Synthesis of a serine-based neuraminic acid C-glycoside

Article abstract of DOI:10.1021/jo026776v

Cell-surface carbohydrates are classified by the nature of their linkages to the protein as either N-linked or O-linked. O- and N-glycans are involved in a number of important biological functions. These activities can be lost on glycoprotein catabolism w

Full text of DOI:10.1021/jo026776v

Syn th esis of a Ser in e-Ba sed Neu r a m in ic Acid C-Glycosid e
Qun Wang and Robert J . Linhardt*
Division of Medicinal and Natural Products Chemistry and Department of Chemical and Biochemical
Engineering, The University of Iowa, Iowa City, Iowa 52242
robert-linhardt@uiowa.edu
Received November 27, 2002
Cell-surface carbohydrates are classified by the nature of their linkages to the protein as either
N-linked or O-linked. O- and N-glycans are involved in a number of important biological functions.
These activities can be lost on glycoprotein catabolism when these glycan linkages are enzymatically
hydrolyzed. The design and synthesis of novel C-linked glycans should provide catabolically stable
glycoproteins useful for understanding and regulating important biological processes. Our efforts
are currently directed toward the synthesis of C-glycosides of ulosonic acids. This paper describes
the first synthesis of a serine-based neuraminic acid C-glycoside. The protecting group chemistry
required for both carbohydrate and peptide syntheses complicates this approach. Different protecting
group strategies were investigated for use in the samarium diiodide mediated C-glycosylation
reaction. The key elements of our synthetic approach involve the following: (i) the substitution of
homoserine for serine in the C-glycosylation reaction to introduce a carbon in place of the O-glycosidic
oxygen, (ii) the use of benzyloxycarbonyl as a homoserine protecting group, compatible with
samarium diiodide mediated C-glycosylation reaction, and (iii) the reduction of the carbonyl group
in homoserine early in the synthesis to improve C-glycosylation yield and to avoid lactone formation.
Using this combined approach, we prepared 4-O-acetyl-4-[2-C-(1-methyl 5-acetamido 4,7,8,9-tetra-
O-acetyl-2,6-anhydro-3,5-dideoxy-D-erythro-L-manno-nononate)]-2S-(benzyloxycarbonyl)amino-1-car-
boxylic acid (1), which will be used in peptide synthesis to prepare glycopeptides containing
catabolically stable C-linked neuraminic acid.
In tr od u ction
in critical biological processes. A carbon-linked analogue
(C-glycopeptide), in which the native O-glycosidic linkage
has been replaced by the enzymatically and chemically
more resistant C-glycosidic linkage, should show en-
hanced stability in vivo and may have applications in
modern medicine for the control of bacterial and viral
diseases, cancer therapy, and treating inflammatory
processes.7-9
Neuraminic acids may be the single most important
monosaccharide components of glycoconjugates, since
they occupy the nonreducing terminal positions, act as
ligands for many glycoprotein receptors, and are often
the first saccharide lost in glycan catabolism. Neuraminic
acid residues in cell membranes and glycoproteins are
also involved in many physiologic and pathophysiologic
phenomena.10,11 Of particular interest to our laboratory
are mucins, important in cell protection and lubrica-
tion.12,13 Mucins are rich in neuraminic acid containing
glycans attached to serine or threonine. The synthesis
Glycoproteins are major components on the surface of
mammalian cells. Many carry O-linked oligosaccharides
(O-glycans), which are conjugated through serine or
threonine residues. Others carry N-linked oligosaccha-
rides (N-glycans), conjugated through an asparagine
residue. The recognition of these O- and N-glycoconju-
gates plays a key role in the transmission of biological
information at the cellular level.1-3 Their numerous
biological functions include roles in cellular recognition,
adhesion, cell-growth regulation, cancer cell metastasis,
and inflammation. Cell-surface glycans also serve as
attachment sites for infectious bacteria, viruses, and
toxins, resulting in pathogenesis.4,5 Anomalies in cell-
surface carbohydrates are often closely associated with
cell transformation, malignancy, and other various patho-
logical conditions, including immunodeficiency syn-
dromes, cancer, and inflammation.6 The synthesis of
structurally defined, catabolically stable glycoconjugates
should provide the opportunity to probe and intervene
(7) Wang, Q.; Wolff, M. W.; Polat, T.; Du, Y.; Linhardt, R. J . Bioorg.
Med. Chem. Lett. 2000, 10, 941.
* Address correspondence to this author at The University of Iowa.
Phone: 319-335-8834. FAX: 319-335-6634.
(1) Saxon, E.; Bertozzi, C. R. Annu. Rev. Cell. Dev. Biol. 2001, 17,
1.
(2) Hughes, R. C. Glycoconj. J . 2001, 17, 567.
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(4) Dwek, R. A. Chem. Rev. 1996, 96, 683.
(5) Varki, A. Glycobiology 1993, 3, 97.
(8) Kuberan, B.; Linhardt, R. J . Curr. Org. Chem. 2000, 4, 653.
(9) Peri, F.; Cipolla, L.; Rescigno, M.; Ferla, B. L.; Nicotra, F.
Bioconjugate Chem. 2001, 12, 325.
(10) Schauer, R. Adv. Carbohydr. Chem. Biochem. 1982, 40, 131.
(11) Varki, A. Glycobiology 1992, 2, 25.
(12) Snyder, J . D.; Walker, W. A. Int. Arch. Allergy Appl. Immunol.
1987, 82, 351.
(13) Sheehan, J . K.; Thornton, D. J .; Somerville, M.; Carlstedt, I.
Am. Rev. Respir. Dis. 1991, 144, S4.
(6) Tsuboi, S.; Fukuda, M. Bioessays 2001, 23, 46.
10.1021/jo026776v CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/20/2003
2668
J . Org. Chem. 2003, 68, 2668-2672

Synthesis of a Serine-Based Neuraminic Acid C-Glycoside
SCHEME 1
F IGURE 1. Serine-based C-glycoside 1 and neuraminic acid-
based glycosylation donor 2.
of serine-based C-glycosides has only been carried out in
a few laboratories and has relied on cross-metathesis of
oxazolidine silyl enol ether,14-16 or the Ramberg-Ba¨ck-
lund rearrangement.17 It is difficult to apply these
methodologies to the synthesis of serine-based neuramin-
ic acid C-glycosides for several reasons: (1) The carboxyl
group at C2 of neuraminic acid sterically restricts gly-
coside formation. (2) The carboxyl function attached at
C2 electronically disfavors oxonium ion formation, an
intermediate for almost all glycosylation reactions. (3)
The lack of a substituent at C3 precludes an assisting
and/or directing effect of an adjacent functional group
resulting in poor stereochemical control. Using a method
for the preparation of C-glycosides of neuraminic acids
with SmI2, pioneered in our laboratory,18 we set out to
synthesize a serine-based C-glycoside of neuraminic acid.
This stable linkage should contribute significantly to
understanding biological recognition and might serve to
enhance or suppress biological events at the molecular
level.
TABLE 1. C-Glycosyla tion w ith 2 u n d er Sm I2
Con d ition s
yield (%)a
entry
R
R′
R′′
Bn
ND
Bn
Allyl
Bn
7
8
6a
6b
6c
6e
6f
H
Pht
H
H
H
Boc
Bn
Fmoc
Cbz
Cbz
ND
ND
ND
38
ND
ND
5
40
0
a
ND ) not detected.
Resu lts a n d Discu ssion
To prepare a C-glycosidic linkage, it was first necessary
to synthesize a neuraminic acid donor 219 and a serine-
based acceptor 6. Homoserine was chosen as the substi-
tute of serine since during the C-glycosylation process
its additional carbon is required to replace the intergly-
cosidic O-linkage. Our strategy to prepare the acceptor
involved the orthogonal protection of amino and carboxyl
groups in homoserine and the oxidation of its hydroxyl
group to an aldehyde acceptor for C-glycosylation.
The initial synthesis of aldehyde acceptor 6 is shown
in Scheme 1. Multiple amino protecting groups (Fmoc,
Pht, Boc, Cbz)20-25 and carboxylic acid protecting groups
(Bn, allyl) were examined in the C-glycosylation reaction.
The resulting acceptors (6a -c) did not undergo C-
F IGURE 2. C-glycosylation products.
glycosylation with glycosyl donor 2 using SmI2 (Table 1).
Failure in all of these syntheses was attributed to the
high reactivity of SmI2, resulting in many side reactions,
including deprotection,26 condensation and reduction of
the aldehyde acceptor, and reduction of Neu5Ac sulfone
2. Cbz-protected homoserine 4e was then converted to
the corresponding allyl ester 5e and benzyl ester 5f,
respectively (Scheme 1). Both 5e and 5f were smoothly
oxidized, using Dess-Martin periodinane, to aldehyde
acceptors 6e and 6f.27 The C-glycosylation reaction of
these two acceptors afforded C-glycoside 7 with concomi-
tant loss of carboxyl protection and subsequent lacton-
ization. Only a small amount of the desired allyl pro-
tected homoserine C-glycoside was obtained (8). This
intramolecular cyclization product is favorable due to the
formation of a stable, five-membered ring, commonly
observed in homoserine-based syntheses28 (Figure 2)
(14) Dondoni, A.; Marra, A. Chem. Commun. 1998, 16, 1741.
(15) Dondoni, A.; Giovannini, P. P.; Marra, A. J . Chem. Soc., Perkin
Trans. 1 2001, 19, 2380.
(16) Dondoni, A.; Mariotti, G.; Marra, A.; Massi, A. Synthesis 2001,
14, 2129.
(17) Ohnishi, Y.; Ichikawa, Y. Bioorg. Med. Chem. Lett. 2002, 12,
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(18) Vlahov, I. R.; Vlahov, P. I.; Linhardt, R. J . J . Am. Chem. Soc.
1997, 119, 1480.
Lactone 7 could be opened by using acetic acid and
water as reaction media, affording the unprotected
serine-based neuraminic acid C-glycoside.29,30 Attempts
(19) Marra, A.; Sinay¨, P. Carbohydr. Res. 1989, 187, 35.
(20) Kuwahara, M.; Arimitsu, M.; Sisido, M. Tetrahedron 1999, 55,
10067.
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43, 2480.
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1983, 13, 311.
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Engl. 2000, 39, 242.
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45, 327.
J . Org. Chem, Vol. 68, No. 7, 2003 2669

Wang and Linhardt
SCHEME 2a
SCHEME 3a
a
Reagents: (i) TBDMSCl, pyridine, (ii) LiBH4, THF, (iii) BnBr,
1.2 equiv of NaH, DMF, 0 °C.
to protect the resulting free amino acid with FmocCl31,32
only afforded the N-Fmoc protected, lactonization prod-
uct.
It was clear that the protection of the amino group and
the presence of an R-COOH group in homoserine com-
plicated samarium-based C-glycosylation. Reduction of
the carboxyl group at the beginning of the synthesis and
its subsequent oxidation following C-glycosylation was
next examined to avoid lactonization and improve the
C-glycosylation yield. The hydroxyl group in Cbz-L-
homoserine allyl ester 5e was protected as the silyl ether
9,33 and the allyl ether was reduced to afford alcohol 1034
(Scheme 2). Surprisingly, benzylation of the newly gener-
ated hydroxyl group in 10 with benzyl bromide and
sodium hydride in DMF at 0 °C afforded aziridine 11 as
a major product. Reducing the reaction temperature and
the amount of NaH failed to prevent the formation of the
undesired aziridine. Benzylation under acidic conditions
with silver oxide and benzyl bromide, however, prevented
the formation of aziridine 11 but afforded the desired 12
in very low yield.35
a
Reagents: (i) dihydropyran, PPTS, CH2Cl2, (ii) TBAF, THF,
(iii) Dess-Martin periodinane, CH2Cl2, (iv) 2, SmI2, THF, (v) Ac2O,
pyridine, (vi) PPTS, EtOH, 50 °C, (vii) RuCl3, NaIO4, CCl4, CH3CN,
H2O, 0 °C.
glycoside 1.37,38 Temperature control and the amount of
the catalyst were critical in the RuCl3-based oxidation
step to prevent lactonization.
Failure of benzyl protection led us to examine tetrahy-
dropyranyl ether (THP) protection, which is easy to
install, stable to most nonacidic reagents, and easy to
remove. THP protection of 10 went smoothly36 and
afforded 13 in 83% yield (Scheme 3). Two isomers were
generated in a ratio of 3:2, and all subsequent synthetic
steps resulted in a mixture of diastereomeric products.
Quantitative tetrabutylammonium fluoride (TBAF) re-
moval of silyl ether afforded 14, which was readily
oxidized to the C-glycosylation aldehyde acceptor 15.
C-Glycosylation of 15 afforded R-C-glycoside 16 in 45%
isolated yield. It is important to note that this yield is
higher than that obtained in the previous C-glycosylation
reactions (Table 1). Acetylation of the newly generated
bridge hydroxyl group afforded fully protected C-glycoside
17. Removal of THP with pyridinium p-toluensulfonate
(PPTS), followed by oxidation of the resulting hydroxyl
group, generated our target, L-homoserine based C-
Exp er im en ta l Section
Proton nuclear magnetic resonance (1H NMR) spectra were
recorded at 360 or 400 MHz and carbon-13 (13C NMR) spectra
were recorded at 400 MHz in CDCl3. Chemical Shifts are
reported in δ units, parts per million (ppm) downfield from
tetramethylsilane (TMS). Mass spectra were obtained in the
electrospray-ionization (ESI) mode. Unless otherwise noted,
reactions were monitored by thin-layer chromatography on
aluminum sheets, Silica Gel 60 F254. The spots were detected
under short-wavelength UV light (254 nm) or by dipping the
plates into Von’s reagent (1.0 g of ceric ammonium sulfate and
24.0 g of ammonium molybdate in 31 mL of sulfuric acid and
470 mL of water), followed by heating. Flash chromatography
was performed with 200-400 mesh Silica Gel 60 under
nitrogen pressure.
l-2-[(Ben zyloxycar bon yl)am in o]-4-h ydr oxybu tyr ic Acid
(4e). Benzyl chloroformate (2.58 mL, 1.5 mmol) was added to
a solution of 3 (1.44 g, 12.0 mmol) in 1 N NaHCO3 (50 mL).
The reaction mixture was stirred at room temperature for 24
h and then extracted with ether (2 × 40 mL). The aqueous
phase was ice cooled, carefully acidified to pH 2-3 with 3 N
HC1, and extracted with EtOAc (4 × 40 mL). The extract was
dried over anhyd Na2SO4, filtered, and evaporated. The
resulting oil (2.65 g, 89%) was directly used in the next step
(31) Hilaire, P. M. S.; Cipolla, L.; Franco, A.; Tedebark, U.; Tilly,
D. A.; Meldal, M. J . Chem. Soc., Perkin Trans. 1 1999, 3559.
(32) Carpino, L. A.; Han, G. Y. J . Org. Chem. 1972, 37, 3404.
(33) J onghe, S. D.; Lamote, I.; Venkataraman, K.; Boldin, S. A.;
Hillaert, U.; Rozenski, J .; Hendrix, C.; Busson, R.; Keukeleire, D. D.;
Calenbergh, S. V.; Futerman, A. H.; Herdewijn, P. J . Org. Chem. 2002,
67, 988.
(34) Laib, T.; Chastanet, J .; Zhu, J . J . Org. Chem. 1998, 63, 1709.
(35) Saotome, C.; Kanie, Y.; Kanie, O.; Wong, C.-H. Bioorg. Med.
Chem. 2000, 8, 2249.
(36) Miyashita, N.; Yoshikoshi, A.; Grieco, P. A. J . Org. Chem. 1977,
42, 3772.
(37) Trost, B. M.; Krueger, A. C.; Bunt, R. C.; Zambrano, J . J . Am.
Chem. Soc. 1996, 118, 6520.
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Commun. 1987, 23, 1792.
2670 J . Org. Chem., Vol. 68, No. 7, 2003

Synthesis of a Serine-Based Neuraminic Acid C-Glycoside
without further purification. 1H NMR (CDCl3): 1.83-1.94 (m,
1H), 2.08-2.14 (m, 1H), 2.83 (br s, 1H), 3.68-3.71 (m, 2H),
4.37-4.41 (m, 1H), 5.07 (s, 2H), 6.58 (br s, 1H), 7.26-7.39 (m,
5H), 11.1 (br s, 1H). HRESIMS: calcd for C12H16NO5 [M + H]+
acceptor 6e, respectively. C-Glycosylation of acceptor 6f by
donor 2 afforded 7 in 40% yield. H NMR (CDCl3): 1.89, 1.94,
1
2.03, 2.09, 2.13, 2.17 (7 × s, 21H), 2.2-2.35 (m, 3H), 2.43 (1H,
dd), 3.80 (s, 3H), 3.89-4.09 (m, 2H), 4.22-4.34 (m, 2H), 4.55
(t, 1H), 4.71 (dd, 1H), 4.87-4.96 (ddd, 1H), 5.11-5.27, (m, 3H),
5.20-5.41 (m, 2H), 6.00 (d, 1H), 7.32-7.37 (m, 5H). 13C NMR
(CDCl3): 173.2, 171.1, 171.0, 170.3, 170.2, 170.0, 169.4, 156.3,
136.1, 128.5, 128.2, 79.7, 72.9, 69.0, 68.4, 67.6, 67.3, 62.8, 53.2,
50.1, 49.8, 33.9, 30.9, 28.3, 23.2, 21.0, 20.9, 20.8, 20.7, 20.6.
HRESIMS: calcd for C32H46N2O16Na [M + Na]+ 731.2276,
found m/z 731.2283 [M + Na]+.
Meth yl 5-Acetam ido-4,7,8,9-tetr a-O-acetyl-2,6-an h ydr o-
3,5-d id eoxy-2-C-[2S-a llyl-2-(b en zyloxyca r b on yl)a m in o
bu ta n oa te)]-D-er yth r o-L-m a n n o-n on on a te (8). 1H NMR
(CDCl3): 1.94-2.24 (m), 2.76-2.82 (m, 1H), 3.72-3.81 (m, 5H),
4.02-4.37 (m, 3H), 4.48-4.75 (m, 3H), 4.87-4.95 (m, 2H),
5.06-5.25 (m, 3H), 5.32-5.41 (m, 2H), 5.88-6.02 (m, 2H),
7.29-7.37 (m, 5H). ESIMS: C35H46N2O17 767 [M + H]+, 789
[M + Na]+.
Allyl-(2S)-2-(b en zyloxyca r b on yl)a m in o-4-[(ter t-b u t -
yld im eth ylsilyl)oxy]bu ta n oa te (9). tert-Butyldimethylsilyl
chloride (1.21 g, 2.43 mmol) was added to a solution of 5f (476
mg, 1.62 mmol) in pyridine (5 mL). The reaction mixture was
stirred at 0 °C for 4 h and evaporated under reduced pressure.
Purification of the residue by silica gel chromatography (80%
hexanes in EtOAc) yielded 9 as an oil (560 mg, 85%). 1H NMR
(CDCl3): 0.041 (s, 3H), 0.037 (s, 3H), 0.88 (s, 9H), 1.95-2.13
(m, 2H), 3.69-3.73 (m, 2H), 4.62 (d, 1H, J ) 5.6 Hz), 4,78 (m,
2H), 5.11 (d, 2H), 5.23 (d, 1H, J ) 10.4 Hz), 5.32 (d, 1H, J )
16.8 Hz), 5.82-5.96 (m, 2H), 7.29-7.35 (m, 5H). 13C NMR
(CDCl3): 171.9, 155.9, 136.4, 131.6, 128.3, 127.8, 127.7, 118.4,
66.7, 65.6, 59.8, 52.5, 33.7, 25.7, 22.5, 5.73, 5.75. ESIMS: 408
[M + H]+, 430 [M + Na]+. HRESIMS: calcd for C21H33NO5-
NaSi [M + Na]+ 430.2026, found m/z 430.2007 [M + Na]+.
Anal. Calcd for C21H33NO5Si: C, 61.88; H, 8.16; N, 3.44.
Found: C, 61.80; H, 8.23; N, 3.47.
254.1035, found m/z 254.1028 [M + H]+. Anal. Calcd for C12H16
-
NO5: C, 56.91; H, 5.97; N, 5.53. Found: C, 56.76; H, 5.90; N,
5.45.
Allyl (2S)-2-(Ben zyloxycar bon yl)am in o Bu tan oate (5e).
NaOH (419 mg, 10.5 mmol) in water (5 mL) was added to a
solution of 4e (2.65 g, 10.5 mmol) in ethanol (30 mL). The
mixture was stirred at room temperature for 24 h and
evaporated to dryness. Dry residue (1.04 g, 3.5 mmol) was
dissolved in 10 mL of DMF and allyl bromide (746 µL, 5.25
mmol) was added. After the reaction mixture was stirred in
the dark for 48 h at room temperature, it was extracted by
EtOAc and water. The organic phase was dried over anhyd
Na2SO4 and evaporated under reduced pressure. The product
(2.95 g, 96%) was obtained from silica gel chromatography,
using solvent gradient (100% hexane to 60% hexane in EtOAc).
1H NMR (CDCl3): 1.71-1.81 (m, 1H), 2.08-2.22 (m, 1H), 3.13
(t, 1H, J ) 6.48 Hz), 3.59-3.76 (m, 2H), 4.51-4.58 (m, 1H),
4.62 (d, 2H, J ) 6.8 Hz), 5.10 (s, 2H), 5.22-5.35 (m, 2H), 5.82-
5.93 (m, 1H), 7.33 (m, 5H). 13C NMR (CDCl3): 172.1, 156.6,
136.0, 131.3, 128.4, 128.1, 128.0 118.8, 67.05, 65.98, 58.6, 51.3,
35.2. HRESIMS: calcd for C15H20NO5 [M + H]+ 294.1353,
found m/z 294.1341 [M + H]+.
Ben zyl (2S)-2-(Ben zyloxyca r bon yl)a m in o Bu t a n oa t e
(5f). N-Cbz-L-homoserine benzyl ester was obtained by the
same procedure as described for 5e except benzyl bromide was
1
added instead of allyl bromide. H NMR (CDCl3): 1.67-1.75
(m, 1H), 2.13-2.20 (m, 1H), 2.75 (br s, 1H), 3.60-3.75 (m, 2H),
4.56-4.62 (m, 1H), 5.12 (d, 2H, J ) 10.4 Hz), 5.18 (s, 2H),
5.67 (d, 1H, J ) 7.5 Hz), 7.32-7.38 (m, 10H). HRESIMS: calcd
for C19H21NO5Na [M + Na]+ 366.1315, found m/z 366.1317 [M
+ Na]+.
Allyl (2S)-2-N-(Ben zyloxycar bon yl)am in o-3-for m yl P r o-
p ion a te (6e). Dess-Martin periodinane (191.2 mg, 0.45 mmol)
was added to a solution of 5e (120 mg, 0.41 mmol) in CH2Cl2
solution. The reaction mixture was stirred for 2 h at room
temperature and evaporated to dryness. The residue was
suspended in diethyl ether (20 mL), extracted with aqueous
NaHCO3/Na2S2O3 solution (100 mL of saturated NaHCO3
containing 25 g of Na2S2O3) and saturated brine, and dried
over anhyd Na2SO4. The organic layer was evaporated and
passed through a silica gel column to obtain 6e with 86% yield.
1H NMR (CDCl3): 2.95 (dd, 1H, J ) 5.1 Hz, 18.5 Hz), 3.04
(dd, 1H, J ) 5.1 Hz, 18.5 Hz), 4.62-4.75 (m, 3H), 5.11 (s, 2H),
5.23-5.27 (m, 2H), 5.82-5.87 (m, 2H), 7.29-7.37 (m, 5H), 9.68
(s, 1H). 13C NMR (CDCl3): 199.0, 170.3, 155.8, 136.0, 131.2,
128.4, 128.1, 128.0, 118.9, 67.0, 66.3, 49.0, 45.6. HRESIMS:
calcd for C15H18NO5Na [M + H]+ 292.1185, found m/z 292.1187
[M + H]+.
2S-2-(Ben zyloxyca r bon yl)a m in o-4-[(ter t-bu tyld im eth -
ylsilyl)oxy]bu ta n -1-ol (10). LiBH4 (180 µL in THF, 0.35
mmol) was added, at 0 °C, to a solution of 9 (120 mg, 0.29
mmol) in THF (2 mL). After being stirred for 1 h at 0 °C and
6 h at room temperature, the reaction was quenched with
saturated aq NaHCO3. The aqueous phase was extracted with
EtOAc and the combined organic phases were washed with
brine, dried over anhyd Na2SO4, and concentrated to dryness
under reduced pressure. The crude product was purified by
silica gel chromatography (33% hexanes in EtOAc) to give
compound 10 in 81% yield. 1H NMR (CDCl3): 0.037 (s, 3H),
0.041 (s, 3H), 0.91 (s, 9H), 1.69-1.95 (m, 2H), 3.40 (br s, 1H),
3.60-3.71 (m, 4H), 3.82-3.91 (m, 1H), 5.12 (s, 2H), 5.65 (s,
1H), 7.29-7.35 (m, 5H). 13C NMR (CDCl3): 156.9, 136.5, 128.5,
128.0, 127.9, 66.7, 65.3, 59.9, 51.8, 33.7, 25.7, 18.1, -5.57,
-5.60. HRESIMS: calcd for
C
18H31NO4NaSi [M + Na]+
376.1920, found m/z 376.1940 [M + Na]+.
Ben zyl (2S)-2-N-(Ben zyloxyca r bon yl)a m in o-3-for m yl
P r op ion a te (6f). Compound 6f was obtained by using the
same method as described for 6e. 1H NMR (CDCl3): 3.02-
3.20 (ddd, 2H), 4.69-4.71 (m, 1H), 5.12 (s, 2H), 5.18 (d, 2H),
2S-2-(Ben zyloxyca r bon yl)a m in o-4-[(ter t-bu tyld im eth -
ylsilyl)oxy]-(1-tetr a h yd r o-2H-p yr a n -2-yloxy)bu ta n ol (13).
A solution of 10 (133 mg, 0.38 mmol) and dihydropyran (51.5
µL, 0.57 mmol) in dry CH2Cl2 (5 mL) containing pyridinium
p-toluenesulfonate (PPTS, 9.5 mg, 0.038 mmol) was stirred for
7 h at room temperature. Then the solution was diluted with
CH2Cl2 and washed with brine to remove the catalyst. After
evaporation of the organic solvent, silica gel chromatography
separation (90% hexanes in EtOAc) gave 165 mg of THP ether
5.68 (d, 1H, J ) 7.6 Hz), 7.31-7.39 (m, 10H), 9.71 (s, 1H). 13
C
NMR (CDCl3): 199.0, 170.5, 155.9, 136.0, 135.0, 128.6, 128.5,
128.3, 128.2, 128.1, 67.7, 67.2, 49.2, 45.8.
Meth yl 5-Acetam ido-4,7,8,9-tetr a-O-acetyl-2,6-an h ydr o-
3,5-d id eoxy-2-C-[L-2-[(ben zyloxyca r bon yl)a m in o]-4-bu ty-
r ola cton e]-D-er yth r o-L-m a n n o-n on on a te (7). Neu5Ac phen-
yl sulfone (2) and 1.2 equiv of electrophile (6e or 6f) were dried
together under high vacuum for 4 h. SmI2 (6 equiv freshly
prepared from Sm and 1,2-diiodoethane, 0.1 M in THF) was
added in one portion at room temperature with vigorous
stirring. After 8 h, the reaction mixture was diluted with ether
and extracted with 1 N HCl, saturated Na2S2O3, NaHCO3, and
brine. The organic layer was dried over anhyd Na2SO4. The
filtrate was concentrated under reduced pressure and purified
on a silica gel column with EtOAc as fluent. The C-glycoside
7 and 8 were obtained as oils in 38% and 5% yield from
1
13 (83%). H NMR (CDCl3): 0.04 (s, 3H), 0.03 (s, 3H), 0.88 (s,
9H), 1.48-1.90 (m, 8H), 3.45-3.55 (m, 2H), 3.68-3.91 (m, 4H),
3.91-3.98 (m, 1H), 4.54-4.58 (m, 1H), 5.09 (s, 2H), 5.30 (d,
0.4H, J ) 7.2 Hz), 5.49 (d, 0.6H, J ) 7.2 Hz), 7.27-7.35 (m,
5H). HRESIMS: calcd for C23H39NO5NaSi [M + Na]+ 460.2495,
found m/z 460.2480 [M + Na]+.
2S-2-(Ben zyloxyca r bon yl)a m in o-1-(tetr a h yd r o-2H-p y-
r a n -2-yloxy)-4-bu ta n ol (14). Tetrabutylammonium fluoride
(180 µL of a 1 M solution in THF, 0.35 mmol) was added to a
solution of 13 (101 mg, 0.23 mmol) in THF (2 mL). The solution
J . Org. Chem, Vol. 68, No. 7, 2003 2671

Wang and Linhardt
1
was stirred at room temperature for 1 h. The reaction media
was then diluted with ether and extracted with water. After
the solution was washed with brine, the organic layer was
dried over anhyd Na2SO4 and purified on a silica gel column
a colorless oil in 95% yield. H NMR (CDCl3): 1.75-2.18 (m,
30H), 2.46-2.52 (m, 1H), 3.44-3.54 (m, 2H), 3.762 (m, 3H),
4.75-4.85 (m, 1H), 5.01-5.85 (m, 6H), 7.30-7.37 (m, 5H).
ESIMS: 862 [M + Na]+. HRESIMS: calcd for C39H54N2O18Na
[M + Na]+ 861.3269, found m/z 861.3262 [M + Na]+.
1
(80% to 50% hexanes in EtOAc) to afford 14 in 94% yield. H
NMR (CDCl3): 1.46-1.88 (m, 8H), 3.20-3.33 (m, 1H), 3.47-
3.55 (m, 2H), 3.62-3.75 (m, 2H), 3.77-3.87 (m, 2H), 3.95-
4.10 (m, 1H), 4.52 (t, 0.7H), 4.58 (t, 0.3H), 5.07-5.16 (m, 2H),
5.24 (d, 0.3H), 5.57 (d, 0.7H), 7.31-7.36 (m, 5H). HRESIMS:
Meth yl 5-Acetam ido-4,7,8,9-tetr a-O-acetyl-2,6-an h ydr o-
3,5-dideoxy-2-C-{4-[4-acetyl-2S-(ben zyloxycar bon yl)am in o-
1-bu ta n ol]}-D-er yth r o-L-m a n n o-n on on a te (18). A solution
of THP ether 17 (144 mg, 0.17 mmol) and PPTS (4.3 mg, 0.017
mmol) in ethanol (4 mL) was stirred at 55 °C (bath temper-
ature) for 5 h. The solvent was removed by evaporation in
vacuo, and the residue was purified by chromatography on a
silica gel column (100% to 95 vol % CH2Cl2 in methanol) to
calcd for
C
17H25NO5Na [M + Na]+ 346.1630, found m/z
346.1630 [M + Na]+.
1-For m yl 3S-(Ben zyloxycar bon yl)am in o-4-(tetr ah ydr o-
2H-p yr a n -2-yloxy)bu ta n ol (15). Compound 14 (370 mg, 1.15
mmol) was added to a solution of the Dess-Martin reagent
(583 mg, 1.37 mmol) in CH2Cl2 (5 mL) at 0 °C. The reaction
mixture was stirred for 30 min at 0 °C, warmed to room
temperature for 6 h, diluted with diethyl ether (15 mL), poured
into sodium thiosulfate in saturated aq NaHCO3 (20 mL), and
stirred for 10 min. The organic layer was washed sequentially
with saturated aqueous NaHCO3, brine, and deionized water,
dried with anhyd Na2SO4, filtered, and concentrated under
reduced pressure. The crude product was purified by silica gel
chromatography (90% to 40% hexanes in EtOAc) to give 15 in
82% yield. 1H NMR (CDCl3): 1.43-1.72 (m, 6H), 2.68 (d, 2H),
3.40-3.45 (m, 2H), 3.72-3.78 (m, 2H), 4.15-4.32 (m, 1H), 4.44
(t, 0.6H), 4.49 (t, 0.4H), 5.03 (s, 2H), 5.24 (d, 0.4H), 5.45 (d,
0.6H), 7.31-7.39 (m, 5H), 9.77 (s, 1H). 13C NMR (CDCl3):
200.5, 200.4, 155.7, 136.3, 136.2, 128.48, 128.45, 128.15,
128.09, 128.02, 99.8, 98.9, 69.2, 68.6, 66.8, 66.7, 63.0, 62.3, 30.4,
30.2, 25.2, 25.1, 19.7, 19.2. HRESIMS: calcd for C17H23NO5-
Na [M + Na]+ 344.1474, found m/z 344.1476 [M + Na]+. Anal.
Calcd for C17H23NO5: C, 63.54; H, 7.21; N, 4.30. Found: C,
63.25; H, 7.56; N, 4.28.
1
afford 17 (102 mg, 92%) with a diastereomer ratio of 6:4. H
NMR (CDCl3): 1.88-2.19 (m), 2.43-2.52 (m, 1H), 2.72 (br s,
0.4 H), 2.89 (br s, 0.6H), 3.65-3.81 (m, 6H), 3.95-4.08 (m, 3H),
4.39 (d, 0.4H), 4.47 (d, 0.6H), 4.79-4.86 (m, 1H), 5.09-5.10
(d, 2H), 5.19-5.35 (m, 5H), 7.30-7.37 (m, 5H). ESIMS: 755
[M + H]+, 777 [M + Na]+, 793 [M + K]+. HRESIMS: calcd for
C34H46N2O17Na [M + Na]+ 777.2694, found m/z 777.2704 [M
+ Na]+.
4-O-Acetyl-4-[2-C-(1-m eth yl 5-a ceta m id o 4,7,8,9-tetr a -
O-acetyl-2,6-an h ydr o-3,5-dideoxy-D-er yth r o-L-ma n n o-n on -
on a te)]-2S-(ben zyloxyca r bon yl)a m in o-1-ca r boxylic Acid
(1). Sodium periodate (31 mg) and ruthenium(III) chloride
hydrate (0.38 mg, 4 mol %) were added to a solution of alcohol
17 (27.5 mg, 0.04 mmol) in CCl4 (250 µL), acetonitrile (250
µL), and water (300 µL) at 0 °C. The reaction mixture was
stirred at 0 °C and changed color to yellow within 10 min. TLC
indicated that there was some starting material left. Another
small portion of RuCl3 (0.3 mg) was added to the solution.
Reaction was completed within another 0.5 h. The reaction
mixture was passed through a pad of Celite and washed with
EtOAc. The combined organic layer was evaporated and the
residue was purified on a silica gel column (100% to 95 vol %
CH2Cl2 in methanol) to afford 1 (13 mg, 47%). Lactone
compound 7 (7 mg) was also isolated from the residue. 1H NMR
(CDCl3): 1.88-2.09 (m), 2.24-2.36 (m, 1H), 2.44-2.52 (m, 1H),
3.74 (s, 1.5H), 3.78 (s, 1.5H), 3.86-3.96 (m), 4.04-4.14 (m),
4.33-4.39 (m), 4.78-4.88 (m), 5.03-5.16 (m), 5.27-5.35 (m),
7.30-7.37 (m, 5H). ESIMS: 769 [M + H]+, 791 [M + Na]+.
HRESIMS: calcd for C34H44N2O18Na [M + Na]+ 791.2487,
found m/z 791.2490 [M + Na]+.
Meth yl 5-Acetam ido-4,7,8,9-tetr a-O-acetyl-2,6-an h ydr o-
3,5-d id eoxy-2-C-{1-[3S-(ben zyloxy ca r bon yl)a m in o-4-(tet-
r a h yd r o-2H-p yr a n -2-yloxy)-1-bu ta n ol]}-D-er yth r o-L-m a n -
n o-n on on a te (16). C-Glycoside 16 was obtained as an oil in
45% yield from the reaction of 15 and 2, using the general
C-glycosylation procedure described for 7. 1H NMR (CDCl3):
1.75-2.18 (m), 2.40-2.46 (m, 1H), 3.78 (s, 3H), 4.78-4.84 (m,
1H), 5.11 (brs, 2H), 5.34-5.44 (m, 2H), 5.59 (m, 1H), 7.31-
7.40 (m, 5H). HRESIMS: calcd for C37H53N2O17 [M + H]+
797.3344, found m/z 797.3348 [M + H]+.
Meth yl 5-Acetam ido-4,7,8,9-tetr a-O-acetyl-2,6-an h ydr o-
3,5-dideoxy-2-C-{1-[1-acetyl-3S-(ben zyloxycar bon yl)am in o-
4-(tetr a h yd r o-2H-p yr a n -2-yloxy)-1-bu ta n a te]}-D-er yth r o-
L-m a n n o-n on on a te (17). A solution of 16 (100 mg, 0.13
mmol) in anhyd pyridine (2 mL) was reacted with acetic
anhydride (0.5 mL) under nitrogen. After being stirred over-
night at room temperature, the reaction mixture was quenched
with methanol and evaporated under vacuum. The residue was
dried by coevaporation with toluene and purified by chroma-
tography on silica gel (50% hexanes in EtOAc) to afford 17 as
Ack n ow led gm en t. The authors would like to thank
Dr. Nathalie Karst for helpful discussion.
Su p p or tin g In for m a tion Ava ila ble: Spectral data for all
new compounds (1H NMR, 13C NMR) and procedures for the
synthesis of 5b, 6a , 6b, and 6c. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O026776V
2672 J . Org. Chem., Vol. 68, No. 7, 2003