Synthesis of an unusual branched-chain sugar, 5-C-methyl-L-idopyranose for SAR studies of pyranmycins: Implication for the future design of aminoglycoside antibiotics

Article abstract of DOI:10.1016/j.bmcl.2004.06.064

The syntheses of a challenging branched-chain sugar and several l-sugars have been accomplished. Their application in the studies of the antibacterial activity of pyranmycins is reported, which could provide new strategies for the future design of aminoglycoside antibiotics. The syntheses of a challenging branched-chain sugar and several l-sugars have been accomplished. Their application in studies of the antibacterial activity of pyranmycins is reported, which could provide new strategies for the future design of aminoglycoside antibiotics.

Full text of DOI:10.1016/j.bmcl.2004.06.064

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4389–4393
Synthesis of an unusual branched-chain sugar,
5-C-methyl-L-idopyranose for SAR studies of pyranmycins:
implication for the future design of aminoglycoside antibiotics
Jinhua Wang,a Jie Li,a Przemyslaw G. Czyryca,a Huiwen Chang,a
Jeff Kaob and Cheng-Wei Tom Changa,
*
aDepartment of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT 84322-0300, USA
bDepartment of Chemistry, Washington University, One Brookings Drive, St. Louis, MO 63130-4899, USA
Received 24 May 2004; revised 19 June 2004; accepted 21 June 2004
Abstract—The syntheses of a challenging branched-chain sugar and several L-sugars have been accomplished. Their application in
studies of the antibacterial activity of pyranmycins is reported, which could provide new strategies for the future design of amino-
glycoside antibiotics.
Ó 2004 Elsevier Ltd. All rights reserved.
Due to their broad spectrum antibacterial activity, ami-
noglycoside antibiotics have been revived as a focus for
NH2
NH2
I
O
I
HO
HO
O
HO
HO
1'
1'
NH2
5
II
1 NH2
the development of new antibacterial agents to counter-
act the growing problem of drug-resistant infectious
diseases.1,2 Previously, we have reported the structure–
activity relationship (SAR) of a novel class of aminogly-
coside antibiotics, pyranmycins, which has been synthe-
sized via a glycodiversification approach developed in
our laboratory.3 From the SAR results, we noticed that
the presence of 6-deoxy-D-glucopyranose as ring III of
pyranmycins is essential for antibacterial activity. On
the other hand, the presence of a 6-amino-6-deoxy-L-
idopyranose as ring III also manifested significant activ-
ity. Thus, we are interested in a design that combines
both structural features, hypothesizing that such a struc-
ture should lead to improved antibacterial activity (Fig.
1).
NH2
5
4
H2N
O
II
1 NH2
4
H2N
O
H3C
O
O
HO
HO
O
HO
O
OH
+
OH
HO
1"
OH
III
1"
OH
III
H2N
6-amino-6-deoxy-L-
idopyranose
6-deoxy-D-glucopyranose
MIC = 9 µM
MIC = 9 µM
NH2
I
O
HO
HO
1'
NH2
5
II
1 NH2
4
H2N
H3C
O
O
HO
O
OH
HO
1"
OH
III
new members of
pyranmycin with
branched-chain sugar
H2N
Figure 1. Strategy for the design of branched-chain sugar.
In order to examine our hypothesis, we targeted the syn-
thesis of six pyranose donors that will be used as ring III
of pyranmycins (Fig. 2). Compound 1 is designed to
ensure that the steric hindrance and the quaternary car-
bon at C-5 will not hamper the antibacterial activity.
Compounds 2, 3, and 4 are designed to demonstrate
the importance of the hydroxymethyl group in L-idopyr-
anose. Compounds 5 and 6 are branched-chain sugars
that combine the desired structural components outlined
above. The synthesis of 2, 3, and 4 began from the
known compounds4 7, 9, and 11 via acid-catalyzed
hydrolysis of the isopropylidene groups, acetylation,
and phenylthiol substitution (Scheme 1).
Keywords: Aminoglycoside antibiotics; Branched-chain sugar; Pyran-
mycins; Drug development; Antibacterial.
*
Corresponding author. Tel.: +1-435-797-3545; fax: +1-435-797-
3390; e-mail: chang@cc.usu.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.064

4390
J. Wang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4389–4393
OAc
O
H3C
H3C
H3C
BnO
BnO
O
O
O
AcO
AcO
O
AcO
AcO
5
b
BnO
BnO
HO
a
SPh
BnO
AcO
C(NH)CCl3
AcO
OAc
BnO
OAc
AcO
68%
BnO
84%
OMe
OAc
AcO
AcO
2
AcO
O
17
1
15
16
O
O
H3C
AcO
AcO
H3C
SPh
AcO
SPh
O
O
c
AcO
AcO
AcO
AcO
SPh
SPh
OAc
H3C
H3C
OAc
N3
99%
AcO
AcO
4
3
AcO
N3
6
H3C
5
O
O
AcO
AcO
AcO
SPh
SPh
AcO
Scheme 3. Reagents: (a) TfOH–AcOH–H2O (1/28/5); (b) (1) H2, Pd/C,
MeOH, (2) Sc(OTf)3, Ac2O, CH2Cl2; (c) PhSH, BF3–OEt2, CH2Cl2.
OAc
OAc
N3
AcO
6
5
Ditosylation of 13 followed by LiAlH4 reduction fur-
nished 15 with the deoxygenation at the desired posi-
tion. The 6-O-tosyl group (based on the nomenclature
of D-glucopyranose) of 13 is accessible toward hydride
attack at C-6, resulting in deoxygenation. However,
the tosyl group on the 5-C-hydroxylmethyl group is ster-
ically blocked by the anomeric methoxyl group. In this
case, the hydride attack occurs at the sulfur of the tosyl
group, which results in regeneration of the free hydroxyl
group. Regioselectivity of the deoxygenation was con-
firmed by 2D NOE experiment.
Figure 2. Proposed donors of pyranoses. Compound 6 was proposed
initially. However, we were unable to synthesize it.
H
O
O
HO
O
AcO
AcO
O
a
b
H
OAc
2
O
O
58%
78%
AcO
7
AcO
8
c
H
O
O
AcO
AcO
N3
O
a
b
O
OAc
3
H
66
75%
%
AcO
O
O
The reactions using Ac2O and acid catalysts for convert-
ing 15 into peracetylated glycoside were unsuccessful. A
1,6-anhydro idopyranose, 16, was often obtained
(Scheme 3). Attempts at employing TMSSPh/TMSOTf7
provided the desired phenylthio glycoside with unsatis-
factory yield. Nevertheless, compound 16 was finally
transformed into 17 by a recently reported method8
using Sc(OTf)3 and Ac2O following hydrogenation.
Treatment of 17 with PhSH and BF3–OEt2 gave the de-
sired glycosyl donor, 5. However, all attempts failed to
generate 6.9
N3
9
10
H
O
O
AcO
AcO
a
b
93%
H3C
O
O
O
OAc
4
H
AcO
CH3
41%
O
12
11
Scheme 1. Reagents: (a) (1) TFA–AcOH–H2O, (2) Ac2O, TFA;
(b) PhSH, BF3–OEt2, CH2Cl2; (c) (1) Tf2O, py., (2) NaN3, DMF.
Although the synthesis of the precursor of 5, 5-C-
methyl-L-idopyranose, has been reported, the synthetic
route is difficult to be amended for large-scale produc-
tion as well as glycosylation of 5.5 Therefore, we decided
to develop an alternative approach using compound 136
as the starting material. Treatment of 13 with Ac2O and
H2SO4 afforded 14, which was converted into the corre-
sponding glycosyl trichloroacetimidate 1 (Scheme 2).
Glycosylation of the 30,40,6-tri-O-benzyltetraazidoneam-
ine acceptor2e using glycosyl trichloriacetamidate and
N3
O
BnO
AcO
BnO
AcO
BnO
N3
O
AcO
BnO
N3
N3
a
O
O
O
AcO
AcO
BnO
AcO
OC(NH)CCl3
OAc
AcO
1
18a
6
HO
BnO
BnO
AcO
BnO
5
O
O
N3
O
a
b
1
AcO
O
BnO
OMe
OAc
AcO
14%
82%
BnO
HO
AcO
R1
BnO
N3
R1
AcO
AcO
14
O
13
N3
N3
O
AcO
AcO
b
SPh
O
BnO
c
24%
OAc
R2
R2
OAc
H
H3C
2-5
18b-e
CH3
O
BnO
O
BnO
BnO
BnO
BnO
OMe
R1
-
H
H
H
CH3
R2
-
CH2OAc
CH2N3
CH3
Products
18a
18b
18c
18d
Yield (%)
HO
15
56
63
22
48
78
15
H
H
H
OBn
OMe
NOE
OH
18e
CH2OAc
5
Scheme 2. Reagents: (a) Ac2O, H2SO4; (b) (1) H2NNH2–HOAc,
DMF, (2) CCl3CN, DBU, CH2Cl2; (c) (1) TsCl, py., (2) LiAlH4, THF.
Scheme 4. Reagents: (a) 30,40,6-tri-O-benzyltetraazidoneamine, BF3–
OEt2, CH2Cl2; (b) NIS–TfOH, CH2Cl2.

J. Wang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4389–4393
4391
N3
N3
O
N3
O
BnO
BnO
BnO
BnO
H3C
BnO
BnO
N3
N3
N3
H3C
AcO
AcO
O
O
O
a
N3
N3
O
O
O
BnO
83%
BnO
2"
OBn
OAc
6"
BnO
AcO
6"
18e
19
N3
N3
O
O
BnO
BnO
BnO
N3
BnO
N3
H3C
BnO
BnO
H3C
BnO
BnO
N3
N3
b
O
N3
O
O
O
O
O
25%
N3
BnO
BnO
OBn
OBn
N3
HO
21
20
Scheme 5. Reagents: (a) (1) NaOMe, MeOH, (2) BnBr, NaH, TBAI, THF; (b) (1) TMSOTf, Ac2O, (2) NaOMe, MeOH.
phenylthio glycosides was carried out via BF3–OEt2 and
NIS/TfOH activation (Scheme 4). In an effort to prepare
one of the designed branched-sugar-containing pyran-
mycins, compound 18e was converted into 19 via
hydrolysis and perbenzylation (Scheme 5). The O-600
benzyl group on 20 was selectively deprotected. How-
ever, all the attempts to make 21 were unsuccessful.10
Synthesis of final products from compounds 18a–e was
performed using reported procedures (Scheme 6).3
concentration (MIC) (Table 1).11 Despite not having 5-
C-methyl-6-aminoidopyransoe available, we have re-
ported the synthesis and antibacterial activity of
TC020.3 When combining TC020 and TC036, we can
still evaluate the effect of branched-chain sugar as in
the case of TC054. Compound TC029 is active against
both strains of the tested bacteria. The antibacterial
activity of TC036 is much better than the deoxygenated
member, TC037, which is consistent with our previous
finding: a C-600 aminomethyl (or hydroxylmethyl) group
is important for the activity of pyranmycins with ring III
L-sugar (Table 1). However, to our surprise, TC054,
which has a design from combining two structural fea-
tures with superior antibacterial activity, individually,
manifested only low activity. To provide the possible
solution for this unexpected result, we evaluated the
binding of TC054 and TC036 toward the target rRNA
fragment using molecular modeling (Fig. 3).12 From
the molecular modeling structures, we did not find sig-
nificant structural perturbation due to the introduction
The synthesized new members of the pyranmycin family
were assayed against Escherichia coli (ATCC 25922),
and Staphylococcus aureus (ATCC 25923) using neomy-
cin B as the control to generate the minimum inhibitory
NH3
O
HO
R1
HO
H3N
O
NH3
NH3
O
a
O
HO
HO
x(Cl-)n
18a-e
HO
R2
OH
Products
TC029
TC036
TC037
TC054
TC020
TC010
R1
CH2OH
H
R2
CH2OH
CH2OH
CH3
CH2OH
H
CH2NH3
Yield (%)
39
69
64
50
Ref. 3a
62
H
CH3
CH3
H
+
Scheme 6. Reagents: (a) (1) K2CO3, MeOH, rt; (2) PMe3, THF, 0.1M
NaOH, (3) H2, Pd/C, HOAc–H2O (1/1), (4) Dowex 1X8 (Cl-form).
Table 1. MIC13 and binding scores12 of the synthesized pyranmycins
Compounds
E. coli
S. aureusa
Binding scoreb
Neomycin B
TC029
TC036
2
22
11
91
87
19
9
0.3
7
ꢀ474.30
ꢀ314.90
ꢀ320.00
ꢀ320.40
ꢀ317.60
ꢀ314.50
ꢀ394.00
3
TC037
TC054
15
27
13
2
TC020
TC010
Figure 3. Binding of TC036 and TC054 toward the A-site of 16S
rRNA (TC036: blue, TC054: pink). The hydrogen atoms are omitted
for clarity except for those attached to the C-600 of TC054 (highlighted
in red).
a Unit: lM.
b The tendency in binding: the lower the number, the better the bind-
ing.

4392
J. Wang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4389–4393
2. For recent publications in the synthesis of aminoglycoside
antibiotics: (a) Ryu, D. H.; Tan, C.-H.; Rando, R. R.
Bioorg. Med. Chem. Lett. 2003, 13, 901–903; (b) Seeberger,
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E. Synlett 2003, 1323; (c) Hanessian, S.; Kornienko, A.;
Swayze, E. E. Tetrahedron 2003, 59, 995–1007; (d)
Tanaka, H.; Nishida, Y.; Furuta, Y.; Kobayashi, K.
Bioorg. Med. Chem. Lett. 2002, 12, 1723–1726; (e)
Greenberg, A. W.; Priestley, S.; Sears, P.; Alper, P.;
Rosenbohm, C.; Hendrix, M.; Wong, C.-H. J. Am. Chem.
Soc. 1999, 121, 6527–6541; (f) Hanessian, S.; Tremblay,
M.; Swayze, E. E. Tetrahedron 2003, 59, 983–993; (g)
Hanessian, S.; Tremblay, M.; Kornienko, A.; Moitessier,
N. Tetrahedron 2001, 57, 3255–3265; (h) Ding, Y.;
Hofstadler, A. S.; Swayze, E. E.; Risen, L.; Griffey, H.
R. Angew. Chem., Int. Ed. 2003, 42, 3409–3412; (i) Ding,
Y.; Hofstadler, A. S.; Swayze, E. E.; Risen, L.; Griffey, H.
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S.; Chen, C.-H.; Lu, L.-D.; Kulkarni, S. S.; Wong, C.-H.;
Hung, S.-C. Org. Lett. 2004, 6, 585–588; (k) Fridman, M.;
Belakhov, V.; Yaron, S.; Baasov, T. Org. Lett. 2003, 5,
3575–3578.
3. (a) Chang, C.-W. T.; Hui, Y.; Elchert, B.; Wang, J.; Li, J.;
Rai, R. Org. Lett. 2002, 4, 4603–4606; (b) Wang, J.; Li, J.;
Tuttle, D.; Takemoto, J.; Chang, C.-W. T. Org. Lett.
2002, 4, 3997–4000; (c) Elchert, B.; Li, J.; Wang, J.; Hui,
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M.; Chang, C.-T. J. Org. Chem. 2004, 69, 1513–1523.
4. Hung, S.-C.; Puranik, R.; Chi, F.-C. Tetrahedron Lett.
2000, 41, 77–80.
5. (a) Ng, C.; Stevens, J. D. Methods Carbohydr. Chem. 1976,
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6. Mazur, A. W.; Hiller, G. D. J. Org. Chem. 1997, 62,
4471–4475.
of the 5-C methyl group on TC054. The binding score of
TC054 also cannot explain the unexpected decreased in
antibacterial activity (Table 1).
In light of the finding in the activity of the newly synthe-
sized pyranmycins, we have learned valuable lessons.
First, a traditional strategy for drug development in-
volves identifying lead structural components (pharma-
cophores). Then, by combining these components on
the same scaffold, it is expected that an additional effect
from these components may lead to improved activity.14
Our results demonstrate that such an addition effect may
not always occur. As a result, synthetic methodologies
that can furnish compounds with more structural as-
pects for identifying leads are essential.
Second, as indicated in our molecular modeling results,
the binding (or fitting) of TC054, which has a ring III
branched-chain sugar, toward the rRNA target, is al-
most identical to that of the TC036. Therefore, there
must be other factors that could cause the dramatic de-
crease in antibacterial activity of TC054. Since amino-
glycosides exert their antimicrobial activity by binding
toward a cytosolic target (decoding region at A-site of
the 16S rRNA), understanding the process of how ami-
noglycosides are recruited by bacteria is essential. Addi-
tionally, from our experience3,15 and structures of other
unusual sugars in naturally occurring antibiotics,16–18
there are prevalent examples for the existence of methyl,
methoxy, methylamino, or dimethylamino groups,
which play key roles in the activity of these antibiotics.
As compared to hydroxyl and amino groups, the pres-
ence of a methyl group in the forms of methoxy, methyl-
amino, or dimethylamino groups will reduce the
solvation effect that may otherwise prevent the antibiot-
ics from entering the targeted sites. Perhaps, solvation is
another factor that deserves more investigation.
7. Zottola, M.; Rao, B. V.; Fraser-Reid, B. J. Chem. Soc.,
Chem. Commun. 1991, 969–970.
8. Lee, J.-C.; Tai, C.-A.; Hung, S.-C. Tetrahedron Lett. 2002,
43, 851–855.
9. Hydrolysis of the acetyl groups of 5, followed by regio-
selective tosylation at O-6 and azide substitution afforded
presumably an undesired 2,6-anhydro-L-idopyranose.
Attempts to use phenylthio 2,3,4-tri-O-benzyl-L-idopyr-
anoside were hampered by the low stereoselectivity of
glycosylation.
10. We obtained a complex mixture with a degraded disac-
charide as the major component when Tf2O/pyridine was
used for triflation followed by NaN3 for azide substitu-
tion. Hydrolysis of the acetyl groups of 18e, followed by
regioselective tosylation at O-600 and azide substitution
afforded presumably an undesired 200,600-anhydro-L-ido-
pyranose as the ring III component.
In conclusion, we have overcome synthetic challenges by
achieving a much more convenient synthesis of a
branched-chain sugar. The syntheses of three L-sugars
are reported. We have also demonstrated the practical
use of these unusual sugars.19 From our antibacterial re-
sults, new directions for the development of aminoglyco-
side antibiotics have also been suggested.
11. Methods for dilution antimicrobial susceptibility testing
for bacteria that grow aerobically. Approved standard M7-
A5, and performance standards for antimicrobial disk
susceptibility tests. Approved standard M2-A7, National
Committee for Clinical Laboratory Standards, Wayne, PA.
12. The score function was based on Amber 96 force-field as
implemented in HyperChem 7.0, and the solvent-accessi-
ble surface methodology to account for the hydration
effects. The program was developed for drug design, not
specifically for the calculation of absolute binding affinity.
Therefore, we look for the tendency in binding rather than
the meaning interpreted from the absolute numbers of
binding: the lower the number, the better the binding.
13. The MIC values were about 2-folds higher than the
reported values against S. aureus, for all the tested
compounds including neomycin B, presumably due to
the variation in the inoculated bacteria concentration.
Acknowledgements
We acknowledge National Institutes of Health
(AI053138), the Utah State University (CURI Grant),
DARPA (DAAD 19-03-1-0050), and National Founda-
tion for Infectious Diseases (New Investigator Matching
Grant) for generous financial support.
References and notes
1. For reviewing: Haddad, J.; Kotra, L. P.; Mobashery, S. In
Glycochemistry Principles, Synthesis, and Applications;
Wang, P. G., Bertozzi, C. R., Eds.; Marcel Dekker: New
York, 2001; pp 353.

J. Wang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4389–4393
4393
However, these values were consistent in duplicated tests
and were normalized to the reported values based on the
MIC of neomycin B for clearer comparison.
D2O) (chloride salt): d 100.0 (s), 95.7 (s), 83.1 (s), 75.9 (s),
75.5 (s), 73.7 (s), 73.1 (s), 72.5 (s), 70.6 (s), 70.2 (s), 69.7 (s),
68.6 (s), 57.1 (s), 53.4 (s), 49.6 (s), 48.9 (s), 40.2 (s), 28.2 (s);
LRFAB m/e 485 ([M4+ꢀ3H+]); HRFAB calcd for
C18H37N4O11 ([M4+ꢀ3H+ ]) m/e 485.2459; measure m/e
485.2454. TC037. 1H NMR (270 MHz, D2O) (acetate
salt): d 5.86 (d, J=3.9Hz, 1H, H-10), 5.13 (d, J=8.2Hz,
1H, H-100), 4.20 (m, 1H), 4.04 (dd, J=9.2Hz, J=9.7Hz,
1H), 3.8–4.0 (m, 2H), 3.79 (dd, J=9.9Hz, J=9.2Hz, 1H),
3.68 (dd, J=9.6Hz, J=5.6Hz, 1H), 3.60 (dd, J=9.6Hz,
J=8.9Hz, 1H), 3.2–3.5 (m, 2H), 3.19 (dd, J=13.5Hz,
J=6.6Hz, 1H), 2.42 (ddd, J=12.5Hz, J=4.3Hz,
J=4.3Hz, 1H, H-2eq), 1.94 (s, 12H,CH3CO2), 1.88 (m,
1H, H-2ax), 1.20 (d, J=6.9Hz, 3H, H-600); 13C NMR
(68MHz, D2O) (acetate salt): d 177.8 (s, CH3CO2), 99.0
(s), 95.9 (s), 81.7 (s), 75.8 (s), 74.1 (s), 73.0 (s), 71.7 (s, two
carbons), 70.9 (s), 70.7 (s), 69.7 (s), 68.4 (s), 53.4 (s), 49.6
(s), 48.8 (s), 40.2 (s), 28.1 (s), 21.2 (s, CH3CO2), 12.1 (s);
LRFAB m/e 469 ([M4+ꢀ3H+]); HRFAB calcd for
C18H37N4O10 ([M4+ꢀ3H+]) m/e 469.2510; measure m/e
469.2501. TC054. 1H NMR (270MHz, D2O) (acetate salt):
d 5.91 (d, J=3.9Hz, 1H, H-10), 5.30 (d, J=8.3Hz, 1H, H-
100), 3.8–4.1 (m, 6H), 3.3–3.8 (m, 10H), 2.51 (ddd,
J=12.5Hz, J=3.9Hz, J=3.9Hz, 1H, H-2eq), 1.92 (ddd,
J=12.5Hz, J=11.9Hz, J=11.9Hz, 1H, H-2ax), 1.33 (s,
3H, H-600); 13C NMR (100MHz, D2O) (acetate salt): d
176.9 (s, CH3CO2), 99.3 (s), 96.5 (s), 82.0 (s), 79.2 (s), 76.7
(s), 76.4 (s), 74.4 (s), 73.3 (s), 72.6 (s), 70.6 (s), 69.8 (s), 68.5
(s), 59.6 (s), 53.7 (s), 49.6 (s), 49.0 (s), 40.1 (s), 28.2 (s, C-2),
22.2 (s, CH3CO2), 20.7 (s); LRFAB m/e 499 ([M4+ꢀ3H+]);
HRFAB calcd for C19H39N4O11 ([M4+ꢀ3H+]) m/e
499.2615; measure m/e 499.2605.
14. Silverman, R. B. The Organic Chemistry of Drug Design
and Drug Action; Academic Press, 1992.
15. Li, J.; Wang, J.; Czyryca, P. G.; Chang, H.; Orsak, T. W.;
Evanson, R.; Chang, C.-W. T. Org. Lett. 2004, 6,
1381–1384.
16. Glycopeptide Antibiotics; Nagarajan, R., Ed.; Marcel
Dekker: New York, 1999.
17. Macrolide Antibiotics; Omura, S., Ed.; Academic Press:
New York, 1984.
18. Hooper, I. R. Aminoglycoside Antibiotics; Springer: New
York, 1982.
19. Selected spectroscopic information: TC029. 1H NMR
(270MHz, D2O) (acetate salt): d 6.03 (d, J=3.9Hz, 1H,
H-10), 5.23 (d, J=8.2Hz, 1H, H-100), 3.6–4.0 (m, 10H),
3.2–3.6 (m, 8H), 2.40 (ddd, J=12.6Hz, J=4.0Hz,
J=4.0Hz, 1H, H-2eq), 1.93 (s, 12H, CH3CO2), 1.8 (m,
1H, H-2ax); 13C NMR (68MHz, D2O) (acetate salt): d
178.1 (s, CH3CO2), 99.3 (s), 95.3 (s), 81.9 (s), 79.8 (s), 75.2
(s), 74.1 (s), 73.2 (s), 72.7 (s), 70.5 (s), 70.0 (s), 69.4 (s), 68.3
(s), 61.8 (s), 57.7 (s), 53.5 (s), 49.4 (s), 48.7 (s), 40.0 (s), 28.1
(s), 21.3 (s, CH3CO2); LRFAB m/e 515 ([M4+ꢀ3H+]);
HRFAB calcd for C19H39N4O12 ([M4+ꢀ3H+]) m/e
515.2564; measure m/e 515.2563. TC036. 1H NMR
(270MHz, D2O) (chloride salt): d 6.02 (d, J=4.0Hz, 1H,
H-10), 5.28 (d, J=7.9Hz, 1H, H-100), 3.8–4.2 (m, 9H), 3.67
(dd, J=9.2Hz, J=9.2Hz, 1H), 3.4–3.6 (m, 6H), 3.30 (dd,
J=13.9Hz, J=6.3Hz, 1H), 2.50 (ddd, J=12.5Hz,
J=3.9Hz, J=3.9Hz, 1H, H-2eq), 1.92 (ddd, J=12.5Hz,
J=12.9Hz, J=12.9Hz, 1H, H-2ax); 13C NMR (100MHz,