Studies toward lipid A: Synthesis of differentially protected disaccharide fragments

Article abstract of DOI:10.1021/jo961668s

Disaccharides containing two glucosamine units are readily synthesized where the two amine functionalities are orthogonally protected as the tetrachlorophthaloyl and pent-4-enoyl moieties. The 1→6 linked disaccharide has the potential to be developed toward a series of biologically interesting lipid A analogs.

Full text of DOI:10.1021/jo961668s

3654
J . Org. Chem. 1997, 62, 3654-3658
Stu d ies tow a r d Lip id A: Syn th esis of Differ en tia lly P r otected
Disa cch a r id e F r a gm en ts†
Sian L. Griffiths,‡ Robert Madsen,‡ and Bert Fraser-Reid*,‡
Paul M. Gross Chemical Laboratory, Department of Chemistry, Duke University,
Durham, North Carolina 27708, and Natural Products and Glycotechnology Research Institute, Inc.
(NPG), Durham, North Carolina 27707
Received August 29, 1996 (Revised Manuscript Received March 6, 1997X)
Disaccharides containing two glucosamine units are readily synthesized where the two amine
functionalities are orthogonally protected as the tetrachlorophthaloyl and pent-4-enoyl moieties.
The 1f6 linked disaccharide has the potential to be developed toward a series of biologically
interesting lipid A analogs.
Ba ck gr ou n d
acid chains present on the molecule may strongly influ-
ence its endotoxic activity.6
We have recently highlighted the use of both pent-4-
enoyl and tetrachlorophthaloyl (TCP) as protecting groups
for amine functionalities.1 In particular when used on
2-deoxy-2-amino glucosamine derivatives, the TCP group
has several attractive features as compared to those of
other commonly used protecting groups, notably the
relatively mild conditions that can be used for its
removal.1a,c,d,2 In this paper we report the further
exploration of pent-4-enoyl-protected glucosamine deriva-
tives and the subsequent generation of disaccharides
containing two glucosamine units where the amine
functionalities are orthogonally protected as the pent-4-
enoyl and TCP moieties.
Recently a lipid A molecule was isolated from Rhod-
bacter sphaeroides which was nontoxic but was a potent
antagonist of LPS-induced responses.7 Christ and co-
workers8,9 synthesized the proposed structure of this lipid
A molecule and thereby disproved the suggested struc-
ture; however, the synthetic material, also a potent
antagonist, was used as the basis for their recently
described lipid A analog E5531 (2).10 To confer stability
onto the molecule the 3 and 3′ acyl linkages were replaced
with ether linkages and a methyl group was added to
the free primary alcohol. E5531 was shown to inhibit
LPS-induced processes in vitro. Additionally, in vivo
studies showed that mice could be protected from a lethal
injection of E. coli when E5531 was administered in
conjunction with a â-lactam antibiotic, and now E5531
is in phase II clinical trials as a potential drug candidate.
The general structure of the lipid A molecule, which
consists of two glucosamine units, requires that the
amino groups be differentiated, and here was seen a
potential application of our amine protecting group
methodology. It was envisaged that an intermediate
such as A could be generated that would allow for each
of the four sites (R1-R4) occupied by a fatty acid chain
to be independently manipulated, thereby giving access
to a series of lipid A analogs from a common intermediate
(Figure 1).
In tr od u ction
Endotoxins are complex lipopolysaccharides (LPS)
situated in the outer membrane of the cell wall of Gram-
negative bacteria.3 They are essential for cell survival,
but during an infection they can be liberated from the
cell wall and through a pathway involving reaction with
the lipopolysaccharide binding protein can cause the
release of agents such as the interleukins. High levels
of LPS therefore cause a huge immune response leading
to such pathological effects as fever, shock, and in
extreme cases organ failure and death.4 Indeed, the
mortality rate of bacterial sepsis (systemic inflammatory
response syndrome) is as high as 60%. The terminal
disaccharide phospholipid of the LPS, known as lipid A
(1 from Escherichia coli) has been shown to be responsible
for its biological activity.3,5 Furthermore it has been
shown that the number and the structure of the fatty
Resu lts a n d Discu ssion
Early attempts to synthesize these disaccharides using
N-pent-4-enoylated glycosyl chlorides as donor molecules
were unsuccesful, in keeping with literature precedent
that these 2-N-acyl-protected compounds are unreactive
due to the formation of the intermediate oxazolines.11 Our
† Supported by a grant from PHS AI 31862 (to B.F.-R.).
‡ Present addresses: S.L.G., Pharmacopeia Inc., Princeton, NJ
08540. R.M., Department of Organic Chemistry, Technical University
of Denmark, Lyngby, Denmark. B.F.-R., Natural Products and Gly-
cotechnology (NPG) Research Institute Inc., 4118 Swarthmore Road,
Durham, NC 27707.
(5) Rietschel, E. T.; Brade, H. Sci. Am. 1992, 267, 4904.
(6) Za¨hringer, U.; Lindner, B.; Rietschel, E. T. Adv. Carbohydr.
Chem. Biochem. 1994, 50, 211.
(7) (a) Qureshi, N.; Honovich, J . P.; Hara, H.; Cotter, R. J .;
Takayama, K. J . Biol. Chem. 1988, 263. (b) Qureshi, N.; Takayama,
K.; Kurtz, R. Infect. Immun. 1991, 59, 441.
(8) Christ, W. J .; McGuinness, P. D.; Asano, O.; Wang, Y.; Mullarkey,
M. A.; Perez, M.; Hawkins, L. D.; Blythe, T. A.; Dubuc, G. R.; Robidoux,
A. L. J . Am. Chem. Soc. 1994, 116, 3637.
X Abstract published in Advance ACS Abstracts, April 15, 1997.
(1) (a) Debenham, J . S.; Madsen, R.; Roberts, C.; Fraser-Reid, B. J .
Am. Chem. Soc. 1995, 117, 3302. (b) Madsen, R.; Roberts, C.; Fraser-
Reid, B. J . Org. Chem. 1995, 60, 7920. (c) Debenham, J . S.; Fraser-
Reid, B. J . Org. Chem. 1996, 61, 432. (d) Debenham, J . S.; Debenham,
S. D.; Fraser-Reid, B. Bioorg. Med. Chem., in press.
(2) Stangier, P.; Hindsgaul, O. Synlett 1996, 179. Castro-Palomino,
J . C.; Schmidt, R. R. Tetrahedron Lett. 1995, 36, 5343. See also N,N-
diacetyl protection which requires more basic conditions (NaOMe/
MeOH) for removal: Castro-Palomino, J . C.; Schmidt, R. R. Tetrahe-
dron Lett. 1995, 36, 6871.
(3) Raetz, C. H. R. Annu. Rev. Biochem. 1990, 59, 129. Raetz, C. H.
R. J . Bacteriol. 1993, 175, 5745.
(9) For other synthetic work on lipid A molecules see: Kusumoto,
S. In Bacterial Endotoxic Lipopolysaccharides; Morrison, D. C., Ryan,
J . L., Eds.; CRC: Boca Raton, FL, 1992; p 81.
(10) Christ, W. J .; Asano, O.; Robidoux, A. L. C.; Perez, M.; Wang,
Y.; Dubuc, G. R.; Gavin, W. E.; Hawkins, L. D.; McGuinness, P. D.;
Mullarkey, M. A.; Lewis, M. D.; Kishi, Y.; Kawata, T.; Bristol, J . R.;
Rose, J . R.; Rossignol, D. P.; Kobayashi, S.; Hishinuma, I.; Kimura,
A.; Asakawa, N.; Katayama, K.; Yamatsu, I. Science 1995, 268, 80.
(4) Parillo, J . E. N. Engl. J . Med. 1993, 328, 1471.
S0022-3263(96)01668-4 CCC: $14.00 © 1997 American Chemical Society

Lipid A: Snthesis of Disaccharide Fragments
J . Org. Chem., Vol. 62, No. 11, 1997 3655
F igu r e 1.
Sch em e 1a
F igu r e 2.
a
Reagents: (i) NaOMe/MeOH; (ii) BzCl, py, -40 °C, 81% from
3; (iii) BnBr, NaH, n-Bu4NI DMF, 80%; (iv) 0.04 M NaOMe, 0
°C, 7 h, 75%.
retrosynthesis therefore led to coupling partners B and
C, where the TCP group was situated on the donor
molecule B, since we have previously shown that TCP-
protected derivatives acted as excellent donor mole-
cules.1a,c,d The use of a pentenyl coupling procedure
involving B is incompatible with the N-pent-4-enoyl
group on C, so instead a Koenigs-Knorr coupling reac-
tion would be used to form the key glycosidic bond (Figure
2). The acceptor molecule 6 was synthesized in three
steps from the previously reported pentenyl glycoside 3.1b
Hydrolysis of the acetate groups and subsequent treat-
ment with benzoyl chloride at low temperature12 gave the
selectively protected 3,6-derivative 4 which could be
crystallized in 81% yield. Benzylation of this material
under acidic conditions using benzyl trichloroacetimidate
following literature procedures12a,13 was attempted, but
the reaction was sluggish and only produced a poor yield
of 4-benzyl ether 5. Instead 4 was benzylated directly
to afford 5 in 80% yield employing sodium hydride and
excess benzyl bromide. Selective removal of the C-6
benzoate was accomplished following Shiba’s procedure12a
to afford coupling partner 6 in 75% yield (Scheme 1).
The TCP-protected tetraacetate derivate 7 was syn-
thesized as previously reported.1d Treatment with HBr
in acetic acid and acetic anhydride overnight afforded the
glycosyl bromide 8 which was used directly in the
coupling reaction. The most efficient promoter for the
glycosidic bond formation was found to be silver triflate,14
which when used in conjunction with activated powdered
4 Å molecular sieves in dichloromethane afforded a
pleasing 76% yield of the disaccharide 9 (Scheme 2). The
trans linkage was ensured by the neighboring group
participation of the TCP group during the coupling
reaction.
With the desired disaccharide 9 in hand attention then
turned to chemoselective deprotection of the the two
amide protecting groups. The N-pent-4-enoyl group was
easily removed from 9 by treatment with iodine (3 equiv)
in a tetrahydrofuran/water mixture1a,b in approximately
8 min, affording the free amine 10 in 71% yield. Longer
exposure to the reaction conditions led to the isolation
of products where the iodine had additionally attacked
the O-pentenyl double bond. The use of other solvent
mixtures for the deprotection such as acetonitrile/water,
which had been shown to give similar yields but with
increased reaction times,1b led to mixtures of products.
The selective removal of the TCP group from 9 proved
to be somewhat more challenging, not however due to
(11) For a review on the synthesis of oligosaccharides of 2-deoxy-
2-amino sugars see: Banoub, J .; Boullanger, P.; Lafont, D. Chem. Rev.
1992, 92, 1167.
(12) (a) Imoto, M.; Yoshimura, H.; Yamamoto, M.; Shimamoto, T.;
Sakaguchi, N.; Kusumoto, S.; Shiba, T. Bull. Chem. Soc. J pn. 1987,
60, 2197. (b) El-Sokkary, R. I.; Silwanis, B. A.; Nashed, M. A.; Paulsen,
H. Carbohydr. Res. 1990, 203, 319.
(13) Wessel, H-P.; Iversen, T.; Bundle, D. R. J . Chem Soc., Perkin
Trans. 1 1985, 2247.
(14) Hanessian, S.; Banoub, J . Carbohydr. Res. 1977, 53, C13.

3656 J . Org. Chem., Vol. 62, No. 11, 1997
Griffiths et al.
Sch em e 2a
Sch em e 4a
a
Reagents: (i) 4, CH2Cl2, AgOTf, -20 °C to rt, 4 Å molecular
sieves, 58%.
accessible. Work toward this objective is currently in
progress and will be reported in due course.
Exp er im en ta l Section
Gen er a l P r oced u r es. All reactions were conducted under
a dry argon atmosphere. THF was distilled from sodium
benzophenone ketyl. Dichloromethane and acetonitrile were
distilled from calcium hydride. Absolute ethanol was stored
over 4 Å molecular sieves. Solutions of compounds in organic
solvents were dried over sodium sulfate. TLC plates were
Kieselgel 60 F254 (Merck Art. 5554). Flash column chroma-
tography was done with silica gel (230-400 mesh, Merck). FAB
mass spectra were recorded using a m-nitrobenzyl alcohol
matrix with xenon as the fast atom. Accurate mass measure-
ments were made using FAB at 10k resolution. Elemental
analyses were conducted by Atlantic Microlab, Inc., P.O. Box
2288, Norcross, GA 30091.
a
Reagents: (i) HBr/Ac2O; (ii) 6, CH2Cl2, AgOTf, -20 °C to rt,
4 Å molecular sieves, 76%.
Sch em e 3a
P en t-4-en yl 3,6-Di-O-ben zoyl-2-d eoxy-2-(p en t-4-en oyl-
a m in o)-â-D-glu cop yr a n osid e (4). To a solution of pent-4-
enyl 3,4,6-tri-O-acetyl-2-deoxy-2-(pent-4-enoylamino)-â-D-glu-
copyranoside (3) (4.5 g, 9.88 mmol) in CH2Cl2 (4 mL) was added
0.04 M NaOMe in MeOH (50 mL), and the mixture was stirred
at rt for 1 h. The reaction was quenched with Amberlite IR-
120(H+), and the solution was filtered through Celite. The
filter cake was further washed with MeOH (15 mL), and the
combined filtrate was concentrated to give a crystalline residue
which was dried under high vacuum overnight. To this
residue dissolved in pyridine (40 mL) at -40 °C was added
benzoyl chloride (2.5 mL, 21.5 mmol), and the reaction mixture
was stirred at this temperature for 2 h. The mixture was
diluted with CHCl3 (150 mL) and washed successively with
water (150 mL), 5% aqueous HCl (2 × 150 mL), and saturated
aqueous NaHCO3 (150 mL) solutions. The organic solutions
were dried (Na2SO4) and concentrated to give a foam which
was crystallized from Et2O to afford 4 (4.28 g, 81%): mp 139-
a
Reagents: (i) I2, THF/H2O, 8 min; (ii) MeCN/THF/EtOH (2:
1:1), ethylenediamine, 60 °C.
removal of the pent-4-enoyl group but because of con-
comitant removal of the C-6 acetate group. Under a
variety of conditions the best yield that could be obtained
for the free amine 11 was 43% along with about 30% of
the disaccharide where both the TCP and the C-6 acetate
group had been removed. However when the corre-
sponding tetrabenzoate derivative 1215 was exposed to
the deprotection conditions, a much more acceptable yield
(64%) of the free amine 13 was obtained (Scheme 3).
140 °C; [R]20 +9.7 (c 1, CHCl3); 1H NMR δ 8.10-8.04 (m, 4H,
D
Ph), 7.62-7.55 (m, 2H, Ph), 7.48-7.40 (m, 4H, Ph), 5.85 (d, J
) 9.2 Hz, NH), 5.77 (m, -CHd), 5.62 (m, -CHd), 5.46 (dd, J
) 8.6, 10.5 Hz, 1H), 5.02-4.84 (m, 3H), 4.77-4.61 (m, 4H),
4.11 (m, 1H), 3.93-3.78 (m, 3H), 3.52 (m, 1H), 3.34 (d, J ) 4.6
Hz, OH), 2.25-2.05 (m, 6H), 1.74-1.65 (m, 2H); 13C NMR
172.6, 167.5, 167.1, 138.0, 136.7, 133.6, 133.3, 130.0, 129.8,
129.7, 129.2, 128.5, 128.4, 115.4, 114.9, 101.0, 76.2, 74.1, 69.7,
69.0, 64.0, 54.3, 36.0, 30.0, 29.4, 28.7. Anal. Calcd for
C30H35NO8: C, 67.02; H, 6.55; N, 2.61. Found: C, 67.18; H,
6.60; N, 2.57.
Finally we briefly explored the synthesis of the corre-
sponding 1f4 linked disaccharide where the previously
synthesized 4 would be used as our acceptor molecule.
Although 4 is both sterically and electronically unreac-
tive, under the same conditions that were used before,
the 1f4 linked ketobioside 14 was obtained in an
acceptable 58% yield (Scheme 4).
P en t -4-en yl 3,5-Di-O-b en zoyl-4-O-b en zyl-2-d eoxy-2-
(p en t-4-en yla m in o)-â-D-glu cop yr a n osid e (5). To an ice-
cooled solution of 4 (2.5 g, 4.65 mmol), Bu4NI (0.3 g, 0.81
mmol), and BnBr (4 mL, 33.6 mmol, 7 equiv) in DMF (20 mL)
was added NaH (0.25 g of a 60% oil dispersion, 6.25 mmol).
The ice bath was removed, and the mixture was stirred at rt
for 20 min. The reaction was quenched by the addition of
AcOH, and the mixture was diluted with CH2Cl2 (70 mL) and
washed with water (70 mL). The organic solution was dried,
concentrated, then taken up in Et2O, and filtered to remove
crystalline impurities. The filtrate was concentrated and
purified by column chromatography (95:5 CH2Cl2/EtOAc) to
In conclusion, we have reported the synthesis of
disaccharides containing two glucosamine units where,
given suitable protection of the hydroxy groups, the two
amine functionalities are orthogonally protected. The
1f6 linked derivative 9 has the potential to be developed
toward a key intermediate for the synthesis of a large
number of biologically interesting lipid A analogs, in
which each of the four fatty acid containing positions in
the naturally occuring molecules would be individually
afford 5 (2.33 g, 80%): Rf ) 0.52; mp 148-149 °C; [R]20 +4.9
D
(c 1, CHCl3); 1H NMR δ 8.10-8.04 (m, 4H, Ph), 7.64-7.57 (m,
2H, Ph), 7.52-7.44 (m, 4H, Ph), 7.16-7.06 (m, 5H, Ph), 5.84-
5.55 (m, 3H), 5.48 (dd, J ) 8.7, 10.5 Hz, 1H), 5.01-4.85 (m,
3H), 4.75 (d, J ) 10.2 Hz, 1H), 4.63-4.50 (m, 5H), 4.28 (m,
(15) Prepared from disaccharide 9 in two steps.

Lipid A: Snthesis of Disaccharide Fragments
J . Org. Chem., Vol. 62, No. 11, 1997 3657
1H), 3.94-3.76 (m, 3H), 3.47 (m, 1H), 2.24-2.03 (m, 6H), 1.71-
1.60 (m, 2H); 13C NMR 172.3, 167.0, 166.3, 138.1, 136.9, 133.7,
133.2, 130.0, 129.8, 129.8, 129.3, 128.7, 128.6, 128.5, 128.4,
128.3, 128.2, 128.0, 115.4, 114.9, 101.5, 75.9, 75.0, 73.1, 68.9,
63.3, 53.8, 36.0, 30.1, 29.4, 28.7. Anal. Calcd for C37H41NO8:
C, 70.80; H, 6.58; N, 2.23. Found: C, 70.93; H, 6.65; N, 2.27.
and the residue was taken up in CHCl3 and washed with brine
(10 mL). The organic layer was dried and concentrated, and
purification by column chromatography (99:1 CH2Cl2/MeOH)
afforded 10 (0.0328 g, 71%) as a white foam: Rf ) 0.46 (97:3
CH2Cl2/MeOH); [R]20 ) +1.0 (c 1.19, CHCl3); 1H NMR δ 8.02
D
(d, J ) 7.4 Hz, 2H), 7.58 (t, J ) 7.4 Hz, 1H), 7.47 (approximate
t, J ) 7.4, 7.8 Hz, 2H), 7.04 (m, 3H), 6.78 (2 overlapping
doublets, J ) 7.9, 7.3, 2H), 5.80-5.68 (m, 2H), 4.42-4.05 (m,
7H), 3.87-3.78 (m, 3H), 3.58-3.49 (m, 3H), 2.92 (m, 1H), 2.14-
2.00 (m, 2H), 2.09 (s, 3H), 2.03 (s, 3H), 1.88 (s, 3H), 1.67 (m,
2H); 13C NMR 170.7, 170.6, 169.3, 166.0, 140.8, 137.9, 136.6,
133.2, 129.7, 129.5, 128.6, 128.1, 127.9, 127.5, 127.5, 126.7,
115.0, 103.9, 97.4, 77.4, 76.6, 74.6, 74.3, 71.8, 70.8, 69.3, 68.5,
68.1, 56.5, 55.2, 30.2, 28.7, 20.7, 20.6, 20.5; HRMS (FAB) calcd
for C45H47Cl4N2O15 [(MH)+] 995.1731, found 995.1712.
P en t -4-en yl 3-O-Ben zoyl-4-O-b en zyl-2-d eoxy-2-(p en t -
4-en oyla m in o)-â-D-glu cop yr a n osid e (6). A solution of 5
(1.1 g, 1.75 mmol) in CH2Cl2 (2 mL) was treated with 0.04 M
methanolic NaOMe (20 mL) at 0 °C for 7 h, and then the
reaction was quenched with Amberlite IR-120(H+). The reac-
tion mixture was filtered through Celite, and the filter cake
was rinsed with a further amount of MeOH (20 mL). The
combined organic solution was concentrated to give a crystal-
line residue which was taken up in hot petroleum ether, cooled,
P en t -4-en yl (3,4,6-Tr i-O-a cet yl-2-d eoxy-2-a m in o-â-D-
glu cop yr a n osyl)-(1f6)-3-O-ben zoyl-4-O-ben zyl-2-d eoxy-
2-(p en t-4-en oyla m in o)-â-D-glu cop yr a n osid e (11). To a
solution of 9 (0.05 g, 0.0463 mmol) in 2:1:1 MeCN/EtOH/THF
(0.5 mL) was added ethylenediamine (6 µL, 0.09 mmol), and
the solution was heated at 60 °C for 2 h. The reaction mixture
was allowed to cool and then concentrated. The residue was
purified by column chromatography (98:2 CH2Cl2/MeOH) to
and then filtered to afford 6 (0.69 g, 75%): mp 150-151 °C;
1
[R]20 -39.4 (c 1, CHCl3); H NMR δ 8.05-8.00 (m, 2H, Ph),
D
7.63-7.56 (m, 1H, Ph), 7.48-7.42 (m, 2H, Ph), 7.19-7.10 (m,
5H, Ph), 5.85-5.71 (m, 2H), 5.61 (m, 1H), 5.46 (dd, J ) 9.2,
10.7 Hz, 1H), 5.04-4.83 (m, 3H), 4.73 (d, J ) 10.1 Hz, 1H),
4.61 (s, 2H), 4.51 (d, J ) 8.3 Hz, 1H), 4.21 (m, 1H), 3.93-3.73
(m, 4H), 3.55-3.42 (m, 2H), 2.23-1.99 (m, 7H), 1.73-1.62 (m,
2H); 13C NMR 172.4, 167.1, 138.0, 137.4, 136.8, 133.6, 129.9,
129.4, 128.6, 128.4, 128.3, 128.0, 115.1, 115.0, 101.5, 76.1, 75.7,
75.0, 74.9, 69.2, 61.6, 53.9, 35.9, 30.0, 29.3, 28.7. Anal. Calcd
for C30H37NO7: C, 68.81; H, 7.12; N, 2.67. Found: C, 68.91;
H, 7.17; N, 2.62.
afford 11 (0.016 g, 43%) as a clear film: Rf ) 0.41 (95:5 CH2Cl2/
1
MeOH); [R]20 ) -15.9 (c 1, CHCl3); H NMR δ 8.02 (dd, J )
D
1.0, 7.4 Hz, 2H), 7.58 (t, J ) 7.4 Hz, 1H), 7.47 (t, J ) 7.6 Hz,
1H), 7.19 (m, 3H), 7.12 (m, 2H), 5.78 (m, 1H), 5.62 (m, 2H),
5.44 (m, 1H), 5.04-4.84 (m, 5H), 4.74 (d, J ) 9.8 Hz, 1H), 4.58
(ABq, 2H), 4.50 (d, J ) 8.1 Hz, 1H), 4.28 (m, 2H), 4.20-4.07
(m, 3H), 3.87 (m, 1H), 3.75-3.70 (m, 3H), 3.63 (m, 1H), 3.46
(m, 1H), 2.96 (dd, J ) 8.4, 9.3 Hz, 1H), 2.19-2.10 (m, 6H),
P en t -4-en yl (3,4,6-Tr i-O-a cet yl-2-d eoxy-2-t et r a ch lo-
r op h th a lim id o-â-D-glu cop yr a n osyl)-(1f6)-3-O-ben zoyl-4-
O-ben zyl-2-d eoxy-2-(p en t-4-en oyla m in o)-â-D-glu cop yr a -
n osid e (9). To 7 (1 g, 1.63 mmol) in acetic anhydride (0.4
mL, 4.24 mmol) was added HBr (45% w/v solution in acetic
acid, 1.5 mL, 8.44 mmol). The reaction mixture was stirred
at rt for 24 h while being protected from light. After being
diluted with CHCl3 (20 mL), the reaction mixture was poured
onto ice/water (20 mL). The organic layer was separated,
washed with H2O (2 × 30 mL) and a saturated aqueous
NaHCO3 solution (30 mL), dried, and concentrated to afford
anomeric bromide 8 as a syrup. To this material, 6 (0.573 g,
1.08 mmol) (dried by azeotroping both together from toluene),
and powdered 4 Å molecular sieves (0.5 g) in CH2Cl2 (5 mL)
at -25 °C was added AgOTf (0.515 g, 2.00 mmol). The reaction
mixture was removed from the cold bath, protected from light,
and stirred at rt for 24 h. After the reaction was quenched
with a saturated aqueous NaHCO3 solution and brine (5 mL
total), the mixture was filtered through Celite. The filter cake
was washed with additional CH2Cl2 (20 mL), and the combined
filtrate was washed with a saturated aqueous NaHCO3 solu-
tion (30 mL), dried, and concentrated. Purification by column
chromatography (10:1 CH2Cl2/EtOAc) afforded 9 (0.883 g,
2.08 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H), 1.69-1.58 (m, 2H); 13
C
NMR 172.1, 170.8, 170.7, 169.8, 166.6, 138.0, 137.1, 136.7,
133.5, 129.8, 129.2, 128.5, 128.4, 128.0, 128.0, 115.3, 114.9,
101.3, 77.1, 75.4, 74.7, 74.5, 71.9, 69.0, 69.0, 68.7, 62.0, 55.6,
53.9, 35.8, 30.0, 29.2, 28.6, 20.9, 20.8, 20.7; HRMS (FAB) calcd
for C42H55N2O14 [(MH)+] 811.3653, found 811.3661.
P en t-4-en yl (3,4,6-Tr i-O-ben zoyl-2-d eoxy-2-a m in o-â-D-
glu cop yr a n osyl)-(1f6)-3-O-ben zoyl-4-O-ben zyl-2-d eoxy-
2-(p en t-4-en oyla m in o)-â-D-glu cop yr a n osid e (13). To pent-
4-enyl (3,4,6-tri-O-benzoyl-2-deoxy-2-tetrachlorophthalimido-
â-D-glucopyranosyl)-(1f6)-3-O-benzoyl-4-O-benzyl-2-deoxy-2-
(pent-4-enoylamino)-â-D-glucopyranoside (12)15 (24.6 mg, 0.019
mmol) in 2:1:1 MeCN/EtOH/THF (0.4 mL) was added ethyl-
enediamine (5.9 µL, 0.088 mmol), and the solution was heated
at 60 °C for 2 h. The reaction mixture was allowed to cool
and then concentrated. The residue was purified by column
chromatography (99:1 CH2Cl2/MeOH) to afford 13 (12.8 mg,
66%) as a clear film: Rf ) 0.41 (95:5 CH2Cl2/MeOH); [R]20
)
D
-20.6 (c 1.28, CHCl3); 1H NMR δ 8.03-7.86 (m, 8H), 7.61-
7.28 (m, 12H), 7.21-7.10 (m, 5H), 5.76 (m, 1H), 5.66-5.42 (m,
4H), 4.97-4.84 (m, 3H), 4.74 (d, J ) 10.2 Hz, 1H), 4.62-4.42
(m, 6H), 4.19 (m, 2H), 4.01 (m, 1H), 3.92-3.82 (3H, m), 3.82-
3.72 (m, 2H), 3.75 (m, 2H), 3.45 (m, 1H), 3.21 (m, 1H), 2.19-
2.01 (m, 6H), 1.70-1.61 (m, 2H); 13C NMR 172.1, 166.6, 166.3,
166.1, 165.4, 138.1, 137.1, 136.7, 133.5, 133.4, 133.1, 129.8,
129.7, 129.2, 129.0, 128.8, 128.5, 128.4, 128.4, 128.4, 128.1,
128.0, 115.3, 114.5, 101.3, 77.2, 76.8, 75.4, 74.7, 74.3, 72.1, 69.7,
69.1, 69.1, 63.2, 56.2, 53.8, 35.8, 30.0, 29.2, 28.7; HRMS (FAB)
calcd for C57H61N2O14 [(MH)+] 997.4123, found 997.4119.
P en t -4-en yl (3,4,6-Tr i-O-a cet yl-2-d eoxy-2-t et r a ch lo-
r op h t h a lim id o-â-D-glu cop yr a n osyl)-(1f4)-3,6-d i-O-b en -
zoyl-2-d e oxy-2-(p e n t -4-e n oyla m in o)-â-D-glu cop yr a n o-
sid e (14). Glycosyl bromide 8 was prepared in the same way
as described for the synthesis of 9 from 7 (57.2 mg, 0.093
mmol). To this material, 4 (25 mg, 0.0465 mmol) (dried by
azeotroping both together from toluene), and powdered 4 Å
molecular sieves (100 mg) in CH2Cl2 (2 mL) at -25 °C was
added AgOTf (26.3 mg, 0.1023 mmol). The reaction mixture
was worked up in the same manner as described for 9.
Purification of the resulting residue by column chromatogra-
phy (85:15 CH2Cl2/EtOAc) afforded 14 (29.5 mg, 58%) as a
76%): Rf ) 0.56 (6:1 CH2Cl2/EtOAc); mp 120-123 °C; [R]20
D
) -3.6 (c 1, CHCl3); 1H NMR δ 7.98 (d, J ) 8.2 Hz, 2H,), 7.59
(t, J ) 7.0 Hz, 1H), 7.40 (t, J ) 7.7 Hz, 2H), 7.12-7.05 (m,
3H), 6.86 (d, J ) 7.5 Hz, 2H), 5.78-5.52 (4H, m), 5.44 (d, J )
8.4 Hz, 1H), 5.35 (dd, J ) 8.8, 10.4 Hz, 1H), 5.21 (t, J ) 9.5
Hz, 1H), 5.01-4.82 (m, 3H), 4.72 (d, J ) 10.2 Hz, 1H), 4.41-
4.30 (m, 5H), 4.20-3.99 (m, 3H), 3.81-3.72 (m, 3H), 3.63 (t, J
) 8.8 Hz, 1H), 3.53 (m, 1H), 3.40 (m, 1H), 2.16-2.06 (m, 6H),
2.10 (s, 3H), 2.03 (s, 3H), 1.89 (s, 3H), 1.65-1.58 (m, 2H); 13C
NMR 172.1, 170.6, 170.5, 169.3, 166.7, 163.4, 162.3, 140.5,
138.0, 136.7, 136.7, 133.5, 129.8, 129.7, 129.2, 128.5, 128.1,
128.0, 127.5, 126.6, 101.1, 97.3, 76.5, 75.7, 74.6, 73.5, 71.8, 70.7,
68.6, 67.6, 61.9, 55.2, 53.7, 35.8, 29.9, 29.2, 28.6, 20.7, 20.6,
20.4. Anal. Calcd for C50H52Cl4N2O16: C, 55.67; H, 4.86; N,
2.60. Found: C, 55.76; H, 4.86; N, 2.54.
P en t -4-en yl (3,4,6-Tr i-O-a cet yl-2-d eoxy-2-t et r a ch lo-
r op h th a lim id o-â-D-glu cop yr a n osyl)-(1f6)-3-O-ben zoyl-4-
O-ben zyl-2-d eoxy-2-a m in o-â-D-glu cop yr a n osid e (10). To
a solution of 9 (0.05 g, 0.046 mmol) in THF (0.5 mL) was added
an equal volume of H2O (0.5 mL). Additional THF (0.5 mL)
was then added until the turbid solution became clear. Iodine
(0.035 g, 0.139 mmol) was then added in one portion at rt,
and the resulting brown solution was stirred for 8 min. The
reaction was quenched by the addition of solid (NH4)2S2O3 until
the brown color disappeared. The mixture was concentrated,
film: Rf ) 0.29 (7:1 CH2Cl2/EtOAc); [R]20 ) +52.7 (c 1.31,
D
CHCl3); 1H NMR δ 8.15-8.02 (m, 2H), 7.69-7.64 (m, 2H),
7.60-7.52 (m, 3H), 7.49-7.38 (m, 3H), 5.76-5.46 (m, 5H), 5.04
(t, J ) 9.7 Hz, 1H), 4.95-4.84 (m, 2H), 4.75 (d, J ) 10.4 Hz),

3658 J . Org. Chem., Vol. 62, No. 11, 1997
Griffiths et al.
4.56 (d, J ) 13.4 Hz, 1H), 4.47 (d, J ) 8.2 Hz, 1H), 4.24-4.10
(m, 4H), 3.88-3.70 (m, 3H), 3.52 (d, J ) 10.7 Hz, 1H), 3.41
(m, 1H), 3.12 (d, J ) 10.0 Hz, 1H), 2.22-2.02 (m, 4H), 2.02 (s,
3H), 1.88 (s, 3H), 1.78 (s, 3H), 1.67-1.50 (m, 4H); 13C NMR
172.3, 170.5, 170.5, 169.0, 166.0, 165.4, 140.4, 137.8, 136.7,
136.6, 129.9, 129.8, 129.7, 129.1, 128.8, 128.6, 128.3, 115.4,
114.8, 101.1, 97.5, 75.7, 74.4, 72.1, 71.6, 70.4, 68.7, 67.2, 62.6,
60.5, 55.9, 54.0, 35.7, 29.8, 29.2, 28.5, 20.7, 20.5, 20.4. Anal.
Calcd for C50H50Cl4N2O17: C, 54.96; H, 4.61; N, 2.56. Found:
C, 55.09; H, 4.58; N, 2.50.
J O961668S