A practical total synthesis of globo-H for use in anticancer vaccines

Article abstract of DOI:10.1021/jo901682p

(Chemical Equation Presented) An improved synthesis of the hexasaccharide MBr1 antigen (globo-H) is reported. Enhanced efficiency in the synthesis was necessary for the scale-up production of globo-H, in order to advance globo-H-based anticancer vaccines

Full text of DOI:10.1021/jo901682p

pubs.acs.org/joc
A Practical Total Synthesis of Globo-H for Use in
Anticancer Vaccines
Insik Jeon,† Karthik Iyer,† and Samuel J. Danishefsky*,†,‡
†Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute
for Cancer Research, 1275 York Avenue, New York, New York
10065, and ‡Department of Chemistry, Columbia University,
Havemeyer Hall, 3000 Broadway, New York, New York 10027
s-danishefsky@ski.mskcc.org
Received August 3, 2009
FIGURE 1. Glycosphingolipid globo-H (1) and representative
globo-H containing anticancer vaccines.
This human breast-cancer-associated antigen was found to
be distinctively overexpressed on the surfaces of a variety of
other epithelial cancer cells such as prostate, ovary, lung,
colon, and small cell lung cancers.3 For this reason, the
globo-H antigen has played an important role in our ongoing
carbohydrate-based cancer vaccine program.4 Of particular
interest to our laboratory was a recent report, indicating that
globo-H and SSEA3;the pentasaccharide precursor of
globo-H;are also overexpressed in breast cancer stem cells.
Interestingly, when co-administered with an immunological
adjuvant (R-galactosylceramide), a globo-H-based vaccine
induces antibodies against both globo-H and SSEA3.5 In the
light of the fact that cancer stem cells are often responsible
for relapse and metastasis of cancerous tissues,6 it is envi-
sioned that globo-H-based therapy might form the basis of
an important new direction in cancer treatment. Indeed, the
globo-H vaccine, 2, synthesized in our laboratories as a
glycoconjugate appended to immunogenic carrier protein,
has shown promise as a potential breast and prostate cancer
vaccine, in preclinical, and even clinical settings.4a-e More
recently, we also disclosed the synthesis and preclinical
evaluation of the unimolecular pentavalent vaccine 3,4f,g
displaying globo-H and other antigens known to be over-
expressed on prostate and breast cancer cell surfaces. The
thought was to construct a single entity antigen system which
reflects the actual degree of carbohydrate heterogeneity
associated with most cancers.7 Nonetheless, the accessibility
An improved synthesis of the hexasaccharide MBr1 antigen
(globo-H) is reported. Enhanced efficiency in the synthesis
was necessary for the scale-up production of globo-H, in
order to advance globo-H-based anticancer vaccines to
clinical trials. The key features of the improved synthesis
include preactivation-based glycosylations and a revised
iodosulfonimidation/rearrangement.
The glycosphingolipid globo-H (1, Figure 1), first isolated
by Hakomori and colleagues from the breast cancer cell line
MCF-7,1 is recognized by the monoclonal antibody MBr1.2
(1) (a) Kannagi, R.; Levery, S. B.; Ishijamik, F.; Hakomori, S.; Schevinsky,
L. H.; Knowles, B. B.; Solter, D. J. Biol. Chem. 1983, 258, 8934–8942.
(b) Bremer, E. G.; Levery, S. B.; Sonnino, S.; Ghidoni, R.; Canevari, S.;
Kannagi, R.; Hakomori, S. J. Biol. Chem. 1984, 259, 14773–14777.
(2) Menard, S.; Tagliabue, E.; Canevari, S.; Fossati, G.; Colnaghi, M. I.
Cancer Res. 1983, 43, 1295–1300.
(3) Livingston, P. O. Semin. Cancer Biol. 1995, 6, 357–366.
(4) (a) Ragupathi, G.; Park, T. K.; Zhang, S.; Kim, I. J.; Graber, L.;
Adluri, S.; Lloyd, K. O.; Danishefsky, S. J.; Livingston, P. O. Angew. Chem.,
Int. Ed. Engl. 1997, 36, 125–128. (b) Allen, J. R.; Allen, J. G.; Zhang, X.-F.;
Williams, L. J.; Zatorski, A.; Ragupathi, G.; Livingston, P. O.; Danishefsky,
S. J. Chem.;Eur. J. 2000, 6, 1366–1375. (c) Ragupathi, G.; Slovin, S. F.;
Adluri, S.; Sames, D.; Kim, I. J.; Kim, H. M.; Spassova, M.; Bornmann, W.
G.; Lloyd, K. O.; Scher, H. I.; Livingston, P. O.; Danishefsky, S. J. Angew.
Chem., Int. Ed. 1999, 38, 563–566. (d) Slovin, S. F.; Ragupathi, G.; Adluri, S.;
Ungers, G.; Terry, K.; Kim, S.; Spassova, M.; Bornmann, W. G.; Fazzari,
M.; Dantis, L.; Olkiewicz, K.; Lloyd, K. O.; Livingston, P. O.; Danishefsky,
S. J.; Scher, H. I. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5710–5715. (e)
Gilewski, T.; Ragupathi, G.; Bhuta, S.; Williams, L. J.; Musselli, C.; Zhang,
X.-F.; Bencsath, K. P.; Panageas, K. S.; Chin, J.; Hudis, C. A.; Norton, L.;
Houghton, A. N.; Livingston, P. O.; Danishefsky, S. J. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 3270–3275. (f) Ragupathi, G.; Koide, F.; Livingston, P. O.;
Cho, Y. S.; Endo, A.; Wan, Q.; Spassova, M. K.; Keding, S. J.; Allen, J.;
Ouerfelli, O.; Wilson, R. M.; Danishefsky, S. J. J. Am. Chem. Soc. 2006, 128,
2715–2725. (g) Zhu, J.; Wan, Q.; Ragupathi, G.; George, C. M.; Livingston,
P. O.; Danishefsky, S. J. J. Am. Chem. Soc. 2009, 131, 4151–4158. (h) Zhu, J.;
Wan, Q.; Yang, G.; Ouerfelli, O.; Danishefsky, S. J. Heterocycles 2009, 79,
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2004, 101, 11937–11942.
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8452 J. Org. Chem. 2009, 74, 8452–8455
Published on Web 10/02/2009
DOI: 10.1021/jo901682p
r
2009 American Chemical Society

Jeon et al.
JOCNote
FIGURE 2. Our previous syntheses of globo-H KLH conjugate (2) and glycosylamino acid (11).
of such complex carbohydrate antigens remained as an
important question. Globo-H isolated from human cancer
tissue collections is typically limited to submilligram levels,
which impedes broader immunological studies, including
more advanced clinical trials. For this reason, globo-H has
been an important synthetic target for our laboratory and
others, including Schmidt,8 Boons,9 Wong,10 Seeberger,11
and Huang.12 As part of our anticancer vaccine program to
advance construct 2 to phase II/III clinical trials and 3 to
phase I clinical trials for breast and prostate cancer, we
required a scale-up synthesis of the globo-H antigen based
on our previously established routes. Our modified route
would need to overcome the yield and efficiency issues
encountered in our small-scale synthesis (vide infra). We
describe herein the development of an improved synthesis of
globo-H, which provides ready access to large quantities of
antigen.
Our earlier total synthesis of globo-H utilized a glycal
building block assembly strategy for the rapid construction
of the complex hexasaccharide.4b,13 Key transformations
include (i) β-glycosylation leading to 4 via DMDO-mediated
R-epoxidation,14 (ii) Mukaiyama15-Nicolaou16 coupling
using fucosyl donor 5 to obtain trisaccharide glycal 6,
(iii) iodosulfonimidation/rearrangement methodology17 to
yield DEF donor 7, (iv) Cp2Zr(OTf)2-mediated construction
of ABC acceptor 8,4b and finally (v) β-selective [3+3] ABC
+ DEF coupling to furnish 9 via sulfonamide participation
(Figure 2). Although this concise and stereoselective strategy
allows rapid access to globo-H, some steps, particularly
those leading to DEF donor 7, still require further optimiza-
tion if we are to secure the globo-H antigen in amounts
required for our ongoing clinical trials with globo-H, both in
monovalent and multivalent settings.
In this regard, we report herein our efforts to seek a much
improved synthesis of the DEF donor 7 and ABC acceptor 8.
As described in our earlier report,4b,13 DEF trisaccharide
glycal 6 was obtained from the coupling of disaccharide glycal
4 and fucosyl donor 518 using Mukaiyama15-Nicolaou16
conditions, that is, SnCl2/AgClO4 (Table 1, entry 1). The
fucosylation had earlier occurred in moderate regioselectivity
mainly at the equatorial (35-47%) rather than the axial
alcohol (∼8%) of 4. In the case of AB þ C coupling using
fluorogalactosyl donor 1419 and AB acceptor 12 under the
same conditions, ABC trisaccharide 13 was generated in 42%
yield with modest anomeric selectivity (R/β = 3:1) (Table 1,
entry 3). However, the yield and selectivity of the AB þ C
coupling could be improved markedly (80%, R/β=10:1) when
the strongly fluorophilic Cp2Zr(OTf)2 was employed as the
promoter (Table 1, entry 4).
Alternatively, the glycosylation employing preactivated
glycosyl donor20 using the promoter p-TolSOTf21 has pro-
ven useful for the construction of stereoselective R- or
β-linkages. In particular, Huang and co-workers have shown
a successful application of this protocol;using thiofucosyl
donor 15 and thiogalactosyl donor 16;to their fascinating
multicomponent one-pot strategy for the synthesis of globo-
H.12 In our system, glycosylation leading to trisaccharide
glycal 6 requires the fucosyl donor to distinguish between the
equatorial C20 and axial C4 hydroxyl groups of the DE
acceptor 4 with high R-selectivity. Gratifyingly, fucosylation
of 4 using thiofucosyl donor 15 under slightly modified
conditions based on Huang’s protocol12 afforded the desired
R-trisaccharide 6 in high yields (78%, entry 2), with no
indication of the presence of C4 fucosylated isomer. In a
similar manner, coupling of thiogalactosyl donor 16 and AB
acceptor 12 proceeded stereoselectively to furnish R-trisac-
charide 13 in 76% yield (Table 1, entry 5). The [2 þ 1]
(8) Lassaletta, J.; Schmidt, R. R. Liebigs Ann. 1996, 1417–1420.
(9) Zhu, T.; Boons, G.-J. Angew. Chem., Int. Ed. 1999, 38, 3495–3497.
(10) Burkhart, F.; Zhang, Z.; Wacowich-Sgarbi, S.; Wong, C.-H. Angew.
Chem., Int. Ed. 2001, 40, 1274–1277.
(11) (a) Bosse, F.; Marcaurelle, L. A.; Seeberger, P. H. J. Org. Chem.
2002, 67, 6659–6670. (b) Werz, D. B.; Castagner, B.; Seeberger, P. H. J. Am.
Chem. Soc. 2007, 129, 2770–2771.
(12) Wang, Z.; Zhou, L.; El-Boubbou, K.; Ye, X.-S.; Huang, X. J. Org.
Chem. 2007, 72, 6409–6420.
(13) (a) Bilodeau, M. T.; Park, T. K.; Hu, S.; Randolph, J. T.; Danishefsky,
S. J.; Livingston, P. O.; Zhang, S. J. Am. Chem. Soc. 1995, 117, 7840–7841.
(b) Park, T. K.; Kim, I. J.; Hu, S.; Bilodeau, M. T.; Randolph, J. T.; Kwon, O.;
Danishefsky, S. J. J. Am. Chem. Soc. 1996, 118, 11488–11500.
(14) Halcomb, R. L.; Danishefsky, S. J. J. Am. Chem. Soc. 1989, 111,
6661–6666.
(15) Mukaiyama, T.; Murai, Y.; Shoda, S. Chem. Lett. 1981, 431–434.
(16) (a) Nicolaou, K. C.; Caulifield, T. J.; Kataoka, H.; Stylianides, N. A.
J. Am. Chem. Soc. 1990, 112, 3693–3695. (b) Nicolaou, K. C.; Hummel, C.
W.; Bockovich, N. J.; Wong, C.-H. J. Chem. Soc., Chem. Commun. 1991,
870–872.
(18) Dejter-Juszynski, M.; Flowers, H. M. Carbohydr. Res. 1973, 28, 61–
64.
(19) Gordon, D. M.; Danishefsky, S. J. Carbohydr. Res. 1990, 206, 361–
366.
(17) Danishefsky, S. J.; Koseki, K.; Griffith, D. A.; Gervay, J.; Peterson,
J. M.; McDonald, F. E.; Oriyama, T. J. Am. Chem. Soc. 1992, 114, 8331–
8333.
(20) Crich, D.; Sun, S. Tetrahedron 1998, 54, 8321–8348.
(21) Martichonok, V.; Whitesides, G. M. J. Org. Chem. 1996, 61, 1702–
1706.
J. Org. Chem. Vol. 74, No. 21, 2009 8453

Jeon et al.
JOCNote
TABLE 1. Coupling Conditions Used To Construct the Trisaccharide 6 and Trisaccharisde 13
entry
donor
acceptor
condition
t (h)
product (yield)
6 (35-47%)a
1
2
3
4
5
5
15
14
14
16
4
4
12
12
12
SnCl2, AgClO4, ether, 2,6-di-tert-butylpyridine
TolSCl, AgOTf, CH2Cl2/Et2O (2:1), 2,6-di-tert-butylpyridine
SnCl2, AgClO4, ether, 2,6-di-tert-butylpyridine
Cp2Zr(OTf)2, toluene/THF (5:1), dark, 2,6-di-tert-butylpyridine
TolSCl, AgOTf, CH2Cl2/Et2O (2:1), 2,6-di-tert-butylpyridine
35
4
35
72
4
6 (78%)b
13 (42%)
13 (80%)
13 (76%)
aApproximately 8% of monofucosylated productat C4 of the galactose was also observed. bTrace amounts of the difucosylated product were detected
by NMR.
couplings using thioglycosyl donors and the promoter
p-TolSOTf gave superior yields with shorter reaction times
than the reactions performed under Mukaiyama conditions.
Importantly, the reactions deliver more consistent results, parti-
cularly in our gram-scale synthesis of the target trisaccharides 6
and 13.
SCHEME 1. Synthesis of globo-H pentenyl glycoside 10 via
DEF donor 7
We next turned our attention to the transformation
of trisaccharide glycal 6 to DEF thiodonor 7 bearing a
2-R-phenylsulfonamido function (Scheme 1). According to
precedent established in our laboratories,17 iodosulfonimi-
dation of glycal 6 using I(sym-coll)2ClO4 and benzenesulfon-
amide followed by treatment with lithium ethanethiolate
afforded DEF thiodonor 7 in 37-40% yields over two
steps.13 The somewhat modest overall conversion is most
likely due to the formation of unidentified impurities asso-
ciated with the initial iodosulfonimidation step. We found,
however, that by tuning reaction conditions, such as tem-
perature and solvent system, we were able to effectively
suppress the formation of byproducts to negligible amounts.
Specifically, when we substituted THF solvent with diethyl
ether and carefully maintained the reaction temperature
at -5 °C for 12 h, we were able to obtain trans-diaxial
iodosulfonamide 17 as almost the exclusive product. With-
out purification, the somewhat labile 17 was immediately
subjected to the next step employing excess lithium ethane-
thiolate to furnish the desired DEF thiodonor 7 in 75% yield.
The DEF thiodonor 7 was then treated with MeOTf22 in the
presence of the PMB-deprotected ABC acceptor 8 to effi-
ciently afford hexasaccharide 9 in 72% yield. Global depro-
tection of 9 followed by peracetylation proceeded smoothly
to afford globo-H pentenyl glycoside 10.
In summary, we have successfully optimized synthetic
steps to DEF donor 7. The DEF donor 7 serves as a key
strategic intermediate for the [3 þ 3] ABC þ DEF coupling
via β-selective sulfonamidogalactosylations leading to the
fully elaborated hexasaccharide skeleton of globo-H. The
improved synthesis reported herein allows facile production
€
€
(22) (a) Lonn, H. Carbohydr. Res. 1985, 139, 105–113. (b) Lonn, H.
J. Carbohydr. Chem. 1987, 6, 301–306.
8454 J. Org. Chem. Vol. 74, No. 21, 2009

Jeon et al.
JOCNote
of globo-H up to gram-scale with quite consistent yields.
Advanced clinical results using synthetic vaccines containing
globo-H, such as 2 and 3, will be reported in due course.
in CH2Cl2 (130 mL) at 0 °C was treated with phosphate buffer
(20 mL, pH 7.2) and 2,3-dichloro-5,6-dicyano-1,4-benzoqui-
none (DDQ) (1.19 g, 5.24 mmol, 1.5 equiv) and stirred at 0 °C
for 5 h. The reaction mixture was diluted with saturated
NaHCO3 and further stirred at rt for 1 h. The mixture was
extracted twice with CH2Cl2, then washed with brine, dried over
MgSO4, and concentrated to dryness. The crude material was
purified by flash column chromatography using 16% EtOAc/
hexane to give 4.06 g (84%) of ABC acceptor 8.
Experimental Section
Ethyl 2,3,4-tri-O-benzyl-r-L-fucopyranosyl-(1f2)-3,4-carbonyl-
6-O-tri-isopropylsilyl-β-D-galactopyranosyl-(1f3)-6-O-triisopro-
pylsilyl-2-deoxy-2-phenylsulfonylamino-1-thio-β-D-galactopyra-
noside (7). A solution of thiofucosyl donor 15 (2.38 g, 4.41 mmol)
4-Pentenyl 2,3,4-tri-O-benzyl-r-L-fucopyranosyl-(1f2)-3,4-
carbonyl-6-O-triisopropylsilyl-β-D-galactopyranosyl-(1f3)-
6-O-triisopropylsilyl-2-deoxy-2-phenylsulfonylamino-β-D-galacto-
pyranosyl-(1f3)-2,4,6-tri-O-benzyl-r-D-galactopyranosyl-(1f4)-
2,3,6-tri-O-benzyl-β-D-galactopyranosyl-(1f4)-2,3,6-tri-O-benzyl-
β-D-glucopyranoside (9). The DEF donor 7 (2.13 g, 1.66 mmol) and
ABC acceptor 8 (1.15 g, 0.831 mmol) were combined, azeotroped
twice with anhydrous benzene, and placed under high vacuum for
5 h. The mixture was then dissolved in CH2Cl2 (20 mL) and Et2O
˚
and freshly activated 4 A molecular sieves (2 g) in CH2Cl2/Et2O
(50 mL/25 mL) was stirred at rt for 1 h, then cooled to -78 °C.
To the cooled solution were added AgOTf (3.4 g, 13.22 mmol)
and 2,6-di-tert-butylpyridine (2.92 mL, 13.92 mmol). After
10 min, freshly distilled p-TolSCl (619 μL, 4.41 mmol) was
added. After 40 min, the characteristic yellow color of p-TolSCl
in the reaction solution disappeared, indicating depletion of
p-TolSCl. A solution of acceptor 4 (2.85 g, 4.41 mmol) in
CH2Cl2/Et2O (6 mL/3 mL) was added dropwise via a syringe.
The reaction mixture was warmed to rt under stirring over 3 h
and stirred for an additional 1 h at rt. The mixture was filtered
over Celite and further washed with CH2Cl2. After evaporation
of solvent in vacuo, the mixture was diluted with ethyl acetate
then washed with a saturated aqueous solution of NaHCO3,
water, and brine and dried over MgSO4. After removal of the
solvent, the crude was purified via silica gel flash chromatogra-
phy using 10-15% EtOAc/hexane to give 3.66 g (78%) of
R-trisaccharide 6. A mixture of trisaccharide glycal 6 (1 g,
0.94 mmol) and benzenesulfonamide (887 mg, 5.64 mmol) was
azeotroped with anhydrous benzene once and further dried
on high vacuum for 1 h. It was dissolved in freshly distilled
˚
(40 mL), treated with freshly activated 4 A molecular sieves (2 g)
and 2,6-di-tert-butylpyridine (734 μL, 3.32 mmol), then cooled to
-78 °C. Methyl triflate (4.0 equiv, 365 μL) was added in one
portion, and the reaction was allowed to warm to rt slowly
overnight. The reaction was quenched by the addition of Et3N
(5 mL) and filtered through Celite with Et2O.Thefiltratewaswashed
with saturated NaHCO3 and brine and dried over MgSO4. Concen-
tration and purification by silica gel chromatography (15-20%
EtOAc/hexane) afforded 1.551 g (72%) of hexasaccharide 9.
4-Pentenyl 2,3,4-tri-O-acetyl-r-L-fucopyranosyl-(1f2)-3,4,6-
tri-O-acetyl-β-D-galactopyranosyl-(1f3)-4,6-O-triacetyl-2-acetyl-
amino-2-deoxy-β-D-galactopyranosyl-(1f3)-2,4,6-tri-O-acetyl-
r-D-galactopyranosyl-(1f4)-2,3,6-tri-O-acetyl-β-D-galactopyr-
anosyl-(1f4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (10). TBAF
(1.0 M in THF, 37.34 mL, 60 equiv) was added to a solution of
the hexasaccharide 9(1.62 g, 0.622 mmol) and acetic acid (2.14 mL,
60 equiv) in THF (35 mL). The reaction was stirred at rt for 3 days,
poured into ice water, and extracted with EtOAc. The organic
extracts were washed with saturated NaHCO3 and brine, dried
over MgSO4, and concentrated. This desilylated intermediate was
purified through a short plug of silica gel with EtOAc. The resulting
triol was dissolved in anhydrous MeOH (20 mL), and sodium
methoxide was added (0.75 mL of a 25% solution in MeOH). The
reaction was stirred at rt for 18 h, neutralized with Dowex-Hþ,
filtered with MeOH washings, and concentrated. THF (5 mL) and
condensed liquid NH3 (∼70 mL) were added at -78 °C to the
resulting white solid. Sodium (∼1.2 g) was added, and the resulting
blue solution was stirred at -78 °C for 2 h. The reaction was
quenched with anhydrous MeOH (20 mL), brought to rt, and
concentrated under a stream of dry N2 to a volume of ∼15 mL. The
reaction was neutralized with Dowex-Hþ, filtered with MeOH
washing, and concentrated to a white solid. The white solid was
dissolved in pyridine (10 mL) and CH2Cl2 (10 mL) and cooled to
0 °C. A few crystals of DMAP were added followed by acetic
anhydride (10 mL). The ice bath was removed and the reaction
stirred at rt overnight. The mixture was diluted with EtOAc and
washed with water, saturated NaHCO3, and brine and dried over
MgSO4. Concentration followed by purification by flash column
chromatography using 60% EtOAc/CH2Cl2 to give 10 as a white
solid (470 mg, 42%).
˚
Et2O (40 mL), and freshly activated 4 A molecular sieves (1 g)
were added. The resulting mixture was stirred at rt for 1 h, then
cooled to -5 °C. To the solution was added I(sym-coll)2ClO4
(925 mg, 1.974 mmol), and the mixture was further stirred at
-5 °C for 12 h. After filtration on Celite pad, the crude was
washed with saturated Na2S2O3 solution, saturated CuSO4, and
brine and dried over MgSO4. After concentration, the crude
material, containing iodosulfonamide 17, was immediately sub-
jected to the next step without purification. To a solution of
EtSH (695 μL, 9.4 mmol) in DMF (5 mL) was added lithium
bis(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 4.7 mL,
4.7 mmol) at -45 °C. After 15 min of stirring, the solution was
transferred dropwise via a cannula to a flask containing iodo-
sulfonamide 17 in DMF (25 mL) at -45 °C. The reaction
mixture was allowed to warm to rt and stirred for a total of
3 h. After dilution with saturated NH4Cl, the crude was ex-
tracted four times with ethyl acetate. The combined extracts
were washed with water and brine and dried over MgSO4.
Concentration and purification by silica gel chromatography
(15-25% EtOAc in hexane) afforded 903 mg (75% over two
steps) of DEF donor 7.
4-Pentenyl 2,4,6-tri-O-benzyl-r-D-galactopyranosyl-(1f4)-
2,3,6-tri-O-benzyl-β-D-galactopyranosyl-(1f4)-2,3,6-tri-O-ben-
zyl-β-D-glucopyranoside (8). A solution of thiogalactosyl donor
˚
16 (3.97 g, 5.86 mmol) and freshly activated 4 A molecular sieves
(4 g) in CH2Cl2/Et2O (100 mL/50 mL) was stirred at rt for 1 h,
then cooled to -78 °C. To the cooled solution were added
AgOTf (4.52 g, 17.6 mmol) and 2,6-di-tert-butylpyridine
(3.9 mL, 17.6 mmol). After 10 min, freshly distilled p-TolSCl
(824 μL, 5.86 mmol) was added. After 10 min, a solution of
acceptor 12 (4.65 g, 4.88 mmol) in CH2Cl2/Et2O (10 mL/5 mL)
was added dropwise. The reaction mixture was warmed to
rt under stirring over 3 h and then stirred for an additional
1 h at rt. After filtration and usual workup as described
previously, the crude was purified via silica gel flash chroma-
tography using 15% EtOAc/hexane to give 5.55 g (76%) of
R-trisaccharide 13. This PMB-protected 13 (5.25 g, 3.49 mmol)
Acknowledgment. Support for this work was provided by
the National Institutes of Health (CA28824). We thank Rebecca
Wilson and Dana Ryan for assistance with the preparation of the
manuscript, and Dr. Dongjoo Lee for helpful discussions.
Supporting Information Available: NMR spectra for 6, 7, 8,
9, and 10. This material is available free of charge via the
Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 74, No. 21, 2009 8455