Synthesis of a common trisaccharide fragment of glycoforms of the outer core region of the Pseudomonas aeruginosa lipopolysaccharide

Article abstract of DOI:10.1016/j.tetlet.2006.03.045

The first synthesis of the common trisaccharide of glycoforms of the outer core region of the Pseudomonas aeruginosa lipopolysaccharide is reported. A fully protected trisaccharide precursor was prepared via a highly efficient α-(1→4)-glucosylation of a β

Full text of DOI:10.1016/j.tetlet.2006.03.045

Tetrahedron Letters 47 (2006) 3583–3587
Synthesis of a common trisaccharide fragment of glycoforms
of the outer core region of the Pseudomonas aeruginosa
lipopolysaccharide
Bozhena S. Komarova,a Yury E. Tsvetkov,a Yuriy A. Knirel,b Ulrich Zahringer,c
¨
Gerald B. Pierd and Nikolay E. Nifantieva,*
aLaboratory of Glycoconjugate Chemistry, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Leninsky Prospect 47, 119991 Moscow, Russia
bCarbohydrate Chemistry, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47,
119991 Moscow, Russia
cResearch Centre Borstel, Centre for Medicine and Biosciences, 23845 Borstel, Germany
dChanning Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Received 9 December 2005; revised 21 February 2006; accepted 9 March 2006
Abstract—The first synthesis of the common trisaccharide of glycoforms of the outer core region of the Pseudomonas aeruginosa
lipopolysaccharide is reported. A fully protected trisaccharide precursor was prepared via a highly efficient a-(1!4)-glucosylation
of a b-(1!3)-linked 6-O-benzyl-2-azido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-a-D-galactopyranoside. In con-
trast, an alternative sequence of glycosylations, which involves b-glucosylation of an a-(1!4)-linked Glc-GalN3 unit, did not lead
to the target trisaccharide backbone. Further O-deacetylation, azido group reduction and debenzylation of the protected trisaccha-
ride precursor gave the corresponding trisaccharide amine. The latter structure was used in the synthesis of a series of trisaccharides
bearing an acetyl group, an L-alanine or an N-acetylated L-alanine residue on its amino group at C-2 of GalN.
Ó 2006 Elsevier Ltd. All rights reserved.
Cystic fibrosis (CF) is a congenital disease wherein there
is a high susceptibility to Pseudomonas aeruginosa infec-
tion in the lungs. It was previously shown1 that the outer
core region of the lipopolysaccharide of P. aeruginosa
influences a critical step in the elimination of this bacte-
rium from a normal host by binding to the CF trans-
membrane conductance regulator (CFTR), the protein
that is missing or is dysfunctional in CF. In order to
ascertain which of the two naturally occurring glyco-
forms2 I and II (Fig. 1) is recognized by the CFTR
receptor on respiratory epithelial cells, we performed a
systematic synthesis of the oligosaccharides representing
these two glycoforms.
α-Rha-(1
6)-α-Glc
1
4
α-Glc-(1
α-Glc-(1
6)-β-Glc-(1
3)-α-GalNAla-(1
inner core
inner core
I
α-Rha
α-Glc
1
1
3
4
6)-β-Glc-(1
3)-α-GalNAla-(1
II
Figure 1. Glycoforms of the outer core region of the Pseudomonas
aeruginosa lipopolysaccharide.
In this letter, we describe the synthesis of a common pre-
cursor for both glycoforms, branched N-(L-alanyl)tri-
saccharide 34, as well as its N-acetyl- 36 and N-(N-
acetyl-L-alanyl)-analogues 35 (Scheme 3). Compounds
34 and 35 were prepared to elucidate the role of the L-
alanine residue and its amino group in the host recogni-
tion of the presence of P. aeruginosa bacterial cells.
*
Corresponding author. Tel.: +7 495 135 87 84; fax: +7 095 135 87
84; e-mail: nen@ioc.ac.ru
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.03.045

3584
B. S. Komarova et al. / Tetrahedron Letters 47 (2006) 3583–3587
A mixture of azidonitrates 1,3 which contained both
a- and b-anomers of 1-O-nitro-2-azido-2-deoxy-D-galac-
topyranose and 1-O-nitro-2-azido-2-deoxy-a-D-talo-
pyranose in a ratio of 3:6:1 was used as the starting
material. No efficient and stereoselective procedure for
conversion of this mixture into a-methyl glycoside has
been reported so far. We found that methanolysis of
2-azido-2-deoxy-D-galactopyranosyl halides 2 or 3 in
the presence of tetraalkylammonium halides in CH2Cl2
gave 4 with high a-stereoselectivity. Thus, treatment of
bromide 24 with MeOH (5 equiv) in the presence of
Bu4NBr (1.5 equiv) in CH2Cl2 afforded an inseparable
mixture of 4 and its b-anomer in a ratio of 5:1, whereas
the reaction of iodide 33 with MeOH (20 equiv) in the
presence of Bu4NI (2 equiv) gave an improvement in
the ratio of a:b = 9:1.
more efficient. Although a large excess (3- to 10-fold)
of 9 was required to complete the conversion of the
acceptor 8, the reaction led to the disaccharide 10
stereospecifically. Liberation of the 3-OH group in the
2-azidogalactoside residue was achieved in two steps
(Scheme 1): first the acyl protecting groups in 10 were
removed with MeONa in MeOH, then the primary
OH group of the resulting structure 11 was selectively
benzoylated to give the disaccharide acceptor 12 (67%
yield in two steps). Direct removal of the acetyl group
from 10 by mild acidic methanolysis6 proved to be slow
(several days) and less efficient (50%).
Glycosylation of the acceptor 12 with peracetylated
glucosyl donors 13, 26 and 27 (Scheme 3, path A) was
studied next. Reactions of 12 with imidate 13 (BF3ÆEt2O
or TMSOTf with MS AW-300), bromide 26 (AgOTf,
MS AW-300 or without MS), or thioglycoside 27
Deacetylation of the latter mixture gave 5 (a:b = 9:1) in
65% overall yield (from 1). Triol 5 was converted into
benzylidene derivative 6; an admixture of the corre-
sponding b-anomer could be easily removed by column
chromatography at this step. The benzylidene derivative
6 was acetylated and the acetal ring in 7 was opened5
regioselectively to give 8.
˚
(NIS/TfOH, MS AW-300 or MS 4 A) resulted in com-
plete decomposition of the starting glycosyl donors; no
formation of the target trisaccharide 28 was detected.
In contrast, glycosylation of 12 with 13 or 26 in the pres-
˚
ence of relatively basic MS 4 A (Scheme 3) gave ortho-
ester 30. Attempts at acid-catalyzed transformation7 of
the orthoester 30 into glycoside 28 failed also. The low
reactivity of the 3-OH group in 12 may be accounted
for by the combined effects of the neighboring elec-
tron-withdrawing azido group8 and the bulky glucosyl
substituent.
a-Glucosylation of the acceptor 8 with per-O-benzylated
glucopyranosyl donors of ethyl thioglycoside (NIS/
TfOH or MeOTf in CH2Cl2 or Et2O), bromide (Bu4NBr
or AgOTf in CH2Cl2), fluoride (SnCl2/AgClO4 in Et2O)
or trichloracetimidate (TMSOTf in CH2Cl2) gave unsatis-
factory results in terms of the yield and stereoselectivity.
Glucosylation with ethyl thioglycoside 9 (Scheme 1)
possessing a 6-O-benzoyl group, which was thought to
be capable of remote anchimeric participation, was
Therefore, an alternative approach to the assembly of
the target trisaccharide was explored where the (1!3)-
bond was constructed first and the resulting disaccha-
ride was 4-O-a-glucosylated. Unlike the disaccharide
RO
OR
AcO
AcO
OAc
OAc
O
a
b
O
O
RO
AcO
AcO
OMe
N3
N3
N3
ONO2
X
4 R = Ac
2 X = Br
c
1
5 R = H
3 X = I
d
Ph
O
HO
O
OBn
OBz
O
O
f
O
N
BnO
BnO
+
SEt
AcO
RO
BnO
N
3 OMe
3 OMe
8
9
6 R = H
g
e
7 R = Ac
OR2
O
BnO
BnO
BnO
O
OBn
O
R1O
N
3 OMe
10 R1 = Ac, R2 = Bz
11 R1, R2 = H
c
h
12 R1 = H, R2 = Bz
Scheme 1. Reagents and conditions: (a) for 2: LiBr, CH3CN (65%); for 3: NaI, CH3CN; (b) from 2: MeOH (5 equiv), Bu4NBr (1.5 equiv), CH2Cl2
(a:b = 5:1; 59% over two steps); from 3: MeOH (20 equiv), Bu4NI (2 equiv), CH2Cl2 (a:b = 9:1; 68% over two steps); (c) MeONa, MeOH; (d)
PhCH(OMe)2 (2 equiv), TsOH (cat.), CH3CN (76% for a-isomer); (e) Ac2O, Py; (f) Me3NÆBH3, AlCl3, H2O, THF (78%); (g) 9 (>3 equiv), NIS
(2.5 equiv), TfOH (0.5 equiv), CH2Cl2, ꢀ5 °C (78% based on 8); (h) BzCl, Py, ꢀ20 °C (67% two steps).

B. S. Komarova et al. / Tetrahedron Letters 47 (2006) 3583–3587
3585
R1O
OR2
O
OAc
OAc
O
a
O
AcO
AcO
AcO
AcO
6
+
O
AcO
N
AcO
3 OMe
O
CCl3
14 R1,R2 = PhCH<
15 R1 = H, R2 = Bn
b
NH
13
Scheme 2. Reagents and conditions: (a) TMSOTf, CH2Cl2, AW-300 (82%); (b) Me3NÆBH3, AlCl3, H2O, THF (86%).
acceptor 12, 3-O-glucosylation of the benzylidene deriv-
ative 6 with the donor 13 (TMSOTf, MS AW-300) pro-
ceeded smoothly and gave disaccharide 14 in 80% yield
(Scheme 2). Reductive opening of the benzylidene acetal
ring in 14 produced the acceptor 15. However, reaction
of 15 with a large excess of the thioglycoside 9, which
previously showed high a-stereoselectivity in reaction
with 8, did not lead to any trisaccharide product
(Scheme 3).
glucosyl donors in the presence of AgOTf and MS
AW-300 was initially studied. The results are summa-
rized in Table 1. Reaction of the trichloroacetimidate
16 (Fig. 2) bearing a participating acetyl group at O-6
resulted in a mixture of the anomeric disaccharides
with good a-stereoselectivity but in moderate yield
(entry 1). Glucosylation with the recently introduced
perbenzylated N-phenyl-trifluoracetimidate 1710 was
much more efficient in terms of yield but the a-stereo-
selectivity was poor (entry 2). Taking into account the
result of the glycosylation with the 6-O-acetylated
trichloracetimidate 16, the anchimeric participation of
remote acyl protecting groups was studied in more detail
with the aim of improving the stereoselectivity.
Consequently, we had to find an effective and a-stereo-
selective method for 4-O-glucosylation of 15. The low
reactivity of the 4-OH in 15 and fast decomposition of
the donor under the conditions used seem to have been
responsible for the low yields of the a-glycosylation with
9 in the presence of NIS–TfOH. Hence, a mild and slow
glycosylation procedure was considered as a preferred
route of synthesis. AgOTf-promoted glycosylation with
trichloroacetimidates9 seemed to satisfy these require-
ments. To verify this, a model glucosylation of the
monosaccharide acceptor 8 with various imidate-type
The concept of remote group participation was vali-
dated with the improvement of the stereoselectivity of
1,2-cis-glycoside formation in L-fuco-,11 D-galacto-,12
and D-manno-series.13 Glycosylation with N-phenyl-tri-
fluoracetimidate 18, which has a 6-O-benzoyl participat-
ing group (entry 3) gave a mixture of 10 and its b-isomer
B
A
OR1
BnO
OBn
OR2
a
O
9
15
15
+
+
promoter
OAc
O
OAc
OBn
O
AcO
AcO
12
+
O
O
AcO
AcO
b
X
21
O
AcO
Y
AcO
N3
OMe
28 R1 = Bn, R2 = Bz
13 X= H, Y = OC(NH)CCl3
C
29 R1, R2 = Ac
OH
26 X= H, Y = Br
R1
O
OR1
OH
27 X= SEt, Y = H
O
OR1
O
e
O
OH
g
O
O
HO
HO
O
HN
H3C
HN
c
R2
d
12
+
OMe
OH
OMe
O
O
B
A
OAc
O
31 R1 = Bn, R2 = N3
32 R1 = H, R2 =NH2
AcO
AcO
t-BuO
f
OBn
33
BnO
OBn
OBz
O
O
O
h
i
O
CH3
OBn
36 R1 = H, R2 = NHAc
O
i
O
O
O
N3
HN
O
OMe
H3C
H2N
HN
OMe
H3C
HN
OMe
30
O
O
34
35
Scheme 3. Reagents and conditions: (a) NIS, TfOH, MS AW-300, CH2Cl2; (b) 21 (1.2 equiv), AgOTf (0.6 equiv), MS AW-300, CH2Cl2–toluene
(6.5:1) (62% for a); (c) 13 (1.2 equiv), AgOTf (1.2 equiv), MS AW-300, CH2Cl2:toluene (1:1); (d) TMSOTf, CH2Cl2; (e) MeONa, MeOH; (f) H2, 20%
Pd(OH)2/C, HCl, MeOH (64%); (g) Boc-L-AlaOSu (1.5 equiv), Amberlyst A-26 (HCO3ꢀ), DMF, H2O (70%); (h) CF3COOH, H2O (67%); (i) Ac2O,
Amberlyst A-26 (HCO3ꢀ), MeOH (95%).

3586
B. S. Komarova et al. / Tetrahedron Letters 47 (2006) 3583–3587
Table 1. Effect of acyl protecting groups in glucosyl donors on the
stereochemical outcome of the glycosylation of acceptor 8
structural analogs was efficiently carried out with the use
of the concept of remote group participation during the
key a-glucosylation step.
Entry Donor Products
a:b ratioa Total yield (%)
1
2
3
4
5
6
16
17
18
19
20
21
23a, 23b
22a, 22b
10 + b-Isomer 6:1
23a, 23b
24a, 24b
25a, 25b
4:1
2:1
48
95
84
90
90
96
Acknowledgements
5:1
4:1
8:1
We thank the DFG # 436 RUS 113/314/0-3, the Rus-
sian Foundation for Basic Research Grant # 03-03-32-
556, the 10th Program of Division of Chemistry and
Material Sciences and the National Institutes of Health
Grant RO1 HL58398, for support of this work.
a The a:b ratio was determined according to signal intensities in the 1H
NMR spectra.
OR2
OAc
References and notes
O
O
BnO
R1O
BnO
BnO
1. Schroeder, T. H.; Lee, M. M.; Yacono, P. W.; Cannon, C.
L.; Gerc¸eker, A. A.; Golan, D. E.; Pier, G. P. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 6907–6912.
2. Bystrova, O. V.; Lindner, B.; Moll, H.; Kocharova, N. A.;
Knirel, Y. A.; Za¨hringer, U.; Pier, G. B. Carbohydr. Res.
2003, 338, 1895–1905.
3. Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57,
1244–1251.
4. Broddefalk, J.; Nilsson, U.; Kihlberg, J. J. Carbohydr.
Chem. 1994, 13, 129–132.
5. Sherman, A. A.; Mironov, Y. V.; Yudina, O. N.;
Nifantiev, N. E. Carbohydr. Res. 2003, 338, 697–703.
6. Byramova, N. E.; Ovchinnikov, M. V.; Backinowsky, L.
V.; Kochetkov, N. K. Carbohydr. Res. 1983, 124, C8-11.
7. Gass, J.; Strobl, M.; Loibner, A.; Kosma, P.; Za¨ringer, U.
Carbohydr. Res. 1993, 244, 69–84.
8. Paulsen, H.; Ho¨lck, J.-P. Carbohydr. Res. 1982, 109, 89–
107.
9. Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J. J.
Carbohydr. Chem. 1993, 12, 131–136.
O
CF3
NPh
BnO
BnO
CCl3
NH
O
17 R1 = Bn, R2 = Bn
18 R1 = Bn, R2 = Bz
19 R1 = Bn, R2 = Ac
20 R1 = Ac, R2 = Bn
21 R1 = Ac, R2 = Ac
16
OR2
O
BnO
R1O
OR2
O
BnO
R1O
BnO
O
O
OBn
O
OBn
BnO
O
AcO
AcO
N
3 OMe
N
3 OMe
R1
R2
22a
23a
24a
25a
22b
Bn
Bn
Ac
Ac
Bn
Ac
Bn
Ac
23b
24b
25b
Figure 2.
10. (a) Yu, B.; Tao, H. Tetrahedron Lett. 2001, 42, 2405–2407;
(b) Adinolfi, M.; Barone, G.; Iadonisi, A.; Schiattarella,
M. Tetrahedron Lett. 2002, 43, 5573–5577.
with an a:b ratio of 6:1. Replacement of the 6-O-benzoyl
group with 6-O-acetyl (entry 4, donor 19) did not
change the proportion of anomers (a:b = 5:1). Participa-
tion of a 3-O-acetyl group seemed to be slightly less effi-
cient than that occurred with the 6-O-acetyl group as the
reaction of 20 with 8 led to a mixture of 24a and 24b
with an a:b ratio of 4:1. Finally, the combined effect
of both 3-O- and 6-O-acetyl groups in donor 21
achieved an a:b ratio of 8:1 (entry 6).14 The most effi-
cient donor 21 was then successfully used for the prepa-
ration of the target, protected trisaccharide 29 (Scheme
3). Reaction of 15 with 21 in the presence of AgOTf
and MS AW-300 afforded 2915 in 62% yield. Trisaccha-
ride 29 was deacetylated and the benzyl groups in the
resulting structure 31 were subjected to catalytic hydro-
genolysis with concomitant reduction of the azido group
to furnish amine 32 in 64% yield. The latter was either
N-alanylated or N-acetylated to provide N-acyl deriva-
tives 33 and 36, respectively. Removal of the N-Boc pro-
tecting group from 33 with aqueous CF3COOH afforded
the target trisaccharide 34 in 60% yield over two steps.
N-Acetylation of 34 with Ac2O yielded another target
structure, N-(N-acetyl-L-alanyl)-derivative 35.15
11. Gerbst, A. G.; Ustuzhanina, N. E.; Grachev, A. A.;
Khatuntseva, E. A.; Tsvetkov, D. E.; Whitfield, D. M.;
Berces, A.; Nifantiev, N. E. J. Carbohydr. Chem. 2001, 20,
821–831.
12. Demchenko, A. V.; Rousson, E.; Boons, G.-J. Tetra-
hedron Lett. 1999, 40, 6523–6526.
13. De Meo, C.; Kamat, M. N.; Demchenko, A. V. Eur. J.
Org. Chem. 2005, 706–711.
14. General procedure for AgOTf promoted glycosylations with
N-phenyltrifluoroacetimidates: To a solution of donor 21
(42.9 mg, 0.07 mmol) and acceptor 8 (21 mg, 0.06 mmol)
in dry CH2Cl2 (0.85 mL), MS AW-300 (80 mg) was added.
The reaction mixture was stirred at room temperature for
1 h after which a solution of AgOTf (9.5 mg, 0.04 mmol)
in toluene (110 lL) was added. After 18 h the reaction was
quenched with pyridine, then filtered through Celite,
washed with Na2S2O3 (1 M) and concentrated. Column
chromatography (CHCl3–Et2O, 20:1) afforded 25a/b
(47 mg, 96%) as a foam, 1H NMR (500 MHz, CDCl3)
for 25a: 7.20–7.40 (15H, m, Ar), 5.59 (1H, t, H-3 B,
J3,2 = J3,4 = 9.7 Hz), 5.22 (1H, dd, H-3 A, J3,4 = 2.7 Hz),
4.86 (1H, d, H-1 A, J1,2 = 3.5 Hz), 4.84 (1H, d, H-1 B,
J1,2 = 3.4 Hz), 4.45–4.60 (4H, m, PhCH2), 4.36 (1H, d,
PhCH2, J = 12.0 Hz), 4.28 (2H, m, H-6 B), 4.23 (1H, d,
PhCH2, J = 12.0 Hz), 4.17 (1H, d, H-4 A, J4,3 = 2.7 Hz),
4.14 (1H, m, H-5 B), 4.00 (1H, t, H-5 A, J5,6A = J5,6B
6.5 Hz), 3.77 (1H, dd, H-6A A, J6A,6B = 10.2 Hz, J6A,5
6.55 Hz), 3.56 (1H, m, H-6B A), 3.52 (1H, t, H-4 B,
=
=
In conclusion, this first synthesis of the common trisac-
charide fragment of both glycoforms of the outer core
region of the P. aeruginosa lipopolysaccharide and some
J4,3 = J4,5 = 9.7 Hz), 3.43 (1H, dd, H-2 B, J2,3 = 10.2 Hz,

B. S. Komarova et al. / Tetrahedron Letters 47 (2006) 3583–3587
3587
J2,1 = 3.4 Hz), 3.40 (3H, s, OMe), 2.10, 2.04, 1.94 (9H, s,
Ac); 13C NMR (125 MHz, CDCl3) for a: 169.6–170.5
(C@O), 137.2–138.1 (ipso-Ph), 127.2–129.5 (Ph), 99.1 (C-1
B), 98.7 (C-1 A), 78.2 (C-2 B), 76.7 (C-4 A), 76.3 (C-4 B),
74.6 (PhCH2), 73.4 (C-3, B), 73.3 (PhCH2), 72.8 (PhCH2),
70.9 (C-3 A), 69.3 (C-5 B), 69.3 (C-5 A), 68.3 (C-6 A), 62.6
(C-6 B), 57.9 (C-2 A), 55.3 (OMe), 20.8–21.0 (Ac). Anal.
Calcd (%) for C40H47N3O12: C, 61.77; H, 6.09. Found: C,
61.86; H, 6.24.
Found: C, 58.70; H, 5.99; N, 3.85. Compound 34: [a]D 36
(c 0.5, H2O); 1H NMR (500 MHz, D2O): 5.00 (1H, d, H-1
C, J1,2 = 3.5 Hz), 4.82 (1H, d, H-1 A, J1,2 = 3.5 Hz), 4.46
(1H, d, H-1 B, J1,2 = 7.5 Hz), 4.45 (1H, dd, H-2 A,
J2,1 = 3.5 Hz, J2,3 = 11.5 Hz), 4.31 (1H, d, H-4 A,
J4,3 = 2.5 Hz), 4.22 (1H, m, H-5 C), 4.13 (1H, dd, H-3
A, J3,2 = 11.5 Hz, J3,4 = 2.5 Hz), 4.04 (2H, m, H-5 A,
0
Ala), 3.89 (1H, d, H-6 B, J6;6 ¼ 12:5 Hz), 3.88 (1H, m, H-
6 A), 3.85 (2H, m, H-60 A, H-6 C), 3.82 (1H, t, H-3 C,
J3,2 = J3,4 = 9.5 Hz), 3.77 (1H, dd, H-60 C, J6 ;5 ¼ 1:3 Hz,
0
15. Data for compounds 29, 34, 35, 36. Compound 29: [a]D 81
(c 1, CHCl3); 1H NMR (500 MHz, CDCl3): 7.10–7.40
(15H, m, Ar), 5.67 (1H, t, H-3 C, J3,2 = J3,4 = 9.7 Hz),
5.27 (1H, t, H-3 B, J3,2 = J3,4 = 9.6 Hz), 5.25 (1H, d, H-1
J6 ;6 ¼ 12:7 Hz), 3.71 (1H, dd, H-60 B, J6 ;5 ¼ 4:0 Hz,
0
0
0
J6 ;6 ¼ 12:5 Hz), 3.52 (1H, t, H-4, J4,3 = J4,5 = 9.5 Hz),
3.42 (1H, m, H-3 B), 3.38 (3H, s, OMe), 3.37 (2H, m, H-4
B, H-5 B), 3.17 (1H, dd, H-2 B, J2,1 = 7.5 Hz,
J2,3 = 9.5 Hz), 1.54 (3H, d, Ala, J = 7.1 Hz); 13C NMR
(125 MHz, D2O): 172.3 (C@O), 105.4 (C-1 B), 100.0 (C-1
C), 99.2 (C-1 A), 76.9 (C-5 B), 76.7 (C-4 A), 76.6 (C-3 B),
76.4 (C-3 A), 74.0 (C-2 A), 73.6 (C-3 C), 72.9 (C-2 C), 72.6
(C-5 A, C-5 C), 70.9 (C-4 B), 70.2 (C-4 C), 61.8 (C-6 B),
61.4 (C-6 A), 61.2 (C-6 C), 56.3 (OMe), 50.5 (C-2 A, CH
Ala), 17.7 (Me, Ala); HRMS C22H40N2O16 [M+H]+;
calculated: 589.246, found: 589.233. Compound 35:
[a]D 70 (c 1, H2O); 1H NMR and 13C NMR spectra of
35 are identical to those of 34 except for the downfield
shift of the signal corresponding to the a-proton of
alanine (d 4.06!4.44) and the presence of a singlet at d
2.00 (3H) in the 1H NMR spectrum and signals at d
22.8 and 176.7 in the 13C NMR spectrum corresponding
to the acetyl group; HRMS C24H42N2O17 [M+Na]+;
calculated: 653.238, found: 653.230. Compound 36:
[a]D 52 (c 0.5, H2O); 1H NMR and 13C NMR spectra
of 36 were identical to those of 34 except for the
absence of the signals corresponding to alanine and the
presence of a singlet at d 2.00 (3H) in the 1H NMR
spectrum and signals at d 23.2 and 176.1 in the 13C NMR
spectrum corresponding to the acetyl group; HRMS
C21H37NO16 [M+Na]+; calculated: 560.219, found:
560.218.
C, J1,2 = 3.4 Hz), 5.16 (1H, t, H-4 B, J4,3 = J4,5
=
10.0 Hz), 5.12 (1H, m, H-2 B), 4.90 (1H, d, H-1 A,
J1,2 = 3.2 Hz), 4.82 (1H, d, H-1 B, J1,2 = 8.0 Hz), 4.63
(1H, d, PhCH2, J = 12.2 Hz), 4.61 (2H, s, PhCH2), 4.55
(1H, d, PhCH2, J = 12.1 Hz), 4.46 (1H, dd, H-6 C,
0
J6,5 = 2.8 Hz, J6;6 ¼ 12:2 Hz), 4.43 (1H, d, PhCH2,
J = 12.0 Hz), 4.36 (1H, dd, H-60 C, J6 ;5 ¼ 1:6 Hz,
0
0
J6;6 ¼ 12:2 Hz), 4.31 (1H, m, H-6 B), 4.30 (1H, d, PhCH2,
J = 12.0 Hz), 4.23 (1H, br s, H-4 A), 4.21 (1H, m, H-5 C),
4.20 (1H, dd, H-60 B, J6 ;5 ¼ 1:7 Hz, J6;6 ¼ 11:8 Hz), 4.02
(1H, dd, H-3 A, J3,2 = 10.8 Hz, J3,4 = 2.0 Hz), 3.97 (1H,
m, H-5 A), 3.84 (1H, dd, H-6 A, J6,5 = 5.6 Hz,
0
0
0
J6;6 ¼ 10:2 Hz), 3.75 (1H, m, H-5 B), 3.72 (1H, m, H-2
A), 3.68 (1H, m, H-60 A), 3.54 (1H, t, H-4 C,
J4,3 = J4,5 = 9.6 Hz), 3.54 (1H, dd, H-2 C, J2,1 = 3.4 Hz
J2,3 = 9.6 Hz), 3.46 (3H, s, OMe), 2.13 (3H, s, Ac), 2.01–
2.09 (9H, 3s, Ac), 1.99 (6H, s, Ac); 13C NMR (125 MHz,
CDCl3): 169.0–170.3 (C@O), 137.6–138.3 (ipso-Ph),
127.4–129.5 (Ph), 102.3 (C-1 C), 98.8 (C-1 A), 98.0 (C-1
B), 78.5 (C-2 C), 78.3 (C-4 A), 77.7 (C-3 A), 76.2 (C-4 C),
74.1 (PhCH2), 73.4 (C-3 C), 73.0 (PhCH2), 72.8 (C-3 B),
72.7 (PhCH2), 72.0 (C-5 B), 70.9 (C-2 B), 70.1 (C-5 A),
69.1 (C-6 A), 68.9 (C-5 C), 68.4 (C-4 B), 62.8 (C-6 C), 69.9
(C-6 B), 59.9 (C-2 A), 55.2 (OMe), 20.3–21.3 (Ac). Anal.
Calcd (%) for C52H63N3O21: C, 58.59; H, 5.96; N, 3.94.