent petroleum ether/ethyl acetate from 3:1 to 7:3) to yield 12
1
as a yellow oil (434 mg, R/ꢀ ca. 7:1, 71%). H NMR (300 MHz,
CDCl3): δ 6.05-5.85 (1H, m, -CHdCH2), 5.34 (1H, bd, J ) 17.4
Hz, CH2CHdCHcisHtrans), 5.25 (1H, bs, H-1), 5.19 (1H, bd, J )
10.2 Hz, CHdCHcisHtrans), 4.91-4.48 (6H, 3 × AB, benzyl CH2),
4.15-4.00 (3H), 3.95-3.60 (5H), 3.24 (bs, 1-OH). 13C NMR (50
MHz, CDCl3): δ 138.3 (×2), 137.8, 134.8, 128.2-127.5, 116.4,
92.4, 79.3, 75.0, 74.9, 73.0, 72.4, 71.2, 70.9, 70.8, 69.5. MALDI-
MS: [M + Na]+ calcd 513.23, found 513.18. Anal. Calcd for
C30H34O6: C, 73.45, H, 6.99. Found: C, 73.28; H, 7.04.
Trichloroacetimidoyl 3-O-Allyl-2,4,6-tri-O-benzyl-r-D-man-
nopyranoside (4). To a solution of 12 (488 mg, 0.99 mmol) in
DCM (5 mL) were sequentially added at 0 °C trichloroacetonitrile
(400 µL, 4 mmol) and sodium hydride (60% in oil, 10 mg, 0.25
mmol). The mixture was allowed to warm to room temperature
and concentrated after 40 min. The residue was chromatographed
on neutral alumina (Brockman grade 2, eluent petroleum ether/
ethyl acetate 9:1 with two drops of pyridine for every 100 mL of
eluent) to yield 4 as an oil (560 mg, yield 89%). 1H NMR (CDCl3,
300 MHz): δ 8.69 (1H, s, -NH), 7.53-7.20 (aromatic protons),
6.52 (1H, s, H-1), 6.04-5.91 (1H, m, -OCH2-CHdCH2), 5.37
(1H, dd, J ) 1.2 and 17.4 Hz, CHdCHcisHtrans), 5.24 (1H, dd, J )
10.5 Hz, -OCH2-CHdC HcisHtrans), 4.99-4.57 (6H, 3 × AB, 3
× benzyl CH2), 4.23 (1H, t, J ) 9.6 Hz, H-4), 4.19-4.07 (3H, m,
acetone 6:4 to yield 3 as a yellow oil (556 mg, 43% over two steps).
1
[R]25 +86.4° (c 1.0, CHCl3). H NMR (CDCl3, 300 MHz): δ
D
7.44-6.70 (aromatic protons), 5.50 (1H, d, J1,2 ) 1.8 Hz, H-1),
4.84-4.40 (6H, 3 × AB, 3 × benzyl CH2), 4.19 (1H, m, H-2),
4.06 (1H, dd, J2,3 ) 3.3 Hz, H-3), 3.94 (1H, t, J3,4 ) J4,5 ) 9.9 Hz,
H-4), 3.91-3.84 (1H, m, H-5), 3.74 (1H, dd, J5,6a ) 4.2 Hz, J6a,6b
) 10.8 Hz, H-6a), 3.73 (3H, s, -OCH3), 3.63 (1H, dd, J5,6b ) 1.5
Hz, H-6b), 2.55 (1H, d, J2,OH ) 2.4 Hz, 2-OH). 13C NMR (CDCl3,
50 MHz): δ 154.9, 150.0, 138.3, 138.2, 137.8, 128.4-127.0, 117.7,
114.5, 98.2, 79.9, 75.0, 74.1, 73.2, 72.0, 71.5, 68.8, 68.2, 55.4.
MALDI-MS: [M + Na]+ calcd 579.24, found 579.32. Anal. Calcd
for C34H36O7: C, 73.36, H, 6.52. Found: C, 73.25; H, 6.55.
Methyl 3-O-Allyl-r-D-mannopyranoside (10). Methyl R-man-
nopyranoside (3.118 g, 16.1 mmol) and Bu2SnO (4.402 g, 17.7
mmol) were refluxed in MeOH (80 mL) for 2 h. The obtained
solution was evaporated under vacuum. To the residue suspended
in toluene (100 mL) were sequentially added allyl bromide (14.0
mL, 165 mmol) and TBAI (5.94 g, 16.1 mmol). The mixture was
kept under stirring at 65-70 °C for 20 h and concentrated. Silica
gel flash chromatography of the residue (eluent ethyl acetate/
petroleum ether from 7:3 to 8:2) yielded 10 slightly contaminated
with tetrabutylammonium salts (estimated yield 65-70% on the
basis of NMR integrations). The product thus obtained was directly
H-5 and -OC H2CHdCH2), 4.06 (1H, dd, J1,2 ) 1.8 Hz, J2,3
)
2.7 Hz, H-2), 3.97 (1H, dd, H-3), 3.91 (1H, dd, J5,6a ) 4.5 Hz,
13
J6a,6b ) 11.4 Hz, H-6a), 3.81 (1H, bd, H-6b). C NMR (CDCl3,
75 MHz): δ 160.0, 138.0 (×2), 137.6, 134.4, 128.1-127.1, 116.8,
95.9, 90.7, 78.7, 74.9, 74.5, 73.7, 73.0 (×2), 72.3, 70.8, 68.4. Anal.
Calcd for C32H34Cl3NO6: C, 60.53, H, 5.40. Found: C, 60.41; H,
5.46.
2,4,6-Tri-O-benzyl-D-mannopyranose (13). To a solution of 12
(307 mg, 0.63 mmol) in methanol (2 mL) was added at room
temperature palladium chloride (15 mg, 0.085 mmol). The mixture
was stirred overnight and then concentrated under vacuum. The
residue was then filtered through a short plug of silica gel (eluent
DCM/MeOH 95:5), concentrated, and purified by silica gel flash
chromatography (eluent petroleum ether/ ethyl acetate from 7:3 to
6:4) to provide diol 13 as an oil (R:ꢀ ca. 9:1, 205 mg, yield 72%).
1H NMR (CDCl3, 300 MHz): δ 7.5-7.10 (aromatic protons), 5.31
(1H, s, H-1), 4.89-4.51 (6H, 3 × AB, 3 × benzyl CH2), 4.10-4.00
(2H, m, H-3, H-5), 3.84-3.64 (3H, m, H-2, H-6a, H-6b), 3.60 (1H,
1
submitted to the subsequent step. H NMR (200 MHz, CDCl3): δ
6.05-5.85 (1H, m, -CHdCH2), 5.29 (1H, dq, J ) 1.4 and 18.8
Hz, -CHdCHcisHtrans), 5.17 (1H, bd,
J
)
10.4 Hz,
-CHdCHcisHtrans), 4.73 (1H, bs, H-1), 4.25-3.70 (6H), 3.65-3.40
(2H), 3.31 (3H, s, -OCH3).
Methyl 3-O-Allyl-2,4,6-tri-O-benzyl-r-D-mannopyranoside
(11). To a solution of compound 10 (estimated mass ca. 2.5 g, ca.
11 mmol) in dry DMF (13 mL) were sequentially added at 0 °C
benzyl bromide (6.4 mL, 54 mmol) and sodium hydride (60% in
oil, 1.75 g, 73 mmol). The mixture was allowed to warm to room
temperature, and after 3 h MeOH (ca. 1 mL) was added. The
mixture was diluted with DCM and the organic phase washed with
water. The aqueous phase was re-extracted with DCM and the
collected organic phases were dried with anhydrous Na2SO4 and
concentrated in vacuo to give a residue which was purified by silica
gel flash chromatography (eluent petroleum ether/ethyl acetate 85:
13
t, J3,4 ) J4,5 ) 9.6 Hz, H-4), 2.92 (1-OH). C NMR (CDCl3, 75
MHz) 138.5, 137.9 (×2), 128.4-127.6, 91.5, 79.0, 78.6, 74.8, 73.4,
72.9, 71.5, 70.6, 69.7. Anal. Calcd for C27H30O6: C, 71.98, H, 6.71.
Found: C, 71.85; H, 6.77.
(N-Phenyl)trifluoroacetimidoyl 2,4,6-Tri-O-benzyl-r,ꢀ-D-
mannopyranoside (5). To a solution of diol 13 (399 mg, 0.89
mmol) in acetone (3 mL) were sequentially added at 0 °C Cs2CO3
(324 mg, 0.99 mmol) and (N-phenyl)trifluoroacetimidoyl chloride
(220 µL, 1.8 mmol). The mixture was allowed to warm to room
temperature and after 2 h concentrated under vacuum. The residue
was purified by chromatography on neutral alumina (Brockman
grade 2, eluent petroleum ether/ethyl acetate 9:1 with two drops of
pyridine for every 100 mL of eluent) to yield 5 as a yellow oil
(anomeric mixture R/ꢀ ca. 4:1, 492 mg, yield 89%). Data for 5r.
1H NMR (CDCl3, 300 MHz): δ 7.50-6.81 (aromatic protons), 6.42
(1H, bs, H-1), 4.92-4.56 (6H, 3 × AB, 3 × benzyl CH2), 4.08
(1H, m, H-3), 4.03-3.77 (5H), 2.75 (1H, d, J3,OH ) 8.4 Hz, 3-OH).
13C NMR (CDCl3, 75 MHz): δ 143.3, 138.0 (×2), 137.0,
129.0-127.7, 127.4, 124.3, 119.3, 94.5, 75.9, 75.5, 74.9, 73.8, 73.3,
72.7, 71.2, 68.5. Anal. Calcd for C35H34F3NO6: C, 67.62, H, 5.51.
Found: C, 67.50; H, 5.59.
15) to yield 11 as an oil (5.465 g, 53% over two steps). [R]25
D
+29.4° (c 1.0, CHCl3). 1H NMR (200 MHz, CDCl3): δ 6.05-5.85
(1H, m, -CH2CHdCH ), 5.34 (1H, dq, J ) 1.6 and 17.4 Hz,
2
-CHdCHcisHtrans), 5.17 (1H, dq, J ) 10.2 Hz, -CHdCHcisHtrans),
4.78 (1H, bs, H-1), 4.93-4.50 (6H, 3 × AB, benzyl CH2),
4.15-4.05 (2H, m, -OCH2CHdCH2), 4.00-3.85 (2H), 3.85-3.70
(4H), 3.34 (3H, s, -OCH 3). 13C NMR (50 MHz, CDCl3): δ 138.5,
138.4 (×2), 134.9, 128.2-127.5, 116.5, 99.0, 79.8, 75.0, 74.8, 74.5,
73.3, 72.5, 71.6, 71.0, 69.3, 54.7. MALDI-MS: [M + Na]+ calcd
527.24, found 527.30. Anal. Calcd for C31H36O6: C, 73.79, H, 7.19.
Found: C, 73.65; H, 7.23.
3-O-Allyl-2,4,6-tri-O-benzyl-D-mannopyranose (12). A solution
of 11 (446 mg, 0.88 mmol) in 7:1 AcOH/1 M H2SO4 (5.4 mL)
was heated at 110 °C for 70 min. The mixture was then diluted
(N-Phenyl)trifluoroacetimidoyl 2,3,4,6-Tetra-O-benzyl-r-D-
mannopyranosyl-(1f3)-2,4,6-tri-O-benzyl-r-D-mannopyrano-
J. Org. Chem. Vol. 73, No. 12, 2008 4501

Valerio et al.
side (15r). Trichloroacetimidate 14 (26 mg, 0.038 mmol) and
trifluoroacetimidate 5 (17 mg, 0.027 mmol) were coevaporated three
times with toluene (3 × 0.5 mL) and dried under vacuum. After
4Å AW 300 MS were added, the mixture was dissolved with 4:1
toluene/Et2O (1 mL) and cooled to -10 °C. After ca. 15 min of
stirring, a solution of Yb(OTf)3 in dioxane (20 mg/mL, 25 µL, 8.1
µmol) was added and after 30 min the reaction was quenched at
the same temperature with pyridine. The mixture was filtered on a
short plug of neutral alumina and concentrated. The residue was
chromatographed on neutral alumina (Brockman grade 2, eluent
petroleum ether/ethyl acetate 85:15 with two drops of pyridine for
every 100 mL) to yield 15r as an oil (18 mg, yield 58%). 1H NMR
(CDCl3, 300 MHz): δ 7.60-6.75 (aromatic protons), 6.25 (1H, bs,
H-1), 5.25 (1H, s, H-1′), 4.90-4.46 (14H, 7 × AB, 7 × benzyl
CH2), 4.15 (1H, dd, H-3), 3.94 (1H, d, J2,3 ) 3.3 Hz, H-2), 3.77
δ 7.50-7.10 (aromatic benzyl protons), 7.10 and 6.93 (4H, 2 × d,
Jortho ) 9.2 Hz, aromatic p-methoxyphenyl protons), 5.80-5.95 (1H,
m, OCH2-CHdCH2), 5.58 (1H, d, J1,2 ) 1.6 Hz, H-1), 5.26 (1H,
dd, Jtrans ) 1.2 and 16.8 Hz, -OCH 2-CHdCHcisHtrans), 5.24 (2H,
bs, H-1′ and H-1′′), 5.26 (1H, dd, Jcis ) 10.4 Hz, -OCH
2-CHdCHcisHtrans), 4.90-4.30 (18H, 9 × AB, 9 × benzyl CH2),
4.27 (1H, t, H-2), 4.23 (1H, dd, J ) 3.2 and 8.4 Hz), 4.15-4.10
(2H), 3.74 (3H, s, -OCH3), 4.15-3.65 (16 H), 3.56 (1H, bd, J )
9.6 Hz). 13C NMR (CDCl3, 75 MHz) 154.9, 150.1, 138.6-138.2,
135.0, 128.3-127.0, 116.4, 118.0, 114.5, 99.5 (×2), 98.0 (C-1),
79.7, 79.6, 75.3, 75.2, 75.1, 74.8, 74.6, 73.2, 73.1, 71.0, 69.2, 68,8,
55.6. MALDI-MS [M + Na]+ calcld 1483.65, found 1483.85. Anal.
Calcd for C91H96O17: C, 74.77, H, 6.62. Found: C, 74.60; H, 6.69.
p-Methoxyphenyl 2,4,6-Tri-O-benzyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-benzyl-r-D-mannopyranosyl-(1f2)-3,4,6-tri-
O-benzyl-r-D-mannopyranoside (20). PdCl2 (2 mg, 0.011 mmol)
was added to a solution of trisaccharide 19 (110 mg, 0.075 mmol)
in MeOH/DCM 9:1 (6 mL). After 3 h under stirring the mixture
was concentrated under vacuum, resuspended with DCM/MeOH
95:5, filtered on a short plug of silica gel, concentrated, and purified
by silica gel flash chromatography (eluent toluene/acetone 96:4)
13
(1H, bd, H-2′), 4.20-3.60 (9H). C NMR (CDCl3, 75 MHz): δ
143.4, 138.7, 138.4, 138.2, 138.1, 138.0, 137.5, 129.3-119.4, 100.1,
94.7, 79.7, 75.8, 75.5, 74.9, 74.7, 74.5, 74.1, 73.39, 73.38, 72.7,
72.4, 72.2, 69.3, 68.6. MALDI-MS: [M + Na]+ calcd 1166.46,
found 1166.70. Anal. Calcd for C69H68F3NO11: C, 72.42, H, 5.99.
Found: C, 72.40; H, 6.07.
p-Methoxyphenyl 2,3,4,6-Tetra-O-benzyl-r-D-mannopyrano-
syl-(1f3)-2,4,6-tri-O-benzyl-r-D-mannopyranosyl-(1f2)-3,4,6-
tri-O-benzyl-r-D-mannopyranoside (17). Disaccharide donor 15r
(25 mg, 0.022 mmol) and acceptor 3 (9 mg, 0.017 mmol) were
coevaporated three times with toluene (3 × 0.5 mL) and dried under
vacuum. After 4Å AW 300 MS were added, the mixture was
dissolved under argon with 4:1 toluene/Et2O (0.7 mL), cooled to
-60 °C, and stirred for 15 min. A solution of Bi(OTf)3 in dioxane
(20 mg/mL, 17 µL, 0.5 µmol) was then added, and after 20 min of
stirring at -60 °C, the temperature was allowed to raise up to -20
°C over 60 min. Pyridine was added to quench the reaction and
the mixture was filtered on a short plug of silica gel, concentrated,
and purified by silica gel flash chromatography (eluent petroleum
ether/ethyl acetate from 8:2 to 7:3) to yield 17 as an oil (22 mg,
to yield 20 as an oil (98 mg, yield 92%). [R]25 +35.2° (c 1.0,
D
CHCl3). 1H NMR (CDCl3, 400 MHz): δ 7.50-7.10 (benzyl
aromatic protons), 6.99 and 6.73 (4H, 2 × d, Jortho ) 9.2 Hz,
p-methoxyphenyl protons), 5.60 (1H, d, J ) 1.6 Hz, H-1), 5.27
(1H, bs, anomeric proton), 5.23 (1H, d, J ) 1.6 Hz, anomeric
proton), 4.85-4.30 (18 H, 9 × AB, 9 × benzyl CH2), 4.28 (1H, t,
H-2), 4.25 (1H, dd, J ) 3.2 Hz, 9.6 Hz), 4.20-4.10 (2H), 3.74
(3H, s, -OCH3), 4.05-3.60 (14 H, m), 3.52 (1H, bd, J ) 11.6
Hz). 13C NMR (CDCl3, 100 MHz): δ 154.9, 150.2, 138.7, 138.3,
138.2 (x6), 137.7, 128.5-126.9, 117.9, 114.5, 99.6, 98.5, 98.0, 79.6,
78.7, 77.5, 75.1 (×2), 74.9, 74.7, 74.5, 74.2, 73.3, 73.2, 73.1, 72.4,
72.3, 72.1, 72.0, 71.7, 71.6, 69.2, 68.7, 55.6 (-OCH3). MALDI-
MS [M + Na]+ calcld 1443.62, found 1443.78. Anal. Calcd for
C88H92O17: C, 74.35, H, 6.52. Found: C, 74.18; H, 6.61.
1
84% yield). H NMR (CDCl3, 400 MHz): δ 7.40-7.00 (benzyl
p-Methoxyphenyl 3-O-Allyl-2,4,6-tri-O-benzyl-r-D-mannopy-
ranosyl-(1f3)-2,4,6-tri-O-benzyl-r-D-mannopyranosyl-(1f3)-
2,4,6-tri-O-benzyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-benzyl-
r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-benzyl-r-D-mannopyrano-
side (21). Trichloroacetimidate 4 (44 mg, 0.069 mmol) and
trifluoroacetimidate 5 (29 mg, 0.047 mmmol) were coevaporated
three times with anhydrous toluene (3 × 1 mL) and dried under
vacuum for 45 min. The residue was dissolved under argon with
4:1 toluene/Et2O (1.5 mL) in the presence of freshly activated 4Å
AW300 MS and the resulting mixture was cooled to -10 °C and
stirred for 15 min. A solution of Yb(OTf)3 in dioxane (22.5 mg/
mL, 40 µL, 1.4 µmol) was added at that temperature, the mixture
was stirred for 30 min and then cooled to - 60 °C. Acceptor 20
(44 mg, 0.031 mmol) in 4:1 toluene/Et2O (0.4 mL), a solution of
Bi(OTf)3 in dioxane (16.6 mg/mL, 49 µL, 1.2 µmol) were
subsequently added, and the temperature was allowed to raise up
to room temperature over 2 h. The reaction was quenched with
some drops of pyridine and the mixture was filtered on a short
plug of silica gel (eluent DCM/MeOH/acetonitrile 85:10:5), con-
centrated, and purified by silica gel flash chromatography (eluent
petroleum ether/ethyl acetate from 9:1 to 8:2) to yield pentasac-
protons), 6.98 and 6.72 (4H, 2 × d, Jortho ) 9.2 Hz, aromatic
p-methoxyphenyl protons), 5.57 (1H, d, J ) 1.6 Hz, H-1), 5.23
(1H, bs, anomeric proton), 5.22 (1H, d, J ) 1.6 Hz, anomeric
proton), 4.90-4.33 (20H, 10 × AB, 10 × benzyl CH2), 4.26 (1H,
t, H-2), 4.21 (1H, dd, J ) 3.0 and 8.4 Hz), 4.13-4.06 (2H),
4.00-3.80 (7H), 3.73 (3H, s, -OCH3), 3.77-3.62 (6H), 3.56 (1H,
bd, J ) 10.0 Hz). 13 C NMR (CDCl3, 50 MHz) 154.8, 150.1, 138.8,
138.5-138.2, 128.2-127.0, 117.9, 114.5, 99.6, 99.5, 98.0, 80.0,
79.6, 75.5, 75.1, 75.0, 74.7, 74.2, 73.3, 73.2, 72.5, 72.3, 72.2, 72.1,
69.2, 68.9, and 55.6. MALDI-MS: [M + Na]+ calculd 1533.67,
found 1533.56. Anal. Calcd for C95H98O17: C, 75.47, H, 6.53.
Found: C, 75.28; H, 6.62.
p-Methoxyphenyl 3-O-Allyl-2,4,6-tri-O-benzyl-r-D-mannopy-
ranosyl-(1f3)-2,4,6-tri-O-benzyl-r-D-mannopyranosyl-(1f2)-
3,4,6-tri-O-benzyl-r-D-mannopyranoside (19). Trichloroacetim-
idate 4 (123 mg, 0.194 mmol) and trifluoroacetimidate 5 (106 mg,
0.170 mmol) were coevaporated three times with anhydrous toluene
(3 × 1 mL) and dried under vacuum for 1 h. The residue was
dissolved under argon with 4:1 toluene/Et2O (4.7 mL) in the
presence of freshly activated 4Å AW300 MS. The mixture was
then cooled to -10 °C and stirred for 15 min. A solution of
Yb(OTf)3 in dioxane (25 mg/mL, 125 µL, 0.005 mmol) was added
at that temperature, and the mixture was stirred for 30 min and
then cooled to -60 °C. Acceptor 3 (75 mg, 0.135 mmol) in 4:1
toluene/Et2O (0.9 mL) and a solution of Bi(OTf)3 in dioxane (16.6
mg/mL, 160 µL, 0.004 mmol) were subsequently added, and the
temperature was allowed to warm up to 10 °C over 90 min. The
reaction was quenched with some drops of pyridine and the mixture
was filtered on a short plug of silica gel (eluent DCM/MeOH/
acetonitrile 85:10:5), concentrated, and purified by silica gel flash
chromatography (eluent petroleum ether/ethyl acetate from 9:1 to
8:2) to yield trisaccharide 19 as a yellow oil (118 mg, 60% overall
yield). [R]25D +29.7° (c 1.0, CHCl3). 1H NMR (CDCl3, 400 MHz):
charide 21 as an oil (40 mg, yield 56%). [R]25 +25.3° (c 0.8,
D
CHCl3). 1H NMR (CDCl3, 400 MHz): δ 7.40-7.00 (benzyl
protons), 6.98 and 6.73 (4H, 2 × d, Jortho ) 9.2 Hz, aromatic
p-methoxyphenyl protons), 6.00-5.90 (1H, m, -OCH2-CHdCH2),
5.60 (1H, bs, H-1), 5.28 (3H, bs, 3× anomeric protons), 5.25 (1H,
bs, anomeric proton), 5.23 (1H, bd, Jtrans ) 17.6 Hz, -CHdCH
cisHtrans), 5.08 (1H, bd, J ) 10.4 Hz, -CHdCHcisHtrans), 4.75-4.25
(30H, 15 × AB, 15 × benzyl CH2), 4.30-4.20 (3H), 4.15-4.10
(2H), 4.05-3.68 (16H), 3.74 (3H, s, -OCH3), 3.65-3.60 (2H),
3.60-3.55 (3H), 3.55-3.45 (3H), 3.38 (1H, bd, J ) 10.4 Hz). 13
C
NMR (CDCl 3, 100 MHz) 154.9, 150.2, 139.0, 138.7, 138.4 (×4),
138.3 (×4), 138.2 (×4), 138.1, 135.0, 128.5-126.8, 118.0, 116.4,
114.5, 99.9, 99.6, 99.4, 99.3, 98.0, 76.7, 76.6, 78.9, 78.4, 77.8, 77.3,
4502 J. Org. Chem. Vol. 73, No. 12, 2008

Assemblage of the Antitumor PI-88 Pentasaccharide
77.2, 76.9, 76.8, 75.7, 75.2, 75.1, 75.0, 74.7, 74.5, 74.3, 73.3, 73.2,
73.1, 72.7, 72.6, 72.5, 72.3, 72.2, 72.0, 71.9, 71.0, 69.4, 69.3, 69.2,
69.0, 68.8, 55.6. MALDI-MS [M + Na]+ calcld 2349.05, found
2348.93. Anal. Calcd for C145H152O27: C, 74.85, H, 6.58. Found:
C, 74.63; H, 6.66.
Jortho ) 10.2 Hz, p-methoxyphenyl protons), 5.71 (1H, d, J1,2 )
1.6 Hz, H-1), 5.09 (2H, bs, 2 × anomeric protons), 5.07 (1H, d,
J1,2 ) 1.2 Hz, anomeric proton), 5.01 (1H, d, J1,2 ) 1.8 Hz,
anomeric proton), 4.21 (1H, dd, J ) 1.8 and 3.0 Hz), 4.18-4.16
(2H), 4.13 (1H, dd, J ) 1.8 and 3.4 Hz, H-2), 4.08 (1H, dd, J )
3.0 and 8.8 Hz), 4.03-4.01 (2H), 3.98 (1H, m), 3.96 (1H, m), 3.76
(3H, s, -OCH3), 3.92-3.60 (21H). 13C NMR (D2O, 50 MHz)
155.9, 151.0, 120.1, 116.3, 103.6 (×2), 103.5, 103.4, 99.1, 80.3,
80.2, 79.3, 74.8, 74.7, 74.6, 71.6, 71.3, 70.9, 68.1, 68.0, 67.5, 67.4,
62.3, 61.9, 57.0. MALDI-MS: [M + Na]+ calculd 957.31, found
957.45. Anal. Calcd for C37H58O27: C, 47.54, H, 6.25. Found: C,
47.38; H, 6.30.
p-Methoxyphenyl r-D-Mannopyranosyl-(1f3)-r-D-mannopy-
ranosyl-(1f3)-r-D-mannopyranosyl-(1f3)-r-D-mannopyrano-
syl-(1f2)-r-D-mannopyranoside (1). PdCl2 (2 mg, 0.011 mmol)
was added to a solution of pentasaccharide 21 (37 mg, 0.014 mmol)
in MeOH/DCM 9:1 (3 mL). After ca. 60 min under stirring the
mixture was concentrated under vacuum, resuspended with DCM/
MeOH 95:5, filtered on a short plug of silica gel, and concentrated.
The residue was dissolved with several aliquots of MeOH/formic
acid 9:1 (total volume 4 mL) and added under argon to palladium
on charcoal (10%, 50 mg) previously wet with the same solution.
The mixture was ultrasonicated for 2 h, then filtered on a plug of
celite, and concentrated under vacuum. The residue was eluted with
water and methanol on a short column of mixed-bed ion-exchange
resin and concentrated to yield pentasaccharide 1 as a white
Acknowledgment. The authors thank Dr. E. Bedini and Dr.
G. Pieretti for helpful discussions. NMR and MS facilities of
CIMCF are acknowledged.
1
Supporting Information Available: Copies of H and 13C
NMR spectra of all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
amorphous solid (9 mg, 70% over two steps). [R]25 +43.3° (c
D
0.6, H2O). 1H NMR (D2O, 400 MHz): δ 7.08 and 6.93 (4H, 2 × d,
JO8003953
J. Org. Chem. Vol. 73, No. 12, 2008 4503