Synthesis and biological evaluation of novel neamine-nucleoside conjugates potentially targeting to RNAs

Article abstract of DOI:10.1016/j.tet.2009.04.084

Eighteen novel neamine-nucleoside conjugates with ethylenediamine-lysine or ethylenediamine-arginine as the linker were synthesized and their potential binding to A site of 16S RNA and TAR RNA was evaluated using SPR (surface plasmon resonance). Compared with neamine, compounds 10i and 10q show 6.3 and 4.8 times potential in binding to A site of 16S RNA and eight and six times potential in binding to TAR RNA, respectively. According to the data of SPR, it indicates that amino acid residue and nucleobase moieties of the designed neamine-nucleosides conjugates exhibit the important contributions for the binding to A site of 16S RNA and TAR RNA. The molecular docking study on the interaction between the ligands and A site of 16S RNA is in agreement with the experimental data. The novel type of modification may provide a promising way for the development of neamine derivatives effectively targeting to RNAs.

Full text of DOI:10.1016/j.tet.2009.04.084

Tetrahedron 65 (2009) 5228–5239
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and biological evaluation of novel neamine–nucleoside conjugates
potentially targeting to RNAs
*
Yanli Xu, Hongwei Jin, Zhenjun Yang, Liangren Zhang, Lihe Zhang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University 100191, Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Eighteen novel neamine–nucleoside conjugates with ethylenediamine–lysine or ethylenediamine–argi-
nine as the linker were synthesized and their potential binding to A site of 16S RNA and TAR RNA was
evaluated using SPR (surface plasmon resonance). Compared with neamine, compounds 10i and 10q
show 6.3 and 4.8 times potential in binding to A site of 16S RNA and eight and six times potential in
binding to TAR RNA, respectively. According to the data of SPR, it indicates that amino acid residue and
nucleobase moieties of the designed neamine–nucleosides conjugates exhibit the important contribu-
tions for the binding to A site of 16S RNA and TAR RNA. The molecular docking study on the interaction
between the ligands and A site of 16S RNA is in agreement with the experimental data. The novel type of
modification may provide a promising way for the development of neamine derivatives effectively tar-
geting to RNAs.
Received 4 March 2009
Received in revised form 22 April 2009
Accepted 24 April 2009
Available online 3 May 2009
Keywords:
Docking
Interaction
Neamine–nucleoside
SPR
Ó 2009 Elsevier Ltd. All rights reserved.
16S RNA
6'
NH2
1. Introduction
4'
5'
O
HO
H2N
4
II
1'
HO
2
2'
3
With the research advances in the clearance of RNA structure and
its important function in the life cycle of pathogenic microorganisms
and the processing of diseases, RNA has been viewed as an important
small molecular drug target for finding potential drugs.1,2 Amino-
glycoside antibiotic family including neomycin, kanamycin, para-
momycin, and so on has been known to bind potentially to many
important RNAs such as bacterial A site of 16S rRNA,3,4 HIV-1 trans-
activation response element (TAR) RNA,5 HIV-1 Rev responsive ele-
ment (RRE) RNA,6 group I introns,7 and the hammerhead ribozyme.8
But the high toxicity, which mainly resulted from the nonspecific
electrostaticinteractionslimitedtheirclinicuseatalargedegree,9 and
also, aminoglycosides are prone to lead to drug resistance because of
their own structural instability and the modification of aminoglyco-
side-modifying enzymes.10 The detailed study has shown that nea-
mine (Fig.1), as the commonpartof neomycin-class aminoglycosides,
is the minimum motif that can specifically bind to the A site of 16S
3'
NH2
H2N
O
1
I
5
HO
Neamine
6
OH
Figure 1. Structure of neamine.
RNA and affect the translation procedure in protein synthesis.4 It
seems a promising way to keep the neamine core and develop novel
potential neamine derivatives. Up to now, many efforts have been
made to optimize the core structure neamine, among which nea-
mine–peptide or nucleobase conjugates have been proved to be
a convenient and effective strategy to improve the binding to RNAs.
The neamine conjugates with arginine/lysine,11,12 arginine peptide,13
nucleobase,14 aromatic rings,15 peptide nucleic acid,16 and alkyl-
amines17 can enhanceits binding affinityand someof themhave been
showed certain binding selectivity. Until now there are no general
rules for the design of the specific as well as potential RNA-binding
small molecules, and all of the reported aminoglycoside conjugates
were designed by connecting neamine with peptide or nucleobase
directly to one of amino groups on the aminosugar moiety.
Abbreviations: Ac, acetyl; Arg, argnine; BAIB, [bis(acetoxy)-iodo]benzene; BOC,
tert-butoxycarbonyl; DCC, dicyclohexylcarbodiimide; DMF, N,N-dimethyl form-
amide; DMSO, dimethyl sulfoxide; Eda, ethylenediamine; Et, ethyl; Fmoc, 9-flu-
orenylmethyloxycarbonyl; HOBT, 1-hydroxybenzotriazole; Lys, lysine; Pbf,
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Py, pyridine; TEMPO,
2,2,4,6-tetramethyl-1-piperidinyloxyl; rt, room tempreture; THF, tetrahydro-
furanyl; Tf2O, trifluoromethanesulfonic anhydride.
Previously, two types of neamine–nucleoside conjugates were
synthesized from our group by the condensation of azidodi-
saccharide and nucleoside using ethylenediamine as a linker. Results
from the data of SPR evaluation suggested that the nucleobase played
a significant role to bind to A site of 16S RNA and an ethylenediamine
* Corresponding author. Tel.: þ86 10 82801700; fax: þ86 10 82802724.
E-mail address: zdszlh@bjmu.edu.cn (L. Zhang).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.04.084

Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
5229
NH2
NH2
O
O
HO
HO
HO
H2N
HN
H2N
HN
HO
NH2
10 m: R=H, B=T
H2N
O
NH2
H2N
O
OH
OH
10 a: R=H, B=T
10 b: R=OH, B=U
10 c: R=OH, B=A
10 d: R=OH, B=C
10 e: R=OH, B=G
10 f: R=OH, B= I
10 n: R=OH, B=U
10 o: R=OH, B=A
10 p: R=OH, B=C
10 q: R=OH, B=G
10 r: R=OH, B= I
Type III
Type I
NH
NH
O
O
H
N
H
N
H
N
O
O
H2N
B
B
O
H2N
O
NH
OH
R
OH
R
NH2
O
O
N
O
NH2
HO
HO
H2N
HN
N
NH
NH
N
10 g: R=H, B=T
T=
U=
A=
I=
NH2
H2N
O
10 h: R=OH, B=U
10 i: R=OH, B=A
10 j: R=OH, B=C
10 k: R=OH, B=G
10 l: R=OH, B= I
N
O
N
O
N
OH
Type II
NH2
O
N
O
NH2
N
N
N
NH
NH
HN
O
C=
B
G=
N
O
H
N
N
O
N
NH2
N
O
OH
R
Figure 2. Structures of neamine–nucleoside conjugates.
HO
HO
HO
O
linker between the neamine and nucleoside was favorable for this
binding.18 In this report we prepared a new class of neamine–nu-
cleoside conjugates (Fig. 2), in which an ethylenediamine–amino
acid was used as linker for the modification of the 5-hydroxyl group
of neamine by nucleosides. The amino groups on neamine play an
important role in their binding to the target RNAs. Keeping the amino
group free in the neamine molecule and modification of other po-
sitions of neamine may lead the derivatives contributing a various
interaction to RNA. Compounds 10a–r were designed using a flexible
side chain to connect the neamine and nucleosides, which were
expected to recognize the specific site of RNA. Moreover, lysines and
arginines are oftenpresent in the RNA-binding proteins (forexample,
HIV-1 transactiviting (Tat) proteins and regulator of expression of
virion (Rev) proteins),19,20 neamine–arginine and neamine–lysine
conjugates both enhance the inhibition of HIV-1 TAR-Tat interaction
compared with their mother compound neamine.11 Based on these
backgrounds, three types of neamine–nucleoside conjugates were
synthesized: type I (compounds 10a–f) consists in neamine de-
rivatives with ethylenediamine–lysine as the linker and nucleosides
B
B
a
B
b
O
O
O
O
O
OH OH
O
O
3 a: B=A
3 b: B=C
3 c: B=G
3 d: B= I
4 a: B=A
4 b: B=C
4 c: B=G
4 d: B= I
5 a: B=A
5 b: B=C
5 c: B=G
5 d: B= I
Scheme 1. Synthesis of protected 50-carboxylic acid-nucleosides 5a, 5b, 5c, and 5d. Re-
agents and conditions: (a) CH3COCH3, 70% HClO4, rt; (b) BAIB, TEMPO, CH3CN, H2O, rt.
5'
O
HO
4'
5'
HO
B
1'
B
1'
O
O
2'
3'
OAc R
4'
2'
R
3'
OH
1 a: R=H,
B=T
2 a: R=H,
2 b: R=OAc, B=U
B=T
1 b: R=OH , B=U
Figure 3. Structures of protected 50-carboxylic acid-nucleosides 2a and 2b.
connecting at the
the similar structure as type II but the nucleosides are condensed
with the -NH2 group of lysine; type III (compounds 10m–r) is
formed by a number of neamine derivatives with ethylenediamine–
arginine as the linker and nucleoside connecting at the -NH2 group
of arginine. The interactions between compounds 10a–r with the A
site of 16S RNA and TAR RNA were evaluated by dissociation con-
stants (KD values in mM) using SPR method. The dissociation con-
a-NH2 group of lysine; type II (compounds 10g–l) is
prepared from compounds 1a and 1b according to the published
procedures.18 Compounds 5a, 5b, 5c, and 5d were synthesized from
the nucleosides 3a–d. After the protection of 20- and 30-hydroxyl
group by isopropylidene group, the nucleoside intermediates 4a, 4b,
4c, and 4d were obtained in good yield. Compounds 4a–d were
oxidized by BAIB and TEMPO in mild conditions to afford the desired
50-carboxylic acid-nucleosides 5a, 5b, 5c, and 5d in 75–82% yields.
For the synthesis of compounds of types I–III (Schemes 2 and 3),
compound 6 was prepared by the reported procedure and used as
the starting material.18 Compounds 8a and 8b were synthesized by
the condensation reaction of compound 6 with Boc-Lys(Fmoc)-OH
to give compound 7a first. For compound 8a, the Boc-protection
3
a
stants clearly indicated that three types of neamine–nucleoside
conjugates showed the better binding properties to 16S RNA and TAR
RNA than the neamine derivatives reported previously.18
2. Results and discussion
group on
CF3COOH in CH2Cl2 (1:4) and compound 8b was obtained by the
removal of Fmoc-protection group on -NH2 group of lysine moiety
a-NH2 group of lysine was selectively removed by
2.1. Synthesis of neamine–nucleoside conjugates 10a–r
3
using Et2NH in DMF (1:9). Compound 6 was condensed with Fmoc-
Arg(Pbf)-OH to yield 7b, after the removal of the Fmoc group on the
Six protected 50-carboxylic acid-nucleosides 2a, b, 5a–d were
synthesized (Scheme 1, Fig. 3), among which 2a and 2b were
a-NH2 group of arginine using the same procedure as the

5230
Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
Nea-Eda
Nea-Eda
9 a: B=T
9 b: B=U
9 c: B=A
9 d: B=C
9 e: B=G
9 f: B=I
NH
O
7 a: R1=Boc
c
b
NH
8 a: R1=H
H
O
O
N
B
NHR1
O
*
FmocHN
a
FmocHN
N3
O
4
5
*
R
R
HO
HO
N3
Nea-Eda
Nea-Eda
NHBoc
9 g: B=T
9 h: B=U
9 i: B=A
9 j: B=C
9 k: B=G
9 l: B=I
N3
N3
O
O
HN
NH
OH
7 a: R2=Fmoc
8 b: R2=H O
B
f
e
N
H
HN
d
O
6
O
NHBoc
4
5
R2HN
*
*
R
R
NH2
Nea-Eda
g
9 m:B=T
9 n: B=U
9 o: B=A
9 p: B=C
9 q: B=G
9 r: B=I
Nea-Eda
NH
O
7 b: R3=Fmoc
8 c: R3=H
H
i
h
NH
O
H
O
H
N
N
B
PbfHN
O
*
NHR3
PbfHN
N
NH
4
5
*
R
R
NH
Scheme 2. Synthesis of protected neamine–nucleoside conjugates. Reagents and conditions: (a) Boc-Lys(Fmoc)-OH, HOBt, DCC, DMF, 0 ꢀC to rt; (b) CF3COOH/CH2Cl2 (1:4), rt; (c) 50-
carboxylic acid-nucleoside (2a, 2b, 5a, 5b, 5c, and 5d), HOBt, DCC, DMF, 0 ꢀC to rt; (d) Boc-Lys(Fmoc)-OH, HOBt, DCC, DMF, 0 ꢀC to rt; (e) Et2NH/DMF (1:9), rt; (f) 50-carboxylic acid-
nucleoside (2a, 2b, 5a, 5b, 5c, and 5d), HOBt, DCC, DMF, 0 ꢀC to rt; (g) Fmoc-Arg(Pbf)-OH, HOBt, DCC, DMF, 0 ꢀC to rt; (h) Et2NH/DMF (1:9), rt; (i) 50-carboxylic acid-nucleoside (2a,
2b, 5a, 5b, 5c, and 5d), HOBt, DCC, DMF, 0 ꢀC to rt. Note: (1) [Nea-Eda] represents the structure of compound 6; (2) for the nucleoside part, when B¼T, R4*¼OAc, R5*¼H; when B¼U,
R4*¼R5*¼OAc, and when B¼A, C, G, and I, R4* and R5* represents the isopropylidene protection of nucleoside 20- and 30-hydroxyl group.
Type I
Type II
Type III
Three types of desired products 10a–r were obtained in good
yields after removal of all the protection groups (Scheme 3). The
target molecules 10a–r were purified by reversed-phase HPLC (1&
CF3COOH in H2O and CH3OH) and obtained as the appropriate tri-
fluoroacetic salts. The intermediates and final products were
characterized by 1H NMR, 13C NMR, HRMS, and optical rotations.
(1), (5), (6)
(2), (5), (6)
9 a
9 b
9 c
9 d
9 e
9 f
(3), (5), (6)
10 a
10 b
10 c
10 d
10 e
10 f
9 g
9 h
9 i
9 j
9 k
9 l
10 g
10 h
10 i
10 j
10 k
10 l
9 m
9 n
10 m
10 n
10 o
10 p
10 q
10 r
9 o
9 p
9 q
9 r
(1), (4), (6)
(2), (6)
(4), (6)
Scheme 3. Synthesis of neamine–nucleoside conjugates. Reagents and conditions: (1)
Et2NH/DMF (1:9), rt; (2) CF3COOH/H2O/PhSCH3 (94:3:3), rt; (3) CF3COOH/CH2Cl2 (1:4),
rt; (4) CF3COOH/H2O (9:1), 0 ꢀC; (5) NH3/CH3OH, 0 ꢀC. (6) H2S, pyridine/Et3N/H2O
(4:3:2), rt.
2.2. RNA-binding assay and molecular docking study
The binding properties of the neamine–nucleoside conjugates
10a–r to the A site of 16S RNA and TAR RNA (Fig. 4a and b) were
evaluated by the determination of dissociation constants using SPR
Biacore 3000 instrument,21,22 and the dissociation constants (KD
preparation of compound 8b, compound 8c was obtained in 85%
yield. Compounds 8a, 8b, and 8c were condensed with the pro-
tected 50-carboxylic acid-nucleosides 2a, b and 5a–d, respectively,
to provide compounds 9a–r in 70–82% yields.
values in
mM) were listed in Table 1. The dissociation constants
clearly indicate that (1) three types of neamine–nucleoside conju-
gates show the better binding properties to 16S RNA and TAR RNA
than the neamine derivatives reported previously.18 It indicates
that the amino acid–ethylenediamine linker in these compounds
a
G
G
C
G
U
C
A
C
C
C
25
G
C
5
U
Table 1
G
A
The KD values of neamine–nucleoside conjugates binding to A site of 16S and TAR
RNA
20
A
G
U
A
Comp
KD (16S)
m
M
KD (TAR) mM
10
C
G
G
Neamine
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
19
35
39
14
16
18
23
13
13
3
18
9
9
14
14
7
12
4
24
46
49
12
29
21
27
19
27
3
21
15
11
13
12
9
C
U
G 15
U C
b
G
C
C
G
G
30
G
C
C
A
5 U
C
G
A
25
U
U
C
U
C
G
A 20
U
C
G
C
C
10
G
A
U
G
10
4
5
G
G
15
13
Figure 4. (a) Structure of A site of 16S RNA, (b) structure of TAR RNA.

Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
5231
13
mM, respectively). It seems that the
3
-amino side chain of lysine
a
in the structure of the compound of type I does not exhibit the
contribution for the interaction with RNA and the guanidino group
of arginine side chain exhibits more contributions in this in-
teraction. (3) Interestingly, all of the compounds consisting of ad-
enine or guanine (10c, 10g, 10i,10k and 10o,10q) indicate the better
KD values than the others, among which 10i and 10q showed the KD
values of 6.3 and 4.8 times potential in binding to 16S RNA and
eight and six times potential in binding to TAR RNA, respectively (3
and 4 mM to A site of 16S RNA and TAR RNA, respectively). It exhibits
that the nucleobase could play an important role in the recognition
of the designed compounds and target RNA.
To obtain more information about the interaction between the
neamine–nucleoside conjugates and the A site of 16S RNA, molecular
docking study of 10i and 10q was investigated.23 The structure of A
site of 16S RNA fragment was extracted from the protein databank
(PDB code 1PBR) and the software AutoDock 3.0 was used for the
molecular docking calculation of two neamine derivatives. By com-
puter calculation, the final configuration (Fig. 5a and b) with the
lowest docking energy was obtained and used for the analysis. Fig-
ure 5 shows the interaction between the two compounds and A site
of 16S RNA. The electrostatic interactions and hydrogen bonds were
considered as the major interactions, which were responsible for the
ligands binding to the pocket of A site of 16S RNA. For compound 10i,
in addition to the four amino groups of neamine, nucleoside base
adenine can form electrostatic interaction with U18 residue of A site
of 16S RNA, and for compound 10q, besides the four amino groups of
neamine, the NH2 on the guanidino group can form electrostatic
interaction with A21 residue of A site of 16S RNA and the nucleobase
guanine can form hydrogen bond with U23 residue of A site of 16S
RNA. Figure 5a and b indicates that the amino acid–ethylenediamine
linker could lead the neamine–nucleoside conjugates to the binding
pocket of RNA and help improving the binding affinities to the target
RNA. The molecular docking study is in agreement with the exper-
imental data.
U5 PO2
A
C6 PO2
H3N
NH3
O
A7 PO2
NH3
U18 PO2
HO
HO
H3N
O
NH2
N
OH
HN
HN
G17 N7
N
N
NH3
N
O
N
H
O
O
OH OH
b
2.3. Conclusion
Eighteen novel neamine–nucleoside conjugates 10a–r with ethyl-
enediamine–lysine or ethylenediamine–arginine as the linker were
synthesized and their potential binding to16S RNA and TAR RNAwas
evaluated using SPR (surface plasmon resonance). Compared with
neamine, compounds 10i and 10q show 6.3 and 4.8 times potential
in binding to A site of 16S RNA and eight and six times potential in
binding to TAR RNA, respectively. According to the data of SPR, it
indicate that amino acid residue and nucleobase exhibit the
important contributions for the binding of the designed neamine–
nucleosides conjugates to A site of 16S RNA and TAR RNA. The mo-
lecular docking study on the interaction between the ligands and A
site of 16S RNA is in agreement with the experimental data. The
novel type of modification may provide a promising way for the
development of neamine derivatives effectively targeting to RNAs.
G17 PO2
B
G17 PO2
NH3
O
U5 PO2
NH3
HO
HO
H3N
H3N
O
OH
HN
NH
C6 PO2
O
N
U23 O4
N
NH
NH2
O
H
N
H
N
O
O
H3N
N
NH
3. Experimental section
OH OH
A21 PO2
3.1. Synthesis of neamine–nucleosides
Figure 5. Molecular modeling for the interactions between 10i (a, A), 10 q (b, B) and A
site of 16S RNA. Panel (a) and (b) shows the hydrogen bonds only and panel (A) and (B)
indicates the interactions including all the hydrogen bonds and electrostatic
interactions.
3.1.1. General procedures for the synthesis of neamine–nucleoside
conjugates 10a–r
All chemical reagents and solvents were purchased from com-
mercial suppliers. DMF was distilled from P2O5 below 40 ꢀC. Silica
gel 60H (200–300 mesh) manufactured by Qing-dao Haiyang
Chemical Company (China) was used for column chromatography.
1H and 13C NMR spectra were recorded on Bruker 400 MHz in-
strument with TMS as an internal standard. HRMS was obtained at
MDS SCIEX QSTAR and Bruker DALTONICS APEX IV 70e
could increase the binding affinities. (2) Compounds of types II and
III show the binding properties with equal to or better than the
parent neamine (for A site of 16S RNA, the dissociation constants KD
of compounds of type II are 3–18
mM and type III are 4–14
mM and
for TAR RNA compounds of type II, KD¼3–27
m
M and type III KD¼4–

5232
Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
instruments, and the data were reported in m/e (intensity to 100%).
UV absorption spectra were recorded on Cary 300 and optical ro-
tations were obtained by Rudolph Research Analytical Autopol III
Automatic Polarimeter.
3.1.3.4. 50-Carboxylic acid-20,30-O-isopropylidene-inosine(5d). Yield:
75% from 4d. 1H NMR (400 MHz, DMSO-d6):
d 8.23 (s, 1H, H-2), 8.01
(s, 1H, H-8), 6.31 (s, 1H, H-10), 5.45 (d, 1H, J¼5.5 Hz, H-20), 5.40 (d,
1H, J¼5.5 Hz, H-30), 4.70 (s, 1H, H-40), 1.52 (s, 3H, –CH3), 1.34 (s, 3H,
–CH3).
3.1.2. General procedure for the synthesis of 4a–d
To a solution of nucleosides 3a–d (2.0 g) in acetone (120 mL)
was added HClO4 (70%, 0.9 mL), the reaction mixture was kept
stirring for 1.5 h at room temperature before aqueous ammonia
(28%, 1.5 mL) was added. After standing by 3 h, the reaction mix-
ture was filtered and compounds 4a–d were obtained as white
powder.
3.1.4. General procedure for the synthesis of 7a and 7b
DCC (29 mg, 0.14 mmol), HOBt (19 mg, 0.14 mmol), and Fmoc-
Arg(Pbf)-OH or Boc-Lys(Fmoc)-OH (0.14 mmol) were dissolved in
anhydrous DMF (1.5 mL). The mixture was stirred in an ice bath for
0.5 h then compound 6 (55 mg, 0.12 mmol) was added. After 6 h,
the reaction mixture was filtered and concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(CH2Cl2/CH3OH¼100:4) to afford a white foam.
3.1.2.1. 20,30-O-Isopropylidene-adenosine (4a). Yield: 78% from 3a.
1H NMR (400 MHz, DMSO-d6):
d 8.34 (s, 1H, H-8), 8.15 (s, 1H, H-2),
7.34 (s, 2H, –NH2), 6.12 (d, J¼3.0 Hz, H-10), 5.35 (dd, 1H, J¼6.0,
3.0 Hz, H-20), 5.23 (t, 1H, J¼5.5 Hz, –OH), 4.96 (dd, 1H, J¼4.8, 2.5 Hz,
H-30), 4.20–4.22 (m, 1H, H-40), 3.52–3.55 (m, 2H, H-50), 1.55 (s, 3H,
–CH3), 1.33 (s, 3H, –CH3).
3.1.4.1. epi-5-N[[[aN-(tert-Butoxycarbonyl)-3N-(9-fluorenylmethyloxycar-
bonyl)]-lysinyl]aminoethyl]-1,3,20,60-tetraazido neamine (7a). Yield: 74%
from 6, white foam. 1H NMR (400 MHz, CD3OD):
d 7.79 (d, 2H,
J¼7.5 Hz, 2ꢁFmoc-H), 7.65 (d, 2H, J¼7.4 Hz, 2ꢁFmoc-H), 7.39 (t,
2H, J¼7.4 Hz, 2ꢁFmoc-H), 7.31 (t, 2H, J¼7.4 Hz, 2ꢁFmoc-H), 5.11 (d,
1H, J¼3.5 Hz, H-10), 4.35 (d, 2H, J¼6.8 Hz, Fmoc-CH2–), 4.20 (t, 1H,
3.1.2.2. 20,30-O-Isopropylidene-cytidine (4b). Yield: 74% from 3b. 1H
NMR (400 MHz, DMSO-d6):
d
7.72 (d, 1H, J¼7.4 Hz, H-6), 7.27
J¼6.8 Hz, Fmoc-CH–), 3.98–4.02 (m, 2H, H-50 and Lys-
a-CH–), 3.76–
(d, J¼33.0 Hz, 2H, –NH2), 5.77 (d, J¼2.0 Hz, 1H, H-10), 5.71 (d, 1H,
J¼7.4 Hz, H-5), 4.99 (t,1H, J¼5.1 Hz, –OH), 4.85 (dd,1H, J¼6.3, 2.1 Hz,
H-20), 4.74 (dd,1H, J¼6.2, 3.8 Hz, H-30), 4.03 (dd,1H, J¼8.3, 4.3 Hz, H-
40), 3.55–3.59 (m, 2H, H-50), 1.48 (s, 3H, –CH3), 1.28 (s, 3H, –CH3).
3.92 (m, 3H, H-30, 1 and 3), 3.59–3.62 (dd, 1H, J¼10.2, 3.2 Hz, H-4),
3.50–3.54 (dd, 1H, J¼13.3, 2.2 Hz, H-60a), 3.27–3.41 (m, 7H, H-20, 40,
5, 6, 60b and Lys-CH2-), 2.98–3.07 (m, 2H, Eda-CH2–), 3.10–3.14 (m,
2H, Eda-CH2–), 2.18–2.22 (m, 1H, Heq-2), 1.58–1.76 (m, 2H, Lys-CH2-
), 1.48–1.52 (m, 2H, Lys-CH2–), 1.32–1.48 (m, 2H, Lys-CH2–), 1.44 (s,
9H, –CH3ꢁ3), 1.13–1.23 (q, 1H, J¼12.5 Hz, Hax-2). 13C NMR
3.1.2.3. 20,30-O-Isopropylidene-guanosine (4c). Yield: 79% from 3c.
1H NMR (400 MHz, DMSO-d6):
d
10.66 (br, 1H, H-1), 7.91 (s, 1H, H-
(100 MHz, CD3OD): d 178.00 (–C]O), 158.99 (–C]O), 157.90
8), 6.50 (s, 2H, –NH2), 5.92 (d, J¼3.0 Hz, H-10), 5.19 (dd, 1H, J¼6.5,
3.0 Hz, H-20), 5.03 (t, 1H, J¼5.5 Hz, –OH), 4.97 (dd, 1H, J¼6.5, 3.0 Hz,
H-30), 4.10–4.13 (m, 1H, H-40), 3.51–3.54 (m, 2H, H-50), 1.51 (s, 3H,
–CH3), 1.32 (s, 3H, –CH3).
(–C]O), 145.39 (2ꢁFmoc-C), 142.62 (2ꢁFmoc-C), 128.78 (2ꢁFmoc-
C), 128.16 (2ꢁFmoc-C), 126.19 (2ꢁFmoc-C), 120.94 (2ꢁFmoc-C),
95.62 (C-10), 80.66 (Boc-C), 78.97 (C-4), 75.79 (C-40), 73.53 (C-6),
73.13 (C-30), 72.59 (C-5), 67.64 (Fmoc-CH2–), 64.91 (C-50), 60.69 (C-
20), 59.72 (C-1), 59.44 (C-3), 56.50 (Lys- -C), 52.53 (C-60), 50.42
a
3.1.2.4. 20,30-O-Isopropylidene-inosine (4d). Yield: 75% from 3d. 1H
(Eda-CH2– and Fmoc-CH–), 41.29 (Eda-CH2– and Lys-CH2–), 33.68
NMR (400 MHz, DMSO-d6):
d
12.42 (s, 1H, H-1), 8.31 (s, 1H, H-2),
(C-2), 33.09 (Lys-CH2–), 30.52 (Lys-CH2–), 28.75 (3ꢁBoc-CH3),
8.09 (s, 1H, H-8), 6.10 (d, 1H, J¼3.0 Hz, H-10), 5.26 (dd, 1H, J¼6.0,
3.0 Hz, H-20), 5.11 (t, 1H, J¼5.0 Hz, –OH), 4.93 (dd, 1H, J¼6.5, 2.5 Hz,
H-30), 4.20–4.24 (m, 1H, H-40), 3.52–3.55 (m, 2H, H-50), 1.54 (s, 3H,
–CH3), 1.32 (s, 3H, –CH3).
24.09 (Lys-CH2–). HRMS calcd for C40H54N16O10 ([MþH]þ):
20
919.4282, found: 919.4278. [
a
]
0.545 (CH3OH, c 0.099).
D
3.1.4.2. epi-5-N[[[aN-(9-Fluorenylmethyloxycarbonyl)-GN-(2,2,4,6,7-pen-
tamethyl dihydrobenzofuran-5-sulfonyl)]arginyl]aminoethyl]-1,3,20,60-tet-
raazido neamine (7b). Yield: 79% from 6, white foam. 1H NMR
3.1.3. General procedure for the synthesis of 5a–d18
Compounds 4a–d (3 mmol), BAIB (6 mmol), and TEMPO
(0.6 mmol) were mixed and to this mixture was added 2 mL of a 1:1
CH3CN/H2O solution. The reaction mixture was stirred for 4 h at
room temperature. The solvent was removed and the residue was
washed with diethyl ether and acetone to afford compounds 5a–
d as white foam.
(400 MHz, CD3OD):
d
7.78 (d, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 7.66 (t, 2H,
J¼7.7 Hz, 2ꢁFmoc-H), 7.38 (t, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 7.30
(t, 2H, J¼7.3 Hz, 2ꢁFmoc-H), 5.12 (d, 1H, J¼3.0 Hz, H-10), 4.37–4.47
(m, 2H, Fmoc-CH2–), 4.21 (t,1H, J¼6.8 Hz, Fmoc-CH–), 4.06–4.13 (m,
1H, Arg-a
-CH–), 3.98–4.01 (m, 1H, H-50), 3.75–3.94 (3H, H-1, 3 and
30), 3.58–3.61 (dd, 1H, J¼9.2, 2.7 Hz, H-4), 3.50 (dd, 1H, J¼11.3,
2.1 Hz, H-60a), 3.27–3.44 (m, 7H, H-60b, 20, 40, 5, 6, and Arg-CH2–),
3.15–3.18 (m, 2H, Eda-CH2–), 2.96–3.08 (m, 2H, Eda-CH2–), 2.98
(s, 2H, Pbf-CH2–), 2.59 (s, 3H, Pbf-CH3), 2.52 (s, 3H, Pbf-CH3), 2.14–
2.17 (m, 1H, Heq-2), 2.07 (s, 3H, Pbf-CH3), 1.52–1.79 (m, 4H, 2ꢁArg-
CH2–), 1.42 (s, 6H, 2ꢁPbf-CH3), 1.11–1.20 (m, 1H, Hax-2). 13C NMR
3.1.3.1. 50-Carboxylic acid-20,30-O-isopropylidene-adenosine (5a). Yield:
82% from 4a. 1H NMR (400 MHz, DMSO-d6):
d 8.42 (s, 1H, H-8), 8.01 (s,
1H, H-2), 7.25 (s, 2H, –NH2), 6.28 (s, 1H, H-10), 5.34–5.38 (m, 2H, H-20
and 30), 4.60 (s, 1H, H-40), 1.52 (s, 3H, –CH3), 1.34 (s, 3H, –CH3).
(100 MHz, CD3OD):
d 174.81 (–C]O), 159.90 (Pbf-C), 158.46
3.1.3.2. 50-Carboxylic acid-20,30-O-isopropylidene-cytidine (5b). Yield:
(–C]O), 158.17 (–C]NH), 145.39 (2ꢁFmoc-C), 142.63 (2ꢁFmoc-C),
139.43 (Pbf-C), 134.32 (Pbf-C), 133.55 (Pbf-C), 128.80 (2ꢁFmoc-C),
128.19 (2ꢁFmoc-C), 126.22 (2ꢁFmoc-C), 126.05 (Pbf-C), 120.95
(2ꢁFmoc-C), 118.47 (Pbf-C), 95.72 (C-10), 87.65 (Pbf-C), 79.50
(C-4), 75.67 (C-40), 73.53 (C-6), 73.14 (C-30), 72.58 (C-5), 67.85
(Fmoc-CH2–), 64.90 (C-50), 60.66 (C-20), 59.69 (C-1), 59.47
77% from 4b. 1H NMR (400 MHz, DMSO-d6):
d 12.51 (br,1H, –COOH),
7.72 (d, 1H, J¼6.0 Hz, H-6), 7.22 (d, 2H, J¼26.0 Hz, –NH2), 5.67 (d,
J¼7.0 Hz, 1H, H-5), 5.63 (s, 1H, H-10), 5.52 (dd, 1H, J¼6.0, 2.0 Hz, H-
30), 5.06 (d, 1H, J¼6.0 Hz, H-20), 4.47 (d, 1H, J¼2.0 Hz, H-40), 1.45 (s,
3H, –CH3), 1.29 (s, 3H, –CH3).
(C-3), 56.50 (Arg-a
-CH–), 52.52 (C-60), 50.45 (Eda-CH2– and Fmoc-
3.1.3.3. 50-Carboxylic acid-20,30-O-isopropylidene-guanosine (5c). Yield:
CH–), 43.95 (Pbf-CH2–), 41.30 (Eda-CH2– and Arg-CH2–), 33.50 (C-2
and Arg-CH2–), 28.68 (2ꢁPbf-CH3 and Arg-CH2–), 19.63 (Pbf-
CH3), 18.43 (Pbf-CH3), 12.52 (Pbf-CH3). HRMS calcd for
79% from 4c. 1H NMR (400 MHz, DMSO-d6):
d 10.80 (s, 1H, H-1),
8.03 (s, 1H, H-8), 6.55 (s, 2H, –NH2), 6.03 (d, 1H, J¼1.0 Hz, H-10), 5.32
(s, 1H, H-20), 5.15 (d, 1H, J¼5.5 Hz, H-30), 4.48 (s, 1H, H-40), 1.51 (s,
3H, –CH3), 1.36 (s, 3H, –CH3).
C48H62N18O11S ([MþH]þ): 1099.4639, found: 1099.4643. [
(H2O, c 0.101).
a]
20 0.446
D

Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
5233
3.1.5. epi-5-N[[[3N-(9-Fluorenylmethyloxycarbonyl)]-
(Pbf-C), 118.46 (Pbf-C), 95.67 (C-10), 87.69 (Pbf-C), 79.03 (C-4), 75.81
(C-40), 73.55 (C-6), 73.12 (C-30), 72.58 (C-5), 64.92 (C-50), 60.76 (C-20),
lysinyl]aminoethyl]-1,3,20,60-tetraazido neamine (8a)
Compound 7a (50 mg, 0.05 mmol) was dissolved in 20%
CF3COOH in CH2Cl2 (2 mL) and kept stirring for about 1.5 h at room
temperature then Et2O (2 mL) was added and the white solid pre-
cipitated was purified by column chromatography on silica gel with
gradient elution (CH2Cl2/CH3OH¼100:5) and the final pure product
8a was obtained as a white foam (34 mg, yield 76%). 1H NMR
59.80 (C-1), 59.42 (C-3), 55.75 (Arg-a
-CH–), 52.54 (C-60), 50.58 (Eda-
CH2–), 43.98 (Pbf-CH2–), 41.18 (Eda-CH2– and Arg-CH2–), 33.66 (C-2),
33.24 (Arg-CH2–), 28.72 (2ꢁPbf-CH3 and Arg-CH2–), 19.59 (Pbf-CH3),
18.40 (Pbf-CH3), 12.51 (Pbf-CH3). HRMS calcd for C33H52N18O9S
([MþH]þ): 877.3958, found: 877.3955. [
a]
D
20 0.599 (CH3OH, c 0.089).
(400 MHz, CD3OD):
d
7.79 (d, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 7.65 (d, 2H,
3.1.8. General procedure for the synthesis of 9a–r
J¼7.4 Hz, 2ꢁFmoc-H), 7.39 (t, 2H, J¼7.4 Hz, 2ꢁFmoc-H), 7.32 (t,
2H, J¼7.4 Hz, 2ꢁFmoc-H), 5.10 (d, 1H, J¼3.5 Hz, H-10), 4.35 (d, 2H,
J¼6.8 Hz, Fmoc-CH2–), 4.20 (t, 1H, J¼6.8 Hz, Fmoc-CH–), 3.98–4.03
(m, 1H, H-50), 3.78–3.93 (m, 3H, H-1, 3 and 30), 3.60 (dd, 1H, J¼10.2,
3.2 Hz, H-4), 3.52 (dd, 1H, J¼13.3, 2.2 Hz, H-60a), 3.31–3.44 (m, 8H,
DCC (0.13 mmol), HOBt (0.13 mmol), and compounds 2a, 2b, 5a,
5b, 5c or 5d (0.13 mmol) were dissolved in 1.5 mL anhydrous DMF.
The mixture was stirred in an ice bath for 0.5 h then compound 8a,
8b or 8c (0.10 mmol) was added. After 6 h, the reaction mixture was
filtered and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (CH2Cl2/CH3OH¼100:3) to
afford a white foam.
H-60b, 20, 4, 5, 6, Lys-
a-CH– and Lys-CH2–), 3.11–3.14 (m, 2H, Eda-
CH2–), 2.94–3.06 (m, 2H, Eda-CH2–), 2.17–2.22 (m, 1H, Heq-2), 1.36–
1.45 (m, 2H, Lys-CH2–), 1.48–1.74 (m, 4H, 2ꢁLys-CH2–), 1.13–1.22 (q,
1H, J¼12.5 Hz, Hax-2). 13C NMR (100 MHz, CD3OD):
d
177.74
3.1.8.1. Compound 9a. Yield: 80% from 8a, white foam. 1H NMR
(–C]O), 158.94 (–C]O), 145.38 (2ꢁFmoc-C), 142.62 (2ꢁFmoc-C),
128.78 (2ꢁFmoc-C), 128.16 (2ꢁFmoc-C), 126.18 (2ꢁFmoc-C), 120.94
(2ꢁFmoc-C), 95.61 (C-10), 78.94 (C-4), 75.83 (C-40), 73.55 (C-6),
73.12 (C-30), 72.59 (C-5), 67.67 (Fmoc-CH2–), 64.92 (C-50), 60.78 (C-
a
20), 59.79 (C-1), 59.37 (C-3), 56.21 (Lys- -CH–), 52.53 (C-60), 50.53
(Eda-CH2– and Fmoc-CH–), 41.15 (Eda-CH2– and Lys-CH2–), 36.01
(400 MHz, CD3OD):
d
7.84 (s, 1H, H-6), 7.78 (d, 2H, J¼7.6 Hz,
2ꢁFmoc-H), 7.63 (d, 2H, J¼7.4 Hz, 2ꢁFmoc-H), 7.38 (t, 2H, J¼7.4 Hz,
2ꢁFmoc-H), 7.30 (t, 2H, J¼7.4 Hz, 2ꢁFmoc-H), 6.20 (dd, 1H, J¼8.0,
6.1 Hz, H-10), 5.46 (d, 1H, J¼6.1 Hz, H-30), 5.11 (d, 1H, J¼3.6 Hz, H-
1%), 4.51 (d, 1H, J¼1.5 Hz, H-40), 4.34–4.37 (m, 3H, Fmoc-CH2– and
Lys-
a
-CH–), 4.19 (t, 1H, J¼6.8 Hz, Fmoc-CH–), 4.00–4.03 (m, 1H, H-
(Lys-CH2–), 33.67 (C-2), 30.72 (Lys-CH2–), 23.92 (Lys-CH2–). HRMS
5%), 3.75–3.94 (m, 3H, H-3%, 100 and 300), 3.59–3.63 (dd, 1H, J¼10.2,
3.3 Hz, H-400), 3.50–3.54 (dd, 1H, J¼13.2, 2.2 Hz, H-6%a), 3.30–3.48
(m, 7H, H-6%b, 2%, 4%, 500, 600 and Lys-CH2–), 2.98–3.14 (m, 4H,
2ꢁEda-CH2–), 2.56–2.60 (m, 1H, H-20a), 2.33–2.37 (m, 1H, H-20b),
2.15–2.23 (m,1H, Heq-200), 2.10 (s, 3H, –CH3),1.87 (s, 3H, –CH3),1.72–
1.87 (m, 2H, Lys-CH2–), 1.49–1.54 (m, 2H, Lys-CH2–), 1.40–1.46 (m,
20
calcd for C35H46N16O8 ([MþH]þ): 819.3757, found: 819.3759. [
0.707 (CH3OH, c 0.075).
a]
D
3.1.6. epi-5-N[[[aN-(tert-Butoxycarbonyl)]lysinyl]aminoethyl]-
1,3,20,60-tetraazido neamine (8b)
Compound 7a (30 mg, 0.03 mmol) was dissolved in 10% Et2NH
in DMF (1 mL), after stirring for 10 min at room temperature, the
resulting mixture was concentrated and the residue was purified by
column chromatography on silica gel (CH2Cl2/CH3OH¼100:15) to
afford a white foam (19 mg, yield 84%). 1H NMR (400 MHz, CD3OD):
2H, Lys-CH2–), 1.14–1.20 (q, 1H, J¼12.5 Hz, Hax-200). HRMS calcd for
20
C47H58N18O14 ([MþH]þ): 1099.4453, found: 1099.4419. [
a
]
0.422
D
(CH3OH, c 0.133). lmax¼210 nm,
3
¼26,356, lmax¼265 nm, ¼15,585.
3
3.1.8.2. Compound 9b. Yield: 77% from 8a, white foam. 1H NMR
(400 MHz, CD3OD):
d
5.15 (d, 1H, J¼3.5 Hz, H-10), 3.78–4.03 (m, 4H, H-50, 1, 3 and 30),
d
7.95 (d, 1H, J¼8.1 Hz, H-6), 7.78 (d, 2H,
3.61 (dd, 1H, J¼10.2, 3.2 Hz, H-4), 3.52 (dd, 1H, J¼13.3, 2.2 Hz, H-
J¼7.5 Hz, 2ꢁFmoc-H), 7.63 (d, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 7.39 (t, 2H,
J¼7.5 Hz, 2ꢁFmoc-H), 7.30 (t, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 5.94 (d, 1H,
J¼2.6 Hz, H-10), 5.73 (d, 1H, J¼8.0 Hz, H-5), 5.63–5.65 (m, 2H, H-20
and H-30), 5.11 (d, 1H, J¼3.5 Hz, H-1%), 4.61 (d, 1H, J¼2.6 Hz, H-40),
60a), 3.31–3.65 (m, 8H, H-60b, 20, 4, 5, 6, Lys-
a-CH– and Lys-CH2–),
2.98–3.08 (m, 2H, Eda-CH2–), 2.85–2.89 (m, 2H, Eda-CH2–), 2.18–
2.24 (m, 1H, Heq-2), 1.62–1.80 (m, 4H, 2ꢁLys-CH2–), 1.46 (s, 9H,
3ꢁBoc-CH3), 1.29–1.59 (m, 2H, Lys-CH2–), 1.19 (m, 1H, Hax-2). 13C
4.34–4.38 (m, 3H, Fmoc-CH2– and Lys-
a-CH–), 4.20 (t, 1H, J¼7.5 Hz,
NMR (100 MHz, CD3OD):
d
175.06 (–C]O), 157.91 (–C]O), 95.62
Fmoc-CH–), 3.99–4.02 (m, 1H, H-5%), 3.77–3.92 (m, 3H, H-3%, 100
and 300), 3.62 (dd, 1H, J¼10.2, 3.2 Hz, H-400), 3.53 (dd, 1H, J¼13.3,
2.2 Hz, H-6%a), 3.30–3.48 (m, 7H, H-6%b, 2%, 4%, 500, 600 and Lys-
CH2–), 2.99–3.16 (m, 4H, 2ꢁEda-CH2–), 2.18–2.23 (m, 1H, Heq-200),
2.13 (s, 3H, –COCH3), 2.05 (s, 3H, –COCH3), 1.72–1.86 (m, 2H, Lys-
CH2–), 1.47–1.54 (m, 2H, Lys-CH2–), 1.33–1.46 (m, 2H, Lys-CH2–),
(C-10), 80.80 (Boc-C), 78.98 (C-4), 75.76 (C-40), 73.56 (C-6), 73.14 (C-
30), 72.59 (C-5), 64.92 (C-50), 60.69 (C-20), 59.75 (C-1), 59.46 (C-3),
55.93 (Lys-a
-CH–), 52.53 (C-60), 50.41 (Eda-CH2–), 41.43 (Eda-
CH2–), 40.79 (Lys-CH2–), 33.69 (C-2), 32.92 (Lys-CH2–), 30.75 (Lys-
CH2–), 28.75 (3ꢁBoc-CH3), 23.95 (Lys-CH2–). HRMS calcd for
20
C25H44N16O8 ([MþH]þ): 697.3601, found: 697.3601. [
(CH3OH, c 0.081).
a
]
0.370
1.21 (q, 1H, J¼12.5 Hz, Hax-200). HRMS calcd for C48H58N18O16
D
20
([MþH]þ): 1143.4351, found: 1143.4321. [
a
]
0.217 (CH3OH, c
¼13,280.
D
0.216). lmax¼210 nm,
3
¼23,203, lmax¼263 nm,
3
3.1.7. epi-5-N[[[GN-(2,2,4,6,7-Pentamethyldihydrobenzofuran-5-
sulfonyl)]arginyl]aminoethyl]-1,3,20,60-tetraazido neamine (8c)
Compound 7b (68 mg, 0.06 mmol) was dissolved in 10% Et2NH in
DMF (1 mL), after stirring for 10 min at room temperature, the
resulting mixture was concentrated and the residue was purified by
column chromatography on silica gel (CH2Cl2/CH3OH¼100:5) to af-
ford a white foam (46 mg, yield 85%). 1H NMR (400 MHz, CD3OD):
3.1.8.3. Compound 9c. Yield: 73% from 8a, white foam. 1H NMR
(400 MHz, CD3OD): 8.16 (s, 1H, H-8), 8.14 (s, 1H, H-2), 7.79 (d, 2H,
d
J¼7.5 Hz, 2ꢁFmoc-H), 7.64 (d, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 7.39 (t, 2H,
J¼7.5 Hz, 2ꢁFmoc-H), 7.31 (t, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 6.34 (s, 1H,
H-10), 5.67 (dd, 1H, H-20), 5.46 (d, 1H, H-30), 5.08 (d, 1H, J¼3.5 Hz, H-
1%), 4.75 (d, 1H, J¼2.6 Hz, H-40), 4.37 (d, 2H, J¼6.8 Hz, Fmoc-CH2–),
4.21 (t, 1H, J¼6.6 Hz, Fmoc-CH–), 4.00–4.03 (m, 1H, H-5%), 3.80–
d
5.15 (d, 1H, J¼3.5 Hz, H-10), 4.00–4.04 (m, 1H, H-50), 3.80–3.96 (m,
3H, H-1, 3 and 30), 3.61 (dd, 1H, J¼10.2, 3.2 Hz, H-4), 3.52 (dd, 1H,
J¼13.3, 2.2 Hz, H-60a), 3.27–3.46 (m, 8H, H-60b, 20, 40, 5, 6, Arg-CH2–
3.93 (m, 3H, H-3%, 100 and Lys- -CH–), 3.68–3.73 (m, 1H, H-300), 3.58
a
(dd, 1H, J¼10.2, 3.2 Hz, H-400), 3.53 (dd, 1H, J¼13.3, 2.2 Hz, H-6%a),
3.27–3.41 (m, 7H, H-6%b, 2%, 4%, 500, 600 and Lys-CH2–), 2.86–3.23
(m, 4H, 2ꢁEda-CH2–), 2.20 (m, 1H, Heq-200), 1.58 (s, 3H, –CH3), 1.39
(s, 3H, –CH3), 1.11–1.34 (m, 6H, 3ꢁLys-CH2–), 1.21 (q, 1H, J¼12.5 Hz,
and Arg-a-CH–), 3.10–3.25 (m, 2H, Eda-CH2–), 2.96–3.08 (m, 2H,
Eda-CH2–), 3.00 (s, 2H, Pbf-CH2–), 2.58 (s, 3H, Pbf-CH3), 2.52 (s, 3H,
Pbf-CH3), 2.17–2.22 (m,1H, Heq-2), 2.07 (s, 3H, Pbf-CH3),1.56–1.72 (m,
4H, 2ꢁArg-CH2-), 1.45 (s, 6H, 2ꢁPbf-CH3), 1.15 (q, 1H, J¼12.5 Hz, Hax
2). 13C NMR (100 MHz, CD3OD):
177.26 (–C]O), 159.89 (Pbf-C),
158.14 (–C]NH),139.40 (Pbf-C),134.35 (Pbf-C),133.53 (Pbf-C),126.04
-
Hax-200). HRMS calcd for C48H59N21O12 ([MþH]þ): 1122.4725, found:
20
d
1122.4681. [
lmax¼263 nm,
a
]
0.241 (CH3OH, c 0.207). lmax¼210 nm,
¼12,708.
3¼21,240,
D
3

5234
Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
3.1.8.4. Compound 9d. Yield: 75% from 8a, white foam. 1H NMR
(400 MHz, CD3OD):
6%a), 3.25–3.47 (m, 7H, H-6%b, 2%, 4%, 500, 600 and Lys-CH2–), 3.24–
3.28 (m, 2H, Eda-CH2–), 2.97–3.07 (m, 2H, Eda-CH2–), 2.20–2.24 (m,
1H, Heq-200), 2.12 (s, 3H, –CH3), 1.91 (s, 3H, –CH3), 1.50–1.79 (m, 4H,
2ꢁLys-CH2–), 1.45 (s, 9H, 3ꢁBoc-CH3), 1.33–1.50 (m, 2H, Lys-CH2–),
d
7.79 (d, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 7.64 (d, 2H,
J¼7.5 Hz, 2ꢁFmoc-H), 7.59 (d,1H, J¼7.4 Hz, H-6), 7.38 (t, 2H, J¼7.5 Hz,
2ꢁFmoc-H), 7.30 (t, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 5.84 (d, 1H, J¼7.3 Hz,
H-5), 5.60 (s, 1H, H-10), 5.36 (dd, 1H, J¼6.2, 2.0 Hz, H-20), 5.20 (d, 1H,
J¼6.3 Hz, H-30), 5.10 (d, 1H, J¼3.5 Hz, H-1%), 4.59 (d, 1H, J¼2.6 Hz, H-
40), 4.34 (d, 2H, J¼6.3 Hz, Fmoc-CH2–), 4.18–4.23 (m, 2H, Fmoc-CH–
1.21 (q, 1H, J¼12.5 Hz, Hax-200). HRMS calcd for C38H56N18O16
20
([MþH]þ): 1021.4194, found: 1021.4189. [
a
]
D
0.454 (CH3OH, c
¼6062.
0.183). lmax¼207 nm,
3
¼11,253, lmax¼258 nm,
3
and Lys-a
-CH–), 3.74–4.04 (m, 4H, H-5%, 3%, 100 and 300), 3.59 (dd,1H,
J¼10.2, 3.2 Hz, H-400), 3.53 (dd, 1H, J¼13.3, 2.2 Hz, H-6%a), 3.27–3.44
(m, 7H, H-6%b, 2%, 4%, 500, 600 and Lys-CH2–), 2.96–3.14 (m, 4H,
2ꢁEda-CH2–), 2.20 (m, 1H, Heq-200), 1.68–1.73 (m, 2H, Lys-CH2–), 1.50
(s, 3H, –CH3), 1.40–1.58 (m, 2H, Lys-CH2–), 1.32 (s, 3H, –CH3), 1.17–
3.1.8.9. Compound 9i. Yield: 75% from 8b, white foam. 1H NMR
(400 MHz, CD3OD): d 8.24 (s, 1H, H-8), 8.18 (s, 1H, H-2), 6.36 (d, 1H,
J¼0.9 Hz, H-10), 5.63 (dd, 1H, J¼6.0, 2.3 Hz, H-20), 5.52 (d, 1H,
J¼5.6 Hz, H-30), 5.15 (d, 1H, J¼3.5 Hz, H-1%), 4.64 (d, 1H, J¼1.8 Hz,
1.32 (m, 2H, Lys-CH2–),1.16 (q,1H, J¼12.5 Hz, Hax-200). HRMS calcd for
H-40), 3.79–4.04 (m, 5H, H-5%, 3%, 100, 300 and Lys-
a-CH–), 3.62 (dd,
20
C47H59N19O13 ([MþH]þ): 1098.4613, found: 1098.4602. [
a
3
]
0.251
1H, J¼10.2, 3.2 Hz, H-400), 3.53 (dd, 1H, J¼13.3, 2.2 Hz, H-6%a), 3.30–
3.48 (m, 7H, H-6%b, 2%, 4%, 500, 600 and Lys-CH2–), 2.96–3.08 (m, 2H,
Eda-CH2–), 2.76–2.87 (m, 2H, Eda-CH2–), 2.20–2.24 (m, 1H, Heq-200),
1.59 (s, 3H, –CH3), 1.47–1.63 (m, 2H, Lys-CH2–), 1.45 (s, 9H, 3ꢁBoc-
CH3), 1.41 (s, 3H, –CH3), 1.21 (q, 1H, J¼12.5 Hz, Hax-200), 1.00–1.13 (m,
D
(CH3OH, c 0.187). lmax¼210 nm,
3
¼26,875, lmax¼265 nm, ¼13,988.
3.1.8.5. Compound 9e. Yield: 70% from 8a, white foam. 1H NMR
(400 MHz, CD3OD):
d
7.80 (d, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 7.77 (s, 1H,
H-8), 7.66 (d, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 7.40 (t, 2H, J¼7.5 Hz,
2ꢁFmoc-H), 7.32 (t, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 6.21 (s, 1H, H-10), 5.73
(dd, 1H, J¼6.0, 1.8 Hz, H-20), 5.33 (d, 1H, J¼6.0 Hz, H-30), 5.10 (d, 1H,
J¼3.5 Hz, H-1%), 4.74 (d, 1H, J¼1.5 Hz, H-40), 4.36 (d, 2H, J¼6.8 Hz,
Fmoc-CH2–), 4.22 (t, 1H, J¼6.8 Hz, Fmoc-CH–), 3.70–4.02 (m, 5H, H-
4H, 2ꢁLys-CH2–). HRMS calcd for C38H57N21O12 ([MþH]þ):
20
1000.4568, found: 1000.4552.
[
a]
0.334 (CH3OH,
¼7107.
c 0.111).
D
lmax¼209 nm,
3
¼10,295, lmax¼260 nm,
3
3.1.8.10. Compound 9j. Yield: 72% from 8b, white foam. 1H NMR
5%, 3%,100, Lys-
a
-CH- and 300), 3.58 (dd,1H, J¼10.2, 3.2 Hz, H-400), 3.52
(400 MHz, CD3OD):
d
7.65 (d, 1H, J¼7.4 Hz, H-6), 5.87 (d,1H, J¼7.4 Hz,
(dd, 1H, J¼13.3, 2.2 Hz, H-6%a), 3.22–3.44 (m, 7H, H-6%b, 2%, 4%, 500,
600 and Lys-CH2–), 2.90–3.03 (m, 4H, 2ꢁEda-CH2–), 2.18–2.22 (m, 1H,
Heq-200), 1.56 (s, 3H, –CH3), 1.40 (s, 3H, –CH3), 1.29–1.42 (m, 6H,
H-5), 5.64 (s, 1H, H-10), 5.26 (d, 1H, H-20), 5.19 (d, 1H, J¼6.0 Hz, H-30),
5.15 (d, 1H, J¼3.5 Hz, H-1%), 4.49 (d, 1H, J¼2.4 Hz, H-40), 3.81–4.04
(m, 5H, H-5%, 3%, 100, 300 and Lys-
a-CH–), 3.62 (dd, 1H, J¼10.2, 3.2 Hz,
3ꢁLys-CH2–), 1.16 (q, 1H, J¼12.5 Hz, Hax-200). HRMS calcd for
H-400), 3.53 (dd, 1H, J¼13.3, 2.2 Hz, H-6%a), 3.30–3.48 (m, 7H, H-6%b,
2%, 4%, 500, 600 and Lys-CH2–), 2.86–3.30 (m, 4H, 2ꢁEda-CH2–), 2.20–
2.24 (m, 1H, Heq-200), 1.60–1.74 (m, 2H, Lys-CH2–), 1.53 (s, 3H, –CH3),
20
C48H59N21O13 ([MþH]þ): 1138.4674, found: 1138.4605. [
a]
0.377
D
(CH3OH, c 0.159). lmax¼211 nm,
3
¼28,154, lmax¼262 nm, ¼22,941.
3
1.45 (s, 9H, 3ꢁBoc-CH3),1.36 (s, 3H, –CH3),1.21 (q,1H, J¼12.5 Hz, Hax
-
3.1.8.6. Compound 9f. Yield: 78% from 8a, white foam. 1H NMR
(400 MHz, CD3OD): 8.14 (s, 1H, H-2), 8.01 (s, 1H, H-8), 7.79 (d, 2H,
200), 1.14–1.50 (m, 4H, 2ꢁLys-CH2–). HRMS calcd for C37H57N19O13
20
d
([MþH]þ): 976.4456, found: 976.4451. [
a]
0.424 (CH3OH, c 0.203).
D
J¼7.5 Hz, 2ꢁFmoc-H), 7.65 (d, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 7.38 (t, 2H,
J¼7.5 Hz, 2ꢁFmoc-H), 7.29 (t, 2H, J¼7.5 Hz, 2ꢁFmoc-H), 6.35 (s, 1H,
H-10), 5.61 (dd, 1H, J¼6.0, 1.7 Hz, H-20), 5.41 (d, 1H, J¼6.0 Hz, H-30),
5.08 (d, 1H, J¼3.5 Hz, H-1%), 4.77 (d, 1H, J¼1.6 Hz, H-40), 4.35 (d, 2H,
J¼6.9 Hz, Fmoc-CH2–), 4.22 (t, 1H, J¼6.8 Hz, Fmoc-CH–), 3.73–4.03
lmax¼209 nm,
3
¼14,298, lmax¼268 nm, ¼4978.
3
3.1.8.11. Compound 9k. Yield: 74% from 8b, white foam. 1H NMR
(400 MHz, CD3OD):
d
7.83 (s, 1H, H-8), 6.21 (s, 1H, H-10), 5.72 (dd,
1H, J¼6.0, 1.5 Hz, H-20), 5.41 (d, 1H, J¼6.0 Hz, H-30), 5.16 (d, 1H,
(m, 5H, H-5%, 3%, 100, Lys-
a
-CH- and 300), 3.58 (dd, 1H, J¼10.2, 3.2 Hz,
J¼3.5 Hz, H-1%), 4.61 (d, 1H, J¼1.6 Hz, H-40), 3.80–4.05 (m, 5H, H-
H-400), 3.52 (dd,1H, J¼13.3, 2.2 Hz, H-6%a), 3.26–3.44 (m, 7H, H-6%b,
2%, 4%, 500, 600 and Lys-CH2–), 2.86–3.24 (m, 4H, 2ꢁEda-CH2–), 2.18–
2.22 (m, 1H, Heq-200), 1.57 (s, 3H, –CH3), 1.39 (s, 3H, –CH3), 1.21 (q,1H,
5%, 3%, 100, 300 and Lys-
a
-CH–), 3.62 (dd, 1H, J¼10.2, 3.2 Hz, H-400),
3.53 (dd, 1H, J¼13.3, 2.2 Hz, H-6%a), 3.30–3.48 (m, 7H, H-6%b, 2%,
4%, 500, 600 and Lys-CH2–), 2.78–3.09 (m, 4H, 2ꢁEda-CH2–), 2.22 (m,
1H, Heq-200), 1.56 (s, 3H, –CH3), 1.46 (s, 9H, 3ꢁBoc-CH3), 1.41 (s, 3H,
J¼12.5 Hz, Hax-200), 1.11–1.40 (m, 6H, 3ꢁLys-CH2–). HRMS calcd for
20
C48H58N20O13 ([MþH]þ): 1123.4565, found: 1123.4522. [
a]
0.242
–CH3), 1.36–1.64 (m, 4H, 2ꢁLys-CH2–), 1.21 (q, 1H, J¼12.5 Hz, Hax
-
D
(CH3OH, c 0.174). lmax¼211 nm,
3¼23,650, lmax¼255 nm,
3¼12,639.
200), 1.14–1.24 (m, 2H, Lys-CH2–). HRMS calcd for C38H57N21O13
20
([MþH]þ): 1016.4518, found: 1016.4517. [
a]
0.490 (CH3OH, c
¼8490.
D
3.1.8.7. Compound 9g. Yield: 82% from 8b, white foam. 1H NMR
0.165). lmax¼206 nm,
3
¼10,380, lmax¼256 nm,
3
(400 MHz, CD3OD):
d
7.95 (s,1H, H-6), 6.30 (dd,1H, J¼8.0, 6.0 Hz, H-
10), 5.41 (d, H, J¼5.9 Hz, H-30), 5.15 (d, 1H, J¼3.5 Hz, H-1%), 4.44 (d,
3.1.8.12. Compound 9l. Yield: 78% from 8b, white foam. 1H NMR
1H, J¼1.5 Hz, H-40), 3.79–4.04 (m, 5H, H-5%, 3%, 100, 300 and Lys-
a
-
(400 MHz, CD3OD): d 8.22 (s, 1H, H-2), 8.02 (s, 1H, H-8), 6.39 (s, 1H,
CH–), 3.62 (dd, 1H, J¼10.2, 3.2 Hz, H-400), 3.53 (dd, 1H, J¼13.3,
2.2 Hz, H-6%a), 3.25–3.47 (m, 7H, H-6%b, 2%, 4%, 500, 600 and Lys-
CH2–), 3.24–3.28 (m, 2H, Eda-CH2–), 2.97–3.07 (m, 2H, Eda-CH2–),
2.53–2.58 (m, 1H, H-20a), 2.36–2.41 (m, 1H, H-20b), 2.20–2.24 (m,
1H, Heq-200), 2.12 (s, 3H, –COCH3), 1.91 (s, 3H, –CH3), 1.57–1.79 (m,
4H, 2ꢁLys-CH2–), 1.45 (s, 9H, 3ꢁBoc-CH3), 1.38–1.45 (m, 2H, Lys-
CH2–), 1.21 (q, 1H, J¼12.5 Hz, Hax-200). HRMS calcd for C37H56N18O14
H-10), 5.62 (d, 1H, J¼6.0, 1.6 Hz, H-20), 5.47–5.51 (m, 1H, H-30), 5.16
(d, 1H, J¼3.5 Hz, H-1%), 4.66 (d, 1H, J¼1.8 Hz, H-40), 3.84–4.04 (m,
5H, H-5%, 3%, 100, 300 and Lys-
a
-CH–), 3.62 (dd, 1H, J¼10.2, 3.2 Hz, H-
400), 3.53 (dd, 1H, J¼13.3, 2.2 Hz, H-6%a), 3.30–3.48 (m, 7H, H-6%b,
2%, 4%, 500, 600 and Lys-CH2–), 2.78–3.09 (m, 4H, 2ꢁEda-CH2–), 2.20–
2.24 (m, 1H, Heq-200), 1.61 (s, 3H, –CH3), 1.46 (s, 9H, 3ꢁBoc-CH3), 1.41
(s, 3H, –CH3), 1.40–1.64 (m, 4H, 2ꢁLys-CH2–), 1.21 (q, 1H, J¼12.5 Hz,
([MþH]þ): 977.4296, found: 977.4315. [
a]
20 0.497 (CH3OH, c 0.187).
Hax-200), 1.12–1.20 (m, 2H, Lys-CH2–). HRMS calcd for C38H56N20O13
D
20
lmax¼208 nm,
3¼11,903, lmax¼264 nm,
3¼7338.
([MþH]þ): 1001.4409, found: 1001.4382. [
a
]
0.413 (CH3OH, c
D
0.196). lmax¼207 nm,
3¼11,977, lmax¼245 nm,
3¼7761.
3.1.8.8. Compound 9h. Yield: 80% from 8b, white foam. 1H NMR
(400 MHz, CD3OD):
d
8.03 (d, 1H, J¼8.0 Hz, H-6), 6.00 (d, 1H,
3.1.8.13. Compound 9m. Yield: 78% from 8c, white foam. 1H NMR
J¼5.3 Hz, H-10), 5.76 (d, 1H, J¼8.0 Hz, H-5), 5.65 (t, 1H, J¼5.6 Hz, H-
(400 MHz, CD3OD):
d
7.88 (s, 1H, H-6), 6.26 (t, 1H, J¼7.0 Hz, H-10),
20), 5.58 (m, 1H, H-30), 5.15 (d, 1H, J¼3.5 Hz, H-1%), 4.53 (d, 1H,
5.47 (d, 1H, J¼6.0 Hz, H-40), 5.14 (d, 1H, J¼3.5 Hz, H-1%), 4.52–4.56
J¼3.7 Hz, H-40), 3.78–4.04 (m, 5H, H-5%, 3%, 100, 300 and Lys-
a
-CH–),
(m, 1H, H-30), 4.44 (dd, 1H, Arg-
a-CH–), 3.76–4.07 (m, 4H, H-5%, H-
100, 300 and 3%), 3.61 (dd, 1H, J¼10.2, 3.2 Hz, H-400), 3.52 (dd, 1H,
3.62 (dd, 1H, J¼10.2, 3.2 Hz, H-400), 3.53 (dd, 1H, J¼13.3, 2.2 Hz, H-

Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
5235
J¼13.3, 2.2 Hz, H-6%a), 3.30–3.46 (m, 7H, H-6%b, 2%, 4%, 500, 600 and
Arg-CH2–), 3.14–3.20 (m, 2H, Eda-CH2–), 2.96–3.10 (m, 2H, Eda-
CH2–), 3.00 (s, 2H, Pbf-CH2–), 2.63 (m, 1H, H-20a), 2.58 (s, 3H, –CH3),
3.1.8.18. Compound 9r. Yield: 75% from 8c, white foam. 1H NMR
(400 MHz, CD3OD): d 8.22 (s, 1H, H-2), 8.04 (s, 1H, H-8), 6.40 (s, 1H,
H-10), 5.60 (dd, 1H, J¼6.0, 2.4 Hz, H-20), 5.48 (d, J¼6.0 Hz, H-30), 5.12
2.52 (s, 3H, –CH3), 2.37–2.41 (m, 1H, H-20b), 2.17–2.21 (m, 1H, Heq
-
(d, 1H, J¼3.5 Hz, H-1%), 4.75 (d, 1H, J¼2.5 Hz, H-40), 3.72–4.03 (m,
200), 2.13 (s, 3H, –COCH3), 2.07 (s, 3H, –CH3), 1.88 (s, 3H, –CH3), 1.65–
1.88 (m, 2H, Arg-CH2–), 1.50–1.65 (m, 2H, Arg-CH2–), 1.45 (s, 6H,
5H, H–H-5%, 100, 300, 3% and Arg-
a
-CH–), 3.59 (dd, 1H, J¼10.2, 3.2 Hz,
H-400), 3.52 (dd, 1H, J¼13.3, 2.2 Hz, H-6%a), 3.20–3.46 (m, 7H, H-
6%b, 2%, 4%, 500, 600 and Arg-CH2–), 3.01 (s, Pbf-CH2–), 2.92–3.20 (m,
4H, 2ꢁEda-CH2–), 2.58 (s, 3H, –CH3), 2.52 (s, 3H, –CH3), 2.18–2.22
(m,1H, Heq-200), 2.09 (s, 3H, –CH3),1.59 (s, 3H, –CH3), 1.45 (s, 6H, 2ꢁ-
CH3), 1.41 (s, 3H, –CH3), 1.11–1.21 (m, 4H, 2ꢁArg-CH2–), 1.16 (q, 1H,
2ꢁ-CH3), 1.15 (q, 1H, J¼12.5 Hz, Hax-200). HRMS calcd for
20
C45H64N20O15S ([MþH]þ): 1157.4654, found: 1157.4641. [
a
3
]
0.446
D
(CH3OH, c 0.182). lmax¼218 nm,
3
¼35,050, lmax¼257 nm, ¼13,363.
3.1.8.14. Compound 9n. Yield: 76% from 8c, white foam. 1H NMR
J¼12.5 Hz, Hax-200). HRMS calcd for C46H64N22O14S ([MþH]þ):
20
(400 MHz, CD3OD):
d
7.96 (d, 1H, J¼8.0 Hz, H-6), 5.97 (d, 1H,
1181.4766, found: 1181.4763.
[
a]
0.302 (CH3OH,
¼17,367.
c 0.155).
D
J¼5.0 Hz, H-10), 5.73 (d, 1H, J¼8.0 Hz, H-5), 5.64–5.68 (m, 2H, H-20
lmax¼218 nm,
3¼36,739, lmax¼252 nm,
3
and 30), 5.15 (d, 1H, J¼3.5 Hz, H-1%), 4.62 (d, 1H, J¼1.7 Hz, H-40),
4.45 (dd, 1H, J¼8.5, 5.0 Hz, Arg-
a
-CH-), 3.76–4.03 (m, 4H, H-5%, 100,
3.1.9. General procedure for the synthesis of 10a and 10b
300 and 3%), 3.61 (dd, 1H, J¼10.2, 3.2 Hz, H-400), 3.52 (dd, 1H, J¼13.3,
2.2 Hz, H-6%a), 3.28–3.46 (m, 7H, H-6%b, 2%, 4%, 500, 600 and Arg-
CH2–), 3.00 (s, 2H, Pbf-CH2–), 2.95–3.19 (m, 4H, 2ꢁEda-CH2–), 2.59
(s, 3H, Pbf-CH3), 2.52 (s, 3H, Pbf-CH3), 2.08–2.12 (m, 1H, Heq-200),
2.15 (s, 3H, –COCH3), 2.08 (s, 3H, –COCH3), 2.07 (s, 3H, Pbf-CH3),
1.50–1.90 (m, 4H, 2ꢁArg-CH2–), 1.46 (s, 6H, 2ꢁPbf-CH3), 1.15 (q, 1H,
Compound 9a or 9b (0.1 mmol) was dissolved in a solution of
10% Et2NH in DMF (1 mL), after stirring for 10 min at room tem-
perature, the resulting mixture was concentrated then the residue
was added NH3/CH3OH (2 mL) and kept stirring for 4 h in an ice
bath. The solvent was removed and the residue was dissolved in Py/
Et3N/H2O (5 mL, 4:3:2) and slowly bubbled by hydrogen sulfide for
1 h at room temperature then the reaction mixture was concen-
trated in vacuum and the residue was purified by HPLC (1&
CF3COOH in H2O and CH3OH) to afford compounds 10a and 10b as
a salt of trifluoroacetic acid.
J¼12.5 Hz, Hax-200). HRMS calcd for C46H64N20O17S ([MþH]þ):
20
1201.4552, found: 1201.4576.
[
a]
0.483 (CH3OH,
¼20,341.
c 0.126).
D
lmax¼218 nm,
3¼46,608, lmax¼255 nm,
3
3.1.8.15. Compound 9o. Yield: 72% from 8c, white foam. 1H NMR
(400 MHz, CD3OD):
d
8.26 (s, 1H, H-8), 8.15 (s, 1H, H-2), 6.38 (s, 1H,
3.1.9.1. Compound 10a. Yield: 58% from 9a, white foam. 1H NMR
H-10), 5.65 (dd, 1H, J¼6.0, 1.8 Hz, H-20), 5.51 (d, J¼5.6 Hz, H-30), 5.12
(400 MHz, D2O):
d
7.63 (s, 1H, H-6), 6.23 (t, 1H, J¼6.6 Hz, H-10), 5.66
(d, 1H, J¼3.5 Hz, H-1%), 4.73 (d, 1H, J¼1.7 Hz, H-40), 3.71–4.02 (m,
(d, 1H, J¼3.4 Hz, H-1%), 4.77–4.81 (m, 1H, H-30), 4.46 (d, 1H,
5H, H-5%, 100, 300, 3% and Arg-
a
-CH–), 3.61 (dd, 1H, J¼10.2, 3.2 Hz, H-
J¼3.4 Hz, H-40), 4.42–4.45 (m, 1H, H-5%), 4.30–4.33 (m, 3H, H-3%,
400), 3.52 (dd, 1H, J¼13.3, 2.2 Hz, H-6%a), 3.19–3.46 (m, 7H, H-6%b,
2%, 4%, 500, 600 and Arg-CH2–), 3.00 (s, 2H, Pbf-CH2–), 2.86–3.06 (m,
4H, 2ꢁEda-CH2–), 2.59 (s, 3H, –CH3), 2.52 (s, 3H, –CH3), 2.08–2.12
(m,1H, Heq-200), 2.09 (s, 3H, –CH3),1.60 (s, 3H, –CH3),1.45 (s, 6H, 2ꢁ-
CH3),1.41 (s, 3H, –CH3),1.16 (q, J¼12.5 Hz, Hax-200), 0.93–1.36 (m, 4H,
400 and 600), 4.09–4.14 (t, 1H, J¼9.3 Hz, Lys-
a-CH–), 3.47–4.00 (m,
11H, H-2%, 4%, 6%a, 100, 6%b, 300, 500 and 2ꢁEda-CH2–), 2.99 (t, 2H,
J¼8.0 Hz, Lys-CH2–), 2.64–2.75 (m, 2H, Heq-200 and H-20a), 2.40–2.47
(m, 1H, H-20b), 1.92–2.05 (q, 1H, J¼12.5 Hz, Hax-200), 1.92 (s, 3H,
–CH3), 1.67–1.88 (m, 4H, 2ꢁLys-CH2–), 1.40–1.44 (m, 2H, Lys-CH2–).
2ꢁArg-CH2–). HRMS calcd for C46H65N23O13S ([MþH]þ): 1180.4926,
13C NMR (100 MHz, D2O):
d 175.26 (–C]O), 172.79 (–C]O), 166.48
20
found: 1180.4910. [
a
]
0.316 (CH3OH, c 0.105). lmax¼215 nm,
(C-4), 151.51 (C-2), 139.92 (C-6), 110.92 (C-5), 95.00 (C-1%), 89.57
(C-10), 85.19 (C-40), 73.58 (C-30 and 400), 70.28 (C-4%), 69.56 (C-3%
and 600), 68.21 (C-500), 57.50 (C-2%), 53.81 (Eda-CH2–), 52.75 (C-100
D
3¼45,876, lmax¼257 nm,
3
¼25,712.
3.1.8.16. Compound 9p. Yield: 70% from 8c, white foam. 1H NMR
and Lys-a
-CH–), 47.50 (C-300), 46.72 (C-5%), 39.54 (C-6%), 39.00
(400 MHz, CD3OD):
d
7.64 (d, 1H, J¼7.5 Hz, H-6), 5.87 (d, 1H,
(Eda-CH2–), 37.69 (Lys-CH2–), 37.34 (C-20), 30.09 (Lys-CH2–), 27.93
(C-200), 26.23 (Lys-CH2–), 22.30 (Lys-CH2–), 11.33 (–CH3). HRMS
calcd for C30H54N10O11 ([MþH]þ): 731.4046, found: 731.4062.
J¼7.4 Hz, H-5), 5.58 (s, 1H, H-10), 5.22–5.24 (m, 2H, H-20 and H-30),
5.12 (d, 1H, J¼3.6 Hz, H-1%), 4.56 (s, 1H, H-40), 4.32–4.35 (m, 1H, H-
5%), 3.74–4.03 (m, 4H, H-100, 300, 3% and Arg-
a
-CH–), 3.61 (dd, 1H,
[a
]
28.6 1.063 (H2O, c 0.073). lmax¼265 nm,
3¼14,750.
D
J¼10.2, 3.2 Hz, H-400), 3.52 (dd, 1H, J¼13.3, 2.2 Hz, H-6%a), 3.18–3.46
(m, 7H, H-6%b, 2%, 4%, 500, 600 and Arg-CH2–), 2.95–3.24 (m, 4H,
2ꢁEda-CH2–), 3.00 (s, 2H, Pbf-CH2–), 2.58 (s, 3H, –CH3), 2.52 (s, 3H,
–CH3), 2.08–2.12 (m, 1H, Heq-200), 2.09 (s, 3H, –CH3), 1.50–1.80 (m,
4H, 2ꢁArg-CH2–), 1.53 (s, 3H, –CH3), 1.45 (s, 6H, 2ꢁ-CH3), 1.35 (s,
3.1.9.2. Compound 10b. Yield: 55% from 9b, white foam. 1H NMR
(400 MHz, D2O):
d
7.79 (d, 1H, J¼8.0 Hz, H-6), 5.86 (d, 1H, J¼8.0 Hz,
H-5), 5.73 (d, 1H, J¼4.0 Hz, H-10), 5.53 (d, 1H, J¼3.6 Hz, H-1%), 4.61
(m, 1H, H-20), 4.47–4.50 (m, 2H, H-30 and 40), 3.19–4.29 (m, 16H, H-
3H, –CH3), 1.16 (q, 1H, J¼12.5 Hz, Hax-200). HRMS calcd for
400, 5%, 600, Lys-
a
-CH–, 4%, 500, 100, 300, 2%, 3%, 6%a, 6%b and 2ꢁEda-
20
C45H65N21O14S ([MþH]þ): 1156.4813, found: 1156.4816. [
a
3
]
0.313
CH2–), 2.96 (t, 2H, J¼8.0 Hz, Lys-CH2–), 2.55–2.59 (m, 1H, Heq-200),
D
(CH3OH, c 0.163). lmax¼218 nm,
3
¼42,416, lmax¼255 nm,
¼15,459.
1.74–1.86 (m, 3H, Hax-200 and Lys-CH2–),1.62–1.73 (m, 2H, Lys-CH2–),
1.39–1.43 (m, 2H, Lys-CH2–). 13C NMR (100 MHz, D2O):
d 177.26
3.1.8.17. Compound 9q. Yield: 71% from 8c, white foam. 1H NMR
(–C]O), 174.83 (–C]O), 168.91 (C-4), 154.08 (C-2), 147.00 (C-6),
104.78 (C-5), 96.84 (C-1%), 93.97 (C-10), 85.38 (C-40), 75.20 (C-30),
74.92 (C-20 and 400), 72.66 (C-4%), 72.44 (C-3% and 600), 71.05 (C-500),
(400 MHz, CD3OD):
d
7.84 (s, 1H, H-8), 6.24 (s, 1H, H-10), 5.74 (dd,
2H, J¼6.0, 1.8 Hz, H-20), 5.40 (d, 1H, J¼6.0 Hz, H-30), 5.12 (d, 1H,
J¼3.6 Hz, H-1%), 4.73 (d, 1H, J¼1.8 Hz, H-40), 3.75–4.02 (m, 5H, H–
59.33 (C-2%), 56.67 (Eda-CH2–), 55.62 (Lys-a
-CH–), 52.04 (C-100),
H-5%, 100, 300, 3% and Arg-
a
-CH–), 3.59 (dd, 1H, J¼10.2, 3.2 Hz, H-400),
50.65 (C-300), 49.55 (C-5%), 42.33 (C-6%), 41.77 (Eda-CH2–), 41.33
(Lys-CH2–), 32.84 (Lys-CH2–), 30.96 (C-200), 28.96 (Lys-CH2–), 25.01
(Lys-CH2–). HRMS calcd for C29H52N10O12 ([MþH]þ): 733.3839,
3.52 (dd, 1H, J¼13.3, 2.2 Hz, H-6%a), 3.25–3.46 (m, 7H, H-6%b, 2%,
4%, 500, 600 and Arg-CH2–), 2.92–3.20 (m, 4H, 2ꢁEda-CH2–), 3.01 (s,
Pbf-CH2–), 2.58 (s, 3H, –CH3), 2.52 (s, 3H, –CH3), 2.18–2.22 (m, 1H,
Heq-200), 2.09 (s, 3H, –CH3), 1.57 (s, 3H, –CH3), 1.45 (s, 6H, 2ꢁ-CH3),
1.41 (s, 3H, –CH3), 1.11–1.41 (m, 4H, 2ꢁArg-CH2–), 1.16 (q, 1H,
found: 733.3854. [
a
]
28.7 1.148 (H2O, c 0.073). lmax¼259 nm,
3
¼8950.
D
3.1.10. General procedure for the synthesis of 10c–f
J¼12.5 Hz, Hax-200). HRMS calcd for C46H65N23O14S ([MþH]þ):
Compounds 9c–f (0.1 mmol) were dissolved in a solution of 10%
Et2NH in DMF (2 mL), after stirring for 10 min at room temperature,
the mixture was concentrated then the residue was added 90%
20
1196.4875, found: 1196.4874.
[
a]
0.344 (CH3OH,
¼10,768.
c 0.131).
D
lmax¼219 nm,
3¼20,832, lmax¼256 nm,
3

5236
Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
CF3COOH in H2O (2 mL) and stirred for 3 h in an ice bath. The sol-
vent was removed and the residue was dissolved in Py/Et3N/H2O
(5 mL, 4:3:2) and slowly bubbled by hydrogen sulfide for 1 h at
room temperature, the reaction mixture was concentrated in vac-
uum and the residue was purified by HPLC (1& CF3COOH in H2O
and CH3OH) to afford compounds 10c–f as a salt of trifluoroacetic
acid.
and 30), 4.62 (d, 1H, J¼4.4 Hz, H-40), 4.23–4.37 (m, 4H, H-3%, 5%, 600
and 400), 4.00 (t, 1H, J¼8.8 Hz, Lys-
a-CH–), 3.32–3.95 (m, 11H, H-4%,
500, 100, 300, 2%, 6%a, 6%b and 2ꢁEda-CH2–), 2.89 (t, 2H, J¼8.0 Hz, Lys-
CH2–), 2.58–2.63 (m, 1H, Heq-200), 1.88 (q, 1H, J¼12.5 Hz, Hax-200),
1.52–1.79 (m, 4H, 2ꢁLys-CH2–), 1.19–1.26 (m, 2H, Lys-CH2–). 13C
NMR (100 MHz, D2O):
d 177.91 (C]O), 174.90 (C]O), 160.99 (C-6),
151.21 (C-4), 149.08 (C-2), 143.24 (C-8), 126.59 (C-5), 94.61 (C-1%),
92.07 (C-10), 85.82 (C-40), 78.27 (C-30), 76.03 (C-20), 75.69 (C-400),
73.33 (C-4%), 72.35 (C-600), 70.98 (C-3%), 70.52 (C-500), 60.65 (C-2%),
56.56 (Eda-CH2–), 55.51 (Lys-a
-CH–), 53.42 (C-100), 50.83 (C-300),
3.1.10.1. Compound 10c. Yield: 52% from 9c, white foam. 1H NMR
(400 MHz, D2O):
d
8.58 (s, 1H, H-8), 8.46 (s, 1H, H-2), 6.27 (d, 1H,
J¼5.0 Hz, H-10), 5.60 (d, 1H, J¼3.4 Hz, H-1%), 4.67–4.72 (3H, H-20, 30
49.51 (C-5%), 42.31 (C-6%), 41.82 (Eda-CH2–), 40.11 (Lys-CH2–),
32.97 (Lys-CH2–), 30.70 (C-200), 29.01 (Lys-CH2–), 25.07 (Lys-CH2–).
HRMS calcd for C30H52N12O11 ([MþH]þ): 757.3051, found: 757.3970.
and 40), 4.34–4.38 (m, 2H, H-400 and 3%), 4.15–4.25 (m, 2H, H-600 and
5%), 4.06 (t, 1H, J¼9.0 Hz, Lys-
a
-CH–), 3.36–3.98 (m, 11H, H-4%, 500,
100, 300, 2%, 6%a, 6%b and 2ꢁEda-CH2–), 2.96 (t, 2H, J¼7.6 Hz, Lys-
[a
]
28.9 1.058 (H2O, c 0.076). lmax¼251 nm,
3¼13,150.
D
CH2–), 2.61–2.64 (m, 1H, Heq-200), 1.89–1.95 (q, 1H, J¼12.5 Hz, Hax
-
200), 1.66–1.89 (m, 4H, 2ꢁLys-CH2–), 1.35–1.40 (m, 2H, Lys-CH2–). 13C
3.1.11. General procedure for the synthesis of 10g and 10h
Compound 9g or 9h (0.1 mmol) was dissolved in 20% CF3COOH
in CH2Cl2 (2 mL), after stirring for 1.5 h at room temperature, Et2O
(2 mL) was added and the white solid precipitated was added NH3/
CH3OH (2 mL) and stirred for 4 h in an ice bath. The solvent was
removed and the residue was dissolved in Py/Et3N/H2O (5 mL,
4:3:2) and slowly bubbled by hydrogen sulfide for 1 h at room
temperature, the reaction mixture was concentrated in vacuum
and the residue was purified by HPLC (1& CF3COOH in H2O and
CH3OH) to afford compounds 10g and 10h as a salt of trifluoroacetic
acid.
NMR (100 MHz, D2O):
d 176.91 (–C]O), 174.47 (–C]O), 153.59
(C-6), 151.05 (C-2), 148.50 (C-4), 145.70 (C-8), 121.91 (C-5), 95.58 (C-
1%), 91.84 (C-10), 85.86 (C-40), 76.03 (C-30), 75.51 (C-20), 75.33 (C-
400), 72.82 (C-4%), 72.52 (C-600), 71.12 (C-3%), 65.21 (C-500), 59.43
(C-2%), 56.55 (Eda-CH2–), 55.74 (Lys-a
-CH–), 51.94 (C-100), 50.81 (C-
300), 49.69 (C-5%), 42.48 (C-6%), 41.70 (Eda-CH2–), 41.45 (Lys-CH2–),
33.32 (Lys-CH2–), 31.05 (C-200), 29.02 (Lys-CH2–), 25.00 (Lys-CH2–).
HRMS calcd for C30H53N13O10 ([MþH]þ): 756.4111, found: 756.4117.
[
a]
28.7 1.123 (H2O, c 0.076). lmax¼266 nm,
3¼10,800.
D
3.1.10.2. Compound 10d. Yield: 50% from 9d, white foam. 1H NMR
(400 MHz, D2O):
d
8.22 (d, 1H, J¼8.0 Hz, H-6), 6.28 (d, 1H, J¼8.0 Hz,
3.1.11.1. Compound 10g. Yield: 55% from 9g, white foam. 1H NMR
H-5), 5.86 (d, 1H, J¼3.7 Hz, H-10), 5.67 (d, 1H, J¼3.5 Hz, H-1%), 4.55–
(400 MHz, D2O):
d
7.67 (s, 1H, H-6), 6.25 (t, 1H, J¼6.8 Hz, H-10), 5.58
4.58 (m, 2H, H-20 and 30), 4.33–4.49 (m, 5H, H-40, 3%, 5%, 600 and 400),
(d, 1H, J¼3.6 Hz, H-1%), 4.64 (m, 1H, H-30), 4.38 (d, 1H, J¼3.0 Hz, H-
4.14 (t, 1H, J¼9.5 Hz, Lys-
a
-CH–), 3.35–4.06 (m, 11H, H-4%, 500, 100, 300,
40), 3.21–4.31 (m, 18H, H-5%, H-3%, 400, 600, Lys-
a-CH–, 2%, 4%, 6%a,
2%, 6%a, 6%b and 2ꢁEda-CH2–), 3.03 (t, 2H, J¼7.5 Hz, Lys-CH2–),
2.64–2.68 (m, 1H, Heq-200), 2.07 (q, 1H, J¼12.5 Hz, Hax-200), 1.83–1.93
(m, 2H, Lys-CH2–), 1.69–1.81 (m, 2H, Lys-CH2–), 1.45–1.53 (m, 2H,
100, 6%b, 300, 500, Lys-CH2– and 2ꢁEda-CH2–), 2.57–2.60 (m, 1H, Heq
-
200), 2.45–2.52 (m, 1H, H-20a), 2.29–2.35 (m, 1H, H-20b), 1.85 (s, 3H,
–CH3), 1.79–1.92 (m, 3H, Hax-200 and Lys-CH2–), 1.47–1.54 (m, 2H,
Lys-CH2–), 1.32–1.38 (m, 2H, Lys-CH2–). 13C NMR (100 MHz, D2O):
Lys-CH2–). 13C NMR (100 MHz, D2O):
d 177.32 (–C]O), 174.42
(–C]O), 162.32 (C-4), 151.29 (C-2), 148.83 (C-6), 97.65 (C-5), 96.67
(C-1%), 91.00 (C-10), 85.13 (C-40), 75.36 (C-30), 75.11 (C-20), 75.01 (C-
400), 72.70 (C-4%), 72.28 (C-600), 71.12 (C-3%), 65.26 (C-500), 59.68
d 174.50 (–C]O), 173.82 (–C]O), 169.13 (C-4), 154.39 (C-2), 141.48
(C-6), 113.95 (C-5), 94.59 (C-1%), 90.32 (C-10), 87.76 (C-40), 76.35 (C-
30), 74.09 (C-400), 73.22 (C-4%), 72.42 (C-600), 70.75 (C-3%), 70.44 (C-
(C-2%), 56.57 (Eda-CH2–), 55.70 (Lys-
a
-CH–), 52.35 (C-100), 50.81 (C-
500), 61.05 (C-2%), 55.68 (Eda-CH2–), 55.45 (C-100), 53.10 (Lys-
a-CH–),
300), 49.98 (C-5%), 42.44 (C-6%), 41.88 (Eda-CH2–), 41.45 (Lys-CH2–),
50.81 (C-300), 49.43 (C-5%), 42.27 (C-6%), 41.38 (Eda-CH2–), 41.29
33.06 (Lys-CH2–), 30.97 (C-200), 29.00 (Lys-CH2–), 24.97 (Lys-CH2–).
(C-20), 39.88 (Lys-CH2–), 39.78 (Lys-CH2–), 33.06 (C-200), 30.61 (Lys-
HRMS calcd for C29H53N11O11 ([MþH]þ): 732.3999, found:
CH2–), 24.25 (Lys-CH2–), 14.20 (–CH3). HRMS calcd for
28.8
28.3
732.3998. [
a]
1.226 (H2O, c 0.073). lmax¼267 nm,
3¼13,200.
C30H54N10O11 ([MþH]þ): 730.3974, found: 730.4062. [
a]
1.227
D
D
(H2O, c 0.073). lmax¼264 nm, ¼18,350.
3
3.1.10.3. Compound 10e. Yield: 48% from 9e, white foam. 1H NMR
(400 MHz, D2O):
d
8.34 (s, 1H, H-8), 6.12 (d, 1H, J¼3.0 Hz, H-10), 5.60
3.1.11.2. Compound 10h. Yield: 54% from 9h, white foam. 1H NMR
(d, 1H, J¼3.3 Hz, H-1%), 4.94 (t, 1H, J¼5.2 Hz, H-20), 4.86 (m, 1H, H-
(400 MHz, D2O):
d
7.92 (d, 1H, J¼8.0 Hz, H-6), 5.86 (d, 1H, J¼8.0 Hz,
30), 4.62 (d, 1H, J¼1.3 Hz, H-40), 4.35 (m, 1H, H-3%), 4.18–4.36 (m,
H-5), 5.83 (d, 1H, J¼4.4 Hz, H-10), 5.58 (d, 1H, J¼3.5 Hz, H-1%), 4.53
3H, H-400, 5% and 600), 4.07 (t, 1H, J¼9.8 Hz, Lys-
a-CH–), 3.34–3.99
(t, 1H, J¼5.0 Hz, H-20), 4.41 (d, 1H, J¼4.8 Hz, H-30), 4.38 (d, 1H,
(m,11H, H-4%, 500,100, 300, 2%, 6%a, 6%b and 2ꢁEda-CH2–), 2.90 (t, 2H,
J¼7.8 Hz, Lys-CH2–), 2.62 (m, 1H, Heq-200), 1.91 (q, 1H, J¼12.5 Hz,
Hax-200), 1.73–1.76 (m, 2H, Lys-CH2–), 1.57–1.65 (m, 2H, Lys-CH2–),
J¼5.2 Hz, H-40), 3.17–4.36 (m, 18H, H-5%, H-3%, 400, 600, Lys-
a-CH–,
2%, 4%, 6%a, 100, 6%b, 300, 500, Lys-CH2– and 2ꢁEda-CH2–), 2.56–2.62
(m, 1H, Heq-200), 1.83–1.89 (m, 3H, Hax-200 and Lys-CH2–), 1.51–1.58
(m, 2H, Lys-CH2–), 1.32–1.40 (m, 2H, Lys-CH2–). 13C NMR (100 MHz,
1.16–1.29 (m, 2H, Lys-CH2–). 13C NMR (100 MHz, D2O):
d 177.94
(–C]O), 175.10 (–C]O), 159.98 (C-6), 157.06 (C-2), 153.35 (C-4),
140.27 (C-8), 115.77 (C-5), 92.79 (C-1% and 10), 85.49 (C-40), 76.00
(C-30), 75.50 (C-20), 73.88 (C-400), 73.09 (C-4%), 72.33 (C-600), 70.91
D2O): d 173.93 (–C]O), 173.82 (–C]O), 168.85 (C-4), 154.27 (C-2),
145.85 (C-6), 105.00 (C-5), 94.37 (C-1% and 10), 85.24 (C-40), 75.16
(C-30), 75.07 (C-20), 74.25 (C-400), 73.22 (C-4%), 72.39 (C-600 and 3%),
(C-3%), 70.59 (C-500), 60.38 (C-2%), 56.69 (Eda-CH2–), 55.43 (Lys-
a
-
70.76 (C-500), 61.03 (C-2%), 55.69 (Eda-CH2–), 55.44 (Lys-
a-CH–),
CH–), 53.34 (C-100), 50.59 (C-300), 49.27 (C-5%), 42.23 (C-6%), 41.66
52.94 (C-100), 50.83 (C-300), 49.33 (C-5%), 42.25 (C-6%), 41.31 (Eda-
(Eda-CH2–), 40.14 (Lys-CH2–), 32.79 (Lys-CH2–), 30.64 (C-200), 28.99
CH2–), 39.83 (Lys-CH2–), 33.05 (Lys-CH2–), 30.53 (C-200), 24.43 (Lys-
(Lys-CH2–), 24.98 (Lys-CH2–). HRMS calcd for C30H53N13O11
CH2–), 24.20 (Lys-CH2–). HRMS calcd for C29H52N10O12 ([MþH]þ):
29.0
28.4
([MþH]þ): 772.4060, found: 772.4045. [
a
]
4.254 (H2O, c 0.015).
733.3839, found: 733.3834.
[a
]
1.279 (H2O,
c
0.073).
D
D
lmax¼263 nm,
3¼62,250.
lmax¼262 nm,
3
¼14,050.
3.1.10.4. Compound 10f. Yield: 53% from 9f, white foam. 1H NMR
3.1.12. General procedure for the synthesis of 10i–l
(400 MHz, D2O):
d
8.39 (s, 1H, H-2), 8.21 (s, 1H, H-8), 6.21 (d, 1H,
Compounds 9i–l were added a solution of 90% CF3COOH in H2O
(2 mL), and stirred for 3 h in an ice bath. The solvent was removed
J¼4.0 Hz, H-10), 5.56 (d, 1H, J¼4.0 Hz, H-1%), 4.78–4.83 (m, 2H, H-20

Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
5237
and the residue was dissolved in Py/Et3N/H2O (5 mL, 4:3:2) and
slowly bubbled by hydrogen sulfide for 1 h at room temperature,
the reaction mixture was concentrated in vacuum and the residue
was purified by HPLC (1& CF3COOH in H2O and CH3OH) to afford
compounds 10i–l as a salt of trifluoroacetic acid.
91.65 (C-10), 86.30 (C-40), 75.81 (C-30), 75.67 (C-20), 74.45 (C-400),
74.15 (C-4%), 73.35 (C-600), 72.45 (C-3%), 70.83 (C-500), 61.11 (C-2%),
55.75 (Eda-CH2–), 55.51 (Lys-a
-CH–), 53.03 (C-100), 50.94 (C-300),
49.53 (C-5%), 42.33 (C-6%), 41.32 (Eda-CH2–), 39.88 (Lys-CH2–),
33.12 (Lys-CH2–), 30.72 (C-200), 28.33 (Lys-CH2–), 24.31 (Lys-CH2–).
HRMS calcd for C30H52N12O11 ([MþH]þ): 757.3951, found: 757.3981.
3.1.12.1. Compound 10i. Yield: 50% from 9i, white foam. 1H NMR
[a
]
28.8 1.206 (H2O, c 0.076). lmax¼250 nm,
3
¼10,500.
D
(400 MHz, D2O):
d 8.57 (s, 1H, H-8), 8.43 (s, 1H, H-2), 6.21 (d, 1H,
J¼5.6 Hz, H-10), 5.57 (d, 1H, J¼3.6 Hz, H-1%), 4.57–4.59 (m, 2H, H-20
3.1.13. General procedure for the synthesis of 10m and 10n
and 30), 3.14–4.34 (m, 19H, H-40, 5%, H-3%, 400, 600, Lys-
a-CH–, 2%,
Compound 9m or 9n (0.1 mmol) was dissolved in CF3COOH/
PhSCH3/H2O (2 mL, 94:3:3), after stirring for 1 h, the resulting
mixture was concentrated and the residue was added NH3/CH3OH
(2 mL) and kept stirring for 4 h in an ice bath. The solvent was re-
moved and the residue was dissolved in Py/Et3N/H2O (5 mL, 4:3:2)
and slowly bubbled by hydrogen sulfide for 1 h at room tempera-
ture, the reaction mixture was concentrated in vacuum and the
residue was purified by HPLC (1& CF3COOH in H2O and CH3OH) to
afford compounds 10m and 10n as a salt of trifluoroacetic acid.
4%, 6%a, 100, 6%b, 300, 500, Lys-CH2– and 2ꢁEda-CH2–), 2.56–2.61 (m,
1H, Heq-200), 1.81–1.93 (m, 3H, Hax-200 and Lys-CH2–), 1.50–1.57 (m,
2H, Lys-CH2–), 1.32–1.40 (m, 2H, Lys-CH2–). 13C NMR (100 MHz,
D2O):
d 173.74 (–C]O), 173.65 (–C]O), 152.79 (C-6), 150.93 (C-2),
147.29 (C-4), 145.97 (C-8), 121.81 (C-5), 94.42 (C-1%), 91.43 (C-10),
86.13 (C-40), 75.85 (C-30), 75.50 (C-20), 74.37 (C-400), 73.07 (C-4%),
72.49 (C-600), 70.80 (C-3%), 65.18 (C-500), 60.78 (C-2%), 55.72 (Eda-
CH2–), 55.49 (Lys-a
-CH–), 52.82 (C-100), 50.79 (C-300), 49.46 (C-5%),
42.30 (C-6%), 41.34 (Eda-CH2–), 40.18 (Lys-CH2–), 33.05 (Lys-CH2–),
30.65 (C-200 and Lys-CH2–), 24.26 (Lys-CH2–). HRMS calcd for
3.1.13.1. Compound 10m. Yield: 56% from 9m, white foam. 1H NMR
C30H53N13O10 ([MþH]þ): 756.4111, found: 756.4130. [
a
]
1.219
(400 MHz, D2O):
d
7.53 (s, 1H, H-6), 6.14 (t, 1H, J¼6.7 Hz, H-10), 5.56
28.5
D
(H2O, c 0.076). lmax¼260 nm,
3¼19,200.
(d, 1H, J¼3.3 Hz, H-1%), 4.70–4.80 (m, 1H, H-30), 4.39 (d, 1H,
J¼3.6 Hz, H-40), 3.14–4.39 (m, 18H, H-5%, H-3%, 400, 600, Arg-
a-CH–,
3.1.12.2. Compound 10j. Yield: 52% from 9j, white foam. 1H NMR
2%, 4%, 6%a, 100, 6%b, 300, 500, Arg-CH2– and 2ꢁEda-CH2–), 2.59–2.63
(m, 2H, H-20a and Heq-200), 2.32–2.40 (m, 1H, H-20b), 1.84 (s, 3H,
–CH3), 1.83–1.95 (m, 1H, Hax-200), 1.65–1.85 (m, 2H, Arg-CH2–), 1.50–
(400 MHz, D2O):
d
8.28 (d, 1H, J¼8.0 Hz, H-6), 6.18 (d, 1H, J¼3.8 Hz,
H-10), 5.81 (d, 1H, J¼8.0 Hz, H-5), 5.56 (d, 1H, J¼3.5 Hz, H-1%), 4.37–
4.41 (m, 3H, H-20, 30 and 40), 3.08–4.30 (m, 18H, H-5%, H-3%, 400, 600,
1.60 (m, 2H, Arg-CH2–). 13C NMR (100 MHz, D2O):
d 178.13 (–C]O),
Lys-
a
-CH–, 2%, 4%, 6%a, 100, 6%b, 300, 500, Lys-CH2– and 2ꢁEda-CH2–),
175.49 (–C]O), 169.13 (C-4), 159.36 (–C]NH), 154.25 (C-2), 142.48
(C-6), 113.76 (C-5), 94.42 (C-1%), 92.24 (C-10), 87.93 (C-40), 76.26 (C-
30 and 400), 73.33 (C-4%), 72.24 (C-600), 70.92 (C-3%), 69.99 (C-500),
2.56–2.64 (m, 1H, Heq-200), 1.77–1.93 (m, 3H, Hax-200 and Lys-CH2–),
1.47–1.52 (m, 2H, Lys-CH2–), 1.31–1.37 (m, 2H, Lys-CH2–). 13C NMR
(100 MHz, D2O):
d
174.03 (–C]O), 173.65 (–C]O), 161.83 (C-4),
60.72 (C-2%), 56.28 (Eda-CH2–), 55.42 (C-100), 53.68 (Arg-
a-CH–),
150.87 (C-2), 147.93 (C-6), 97.72 (C-5), 94.68 (C-1% and 10), 85.13 (C-
40), 75.73 (C-30), 75.04 (C-20), 73.37 (C-400), 72.34 (C-4%), 70.67 (C-
3% and 600), 69.82 (C-500), 61.38 (C-2%), 55.66 (Eda-CH2–), 55.39
50.71 (C-300), 49.38 (C-5%), 43.12 (C-6%), 42.23 (Eda-CH2–), 40.04
(Arg-CH2–), 39.79 (C-20), 30.50 (C-200 and Arg-CH2–), 27.40 (Arg-
CH2–), 14.05 (–CH3). HRMS calcd for C30H54N12O11 ([MþH]þ):
27.6
(Lys-
a
-CH–), 53.46 (C-100), 50.78 (C-300), 49.28 (C-5%), 42.24 (C-6%),
759.4108, found: 759.4114.
[
a
]
0.443 (H2O,
c
0.076).
D
41.41 (Eda-CH2–), 39.30 (Lys-CH2–), 37.21 (Lys-CH2–), 33.03 (C-200),
30.43 (Lys-CH2–), 24.25 (Lys-CH2–). HRMS calcd for C29H54N11O11
lmax¼265 nm,
3¼10,600.
([MþH]þ): 732.4004, found: 732.3984. [
a]
28.5 1.248 (H2O, c 0.073).
3.1.13.2. Compound 10n. Yield: 54% from 9n, white foam. 1H NMR
D
lmax¼265 nm,
3¼25,900.
(400 MHz, D2O):
d
7.76 (d, 1H, J¼8.0 Hz, H-6), 5.85 (d, 1H, J¼8.0 Hz,
H-5), 5.73 (d, 1H, J¼4.4 Hz, H-10), 5.56 (d, 1H, J¼3.6 Hz, H-1%), 4.58
3.1.12.3. Compound 10k. Yield: 48% from 9k, white foam. 1H NMR
(t, 1H, J¼4.6 Hz, H-20), 4.48–4.52 (m, 2H, H-30 and 40), 3.15–4.32 (m,
(400 MHz, D2O):
d
8.92 (s, 1H, H-8), 6.11 (d, 1H, J¼4.0 Hz, H-10), 5.61
18H, H-5%, H-3%, 400, 600, Arg- -CH–, 2%, 4%, 6%a, 100, 6%b, 300, 500,
a
(d, 1H, J¼3.4 Hz, H-1%), 4.63 (t, 1H, J¼5.0 Hz, H-20), 4.56 (d, 1H,
Arg-CH2– and 2ꢁEda-CH2–), 2.57–2.61 (m, 1H, Heq-200), 1.71–1.90
J¼6.0 Hz, H-40), 3.00–4.16 (m, 19H, H-30, 5%, H-3%, 400, 600, Lys-
a-CH–,
(m, 3H, Arg-CH2– and Hax-200), 1.57–1.66 (m, 2H, Arg-CH2–). 13C
2%, 4%, 6%a, 100, 6%b, 300, 500, Lys-CH2– and 2ꢁEda-CH2–), 2.59–2.64
(m, 1H, Heq-200), 1.82–1.95 (m, 3H, Hax-200 and Lys-CH2–), 1.46–1.54
(m, 2H, Lys-CH2–), 1.24–1.40 (m, 2H, Lys-CH2–). 13C NMR (100 MHz,
NMR (100 MHz, D2O): d 177.66 (C]O), 174.91 (C]O), 168.91 (C-4),
159.46 (–C]NH), 154.19 (C-2), 147.00 (C-6), 104.96 (C-5), 96.81 (C-
1%), 94.41 (C-10), 85.56 (C-40), 75.24 (C-30), 74.93 (C-20), 74.45 (C-
400), 73.05 (C-4%), 72.51 (C-600 and 3%), 71.06 (C-500), 60.18 (C-2%),
56.45 (Eda-CH2–), 55.60 (Arg-a
-CH–), 53.04 (C-100), 50.79 (C-300),
D2O):
d
173.86 (2ꢁ-C]O), 158.80 (C-6), 157.69 (C-2), 152.84 (C-4),
139.59 (C-8), 114.70 (C-5), 94.62 (C-1%), 92.63 (C-10), 85.95 (C-40),
75.92 (C-30), 75.42 (C-20), 74.72 (C-400), 73.29 (C-4%), 72.40 (C-600),
70.74 (C-3%), 65.15 (C-500), 61.20 (C-2%), 55.69 (Eda-CH2–), 55.44
49.58 (C-5%), 43.20 (C-6%), 42.37 (Eda-CH2–), 40.72 (Arg-CH2–),
30.82 (C-200), 30.57 (Arg-CH2–), 27.47 (Arg-CH2–). HRMS calcd for
27.9
(Lys-a
-CH–), 53.11 (C-100), 50.84 (C-300), 49.43 (C-5%), 49.34 (C-6%),
C29H52N12O12 ([MþH]þ): 761.3900, found: 761.3916. [
a]
1.211
D
42.27 (Eda-CH2–), 41.42 (Lys-CH2–), 39.64 (Lys-CH2–), 33.08 (C-200),
(H2O, c 0.076). lmax¼261 nm, ¼13,300.
3
30.56 (Lys-CH2–), 24.28 (Lys-CH2–). HRMS calcd for C30H53N13O11
28.8
([MþH]þ): 772.4021, found: 772.4038. [
a
]
1.089 (H2O, c 0.077).
3.1.14. General procedure for the synthesis of 10o–r
D
lmax¼257 nm,
3
¼16,500.
Compounds 9o–r (0.1 mmol) were dissolved in CF3COOH/
PhSCH3/H2O (2 mL, 94:3:3), after stirring for 1 h, the resulting
mixture was concentrated and the residue was dissolved in Py/
Et3N/H2O (5 mL, 4:3:2) and slowly bubbled by hydrogen sulfide for
1 h at room temperature, the reaction mixture was concentrated in
vacuum and the residue was purified by HPLC (1& CF3COOH in H2O
and CH3OH) to afford compounds 10o–r as a salt of trifluoroacetic
acid.
3.1.12.4. Compound 10l. Yield: 54% from 9l, white foam. 1H NMR
(400 MHz, D2O):
d 8.50 (s, 1H, H-2), 8.23 (s, 1H, H-8), 6.18 (d, 1H,
J¼5.4 Hz, H-10), 5.59 (d, 1H, J¼3.6 Hz, H-1%), 4.78 (m, 1H, H-20),
4.57–4.58 (m, 2H, H-30 and 40), 3.13–4.39 (m, 18H, H-5%, H-3%, 400,
600, Lys-
a
-CH–, 2%, 4%, 6%a, 100, 6%b, 300, 500, Lys-CH2– and 2ꢁEda-
CH2–), 2.57–2.62 (m, 1H, Heq-200), 1.80–1.93 (m, 3H, Hax-200 and Lys-
CH2–), 1.44–1.49 (m, 2H, Lys-CH2–), 1.26–1.34 (m, 2H, Lys-CH2–). 13
NMR (100 MHz, D2O): 173.97 (–C]O), 173.83 (–C]O), 160.75 (C-
6),151.17 (C-4),149.31 (C-2),143.12 (C-8),126.11 (C-5), 94.89 (C-1%),
C
d
3.1.14.1. Compound 10o. Yield: 51% from 9o, white foam. 1H NMR
(400 MHz, D2O):
d 8.52 (s, 1H, H-8), 8.41 (s, 1H, H-2), 6.22 (d, 1H,

5238
Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
J¼5.2 Hz, H-10), 5.56 (d,1H, J¼3.6 Hz, H-1%), 4.64–4.79 (m, 3H, H-20,
3.2. Determination of dissociation constants for the
neamine–nucleoside conjugates binding to A site
of 16S RNA and TAR RNA
30 and 40), 3.08–4.39 (m, 18H, H-5%, H-3%, 400, 600, Arg-
a-CH–, 2%,
4%, 6%a, 100, 6%b, 300, 500, Arg-CH2– and 2ꢁEda-CH2–), 2.59–2.63 (m,
1H, Heq-200), 1.84–1.93 (m, 1H, Hax-200), 1.64–1.81 (m, 2H, Arg-CH2–),
1.45–1.55 (m, 2H, Arg-CH2–). 13C NMR (100 MHz, D2O):
d
177.57
The binding properties of neamine–nucleoside conjugates to A
site of 16S RNA and TAR RNA were evaluated by SPR using Biacore
3000 instrument. Biotinylated A site of 16S and TAR RNA were
purchased from Bioneer corporation of Korea. The biochip and the
streptavidin were purchased from Biacore and Sigma, respectively.
All the glass containers, pipette tips, eppendorf tubes and so on,
which have been used in the experiment were pretreated to RNase
free and keep not contaminated. All the solutions were prepared
(–C]O), 174.44 (–C]O), 159.35 (–C]NH), 152.72 (C-6), 150.80
(C-2),147.21 (C-4),146.09 (C-8),121.85 (C-5), 96.00 (C-1%), 91.96 (C-
10), 85.70 (C-40), 76.07 (C-30), 75.29 (C-20), 73.28 (C-400), 72.29 (C-
4%), 70.88 (C-600 and 3%), 70.28 (C-500), 60.70 (C-2%), 56.17 (Eda-
CH2–), 55.43 (Arg-a
-CH–), 53.42 (C-100), 50.72 (C-300), 49.40 (C-5%),
43.01 (C-6%), 42.24 (Eda-CH2–), 39.81 (Arg-CH2–), 30.63 (C-200 and
Arg-CH2–), 27.24 (Arg-CH2–). HRMS calcd for C30H53N15O10
([MþH]þ): 784.4173, found: 784.4174. [
a]
28.1 1.254 (H2O, c 0.052).
with RNase free water and filtered (pore size: 0.22
For preparing the streptavidin-functionalized (SA) biochip used in
the assay, streptavidin in an acetate buffer solution (200 g/mL,
60 L) flowed through the surface of the carboxy-methyl dextran
mm) before use.
D
lmax¼261 nm, ¼16,392.
3
m
3.1.14.2. Compound 10p. Yield: 52% from 9p, white foam. 1H NMR
m
(400 MHz, D2O):
d
8.11 (d, 1H, J¼8.0 Hz, H-6), 6.20 (d, 1H, J¼8.0 Hz,
(CM-5) sensor chip and was covalently attached to the surface
through the ethanolamine linkers. Biotinylated A site of 16S RNA
and TAR RNA in HBS (HEPES-buffered saline, 10 mM HEPES,
100 mM NaCl, 0.1 mM EDTA, pH 6.8) buffer (0.1 OD/mL, 6 mL) was
flowed through the surface and immobilized on the streptavidin-
coated sensor chip and a solution with different concentrations of
H-5), 5.76 (d, 1H, J¼3.5 Hz, H-10), 5.57 (d, 1H, J¼2.8 Hz, H-1%), 4.50–
4.53 (m, 2H, H-20 and 30), 4.42 (t, 1H, J¼5.6 Hz, H-40), 3.14–4.37 (m,
18H, H-5%, H-3%, 400, 600, Arg-
a
-CH–, 2%, 4%, 6%a, 100, 6%b, 300, 500,
Arg-CH2– and 2ꢁEda-CH2–), 2.57–2.63 (m, 1H, Heq-200), 1.70–1.93
(m, 3H, Hax-200 and Arg-CH2–), 1.56–1.63 (m, 2H, Arg-CH2–). 13C
NMR (100 MHz, D2O):
d
177.74 (–C]O), 174.43 (–C]O), 162.04 (C-
the neamine–nucleoside conjugates (160
mM, 80
mM, 40
mM, 20
mM,
4), 159.41 (–C]NH), 150.94 (C-2), 148.88 (C-6), 97.74 (C-5), 96.75
(C-1% and 10), 85.14 (C-40), 75.28 (C-30 and 20), 75.02 (C-400), 73.17
(C-4%), 72.32 (C-600 and 3%), 70.94 (C-500), 60.51 (C-2%), 56.22 (Eda-
10 M, 5 M, 2.5 M, 1.25 M, 0.625 M, 0.3125
m
m
m
m
m
m
M, ., 0 m
M as
blank control) in HBS buffer (10 mM HEPES, 150 mM NaCl, 3 mM
EDTA, pH 7.4) was flowed through on the surface, the response
units (RUs) for diverse concentrations were obtained and the dis-
sociation constants (KD) were determined by the curve fitting
analysis according to the equation: RUAG¼RUmax[AG]/KDþ[AG],
among which RUmax¼RURNA(MWAG/MWRNA). Where RUAG is the
response unit of the neamine–nucleoside conjugates, [AG] is
the concentration of the neamine–nucleoside conjugates, RUmax is
the expected maximum response unit value of the neamine–nu-
cleoside conjugates. RURNA is the response unit of the immobilized
A site of 16S RNA and TAR RNA on the biochip, MWAG and MWRNA
are the molecular weights of the neamine–nucleoside conjugates
and A site of 16S RNA and TAR RNA.
CH2–), 55.46 (Arg-a
-CH– and C-100), 50.71 (C-300), 49.33 (C-5%),
43.11 (C-6%), 42.25 (Eda-CH2–), 40.10 (Arg-CH2–), 30.59 (C-200 and
Arg-CH2–), 27.22 (Arg-CH2–). HRMS calcd for C29H53N13O11
([MþH]þ): 760.4060, found: 760.4067. [
a]
28.2 1.265 (H2O, c 0.076).
D
lmax¼265 nm, ¼15,500.
3
3.1.14.3. Compound 10q. Yield: 49% from 9q, white foam. 1H
NMR (400 MHz, D2O):
d
8.66 (s, 1H, H-8), 6.08 (d, 1H, J¼3.0 Hz, H-
10), 5.56 (d, 1H, J¼4.0 Hz, H-1%), 4.78–4.87 (m, 2H, H-20 and 30),
4.58 (d, 1H, J¼5.6 Hz, H-40), 3.03–4.38 (m, 18H, H-5%, H-3%, 400,
600, Arg-
a
-CH–, 2%, 4%, 6%a, 100, 6%b, 300, 500, Arg-CH2– and 2ꢁEda-
CH2–), 2.58–2.63 (m, 1H, Heq-200), 1.88 (q, 1H, J¼12.5 Hz, Hax-200),
1.57–1.79 (m, 2H, Arg-CH2–), 1.38–1.50 (m, 2H, Arg-CH2–). 13C
3.3. Procedures for the molecular docking study
NMR (100 MHz, D2O):
d 177.89 (–C]O), 175.00 (–C]O), 159.41
(–C]NH), 159.27 (C-6), 157.41 (C-2), 153.01 (C-4), 140.20 (C-5),
114.52 (C-8), 95.00 (C-1%), 93.36 (C-10), 85.60 (C-40), 76.15 (C-30),
75.36 (C-20), 74.00 (C-400), 73.37 (C-4%), 72.37 (C-600), 70.99 (C-
AutoDock 3.0 program was used for the molecular docking
procedure. The target structure of A site of 16S RNA was extracted
from the protein databank (PDB code 1PBR). The ligands were
minimized with AMBER force field using the steepest decent and
conjugated gradient methods consecutively. The obtained opti-
mized structures were used for the following docking.
The molecular docking calculations were carried out using an
empirical free energy function and Lamarckian Genetic Algorithm.
Each dihedral angle of ligands was chosen to be flexible. The
number of generations, energy evaluation, and docking runs were
set to 370,000, 1,500,000, and 50, respectively, and the kinds of
atomic charges were taken as Kollman-all-atom for 16S RNA and
Gasteiger–Hucel for the ligands.
3%), 70.43 (C-500), 60.71 (C-2%), 56.52 (Eda-CH2–), 55.52 (Arg-
a-
CH–), 53.50 (C-100), 50.78 (C-300), 49.42 (C-5%), 43.19 (C-6%),
42.33 (Eda-CH2–), 40.04 (Arg-CH2–), 30.54 (C-200 and Arg-CH2–),
27.35 (Arg-CH2–). HRMS calcd for C30H53N15O11 ([MþH]þ):
28.9
800.4122, found: 800.4121.
[
a
]
0.991 (H2O, c 0.080).
D
lmax¼252 nm,
3
¼10,050.
3.1.14.4. Compound 10r. Yield: 54% from 9r, white foam. 1H NMR
(400 MHz, D2O):
d 8.35 (s, 1H, H-2), 8.19 (s, 1H, H-8), 6.18 (d, 1H,
J¼5.0 Hz, H-10), 5.54 (d, 1H, J¼3.6 Hz, H-1%), 4.73–4.78 (m, 2H, H-20
and 30), 4.63 (d, 1H, J¼4.0 Hz, H-40), 3.04–4.34 (m, 18H, H-5%, H-3%,
400, 600, Arg- -CH–, 2%, 4%, 6%a, 100, 6%b, 300, 500, Arg-CH2– and
a
2ꢁEda-CH2–), 2.57–2.62 (m, 1H, Heq-200), 1.60–1.91 (m, 3H, Hax-200
Acknowledgements
and Arg-CH2–), 1.41–1.50 (m, 2H, Arg-CH2–). 13C NMR (100 MHz,
D2O): d 177.42 (–C]O), 174.81 (–C]O), 161.06 (–C]NH), 159.42 (C-
This study was supported by the National Natural Science
Foundation of China (20332010) and The Ministry of Science and
Technology of China (2004cb518904).
6), 151.15 (C-4), 148.91 (C-2), 143.38 (C-8), 126.92 (C-5), 94.55
(C-1%), 92.04 (C-10), 85.94 (C-40), 75.95 (C-30), 75.61 (C-20), 74.31 (C-
400), 73.20 (C-4%), 72.46 (C-600 and 3%), 71.00 (C-500), 60.47 (C-2%),
56.29 (Eda-CH2–), 55.57 (Arg-a
-CH–), 53.06 (C-100), 50.84 (C-300),
49.57 (C-5%), 43.07 (C-6%), 42.35 (Eda-CH2–), 40.37 (Arg-CH2–),
Supplementary data
30.71 (C-200 and Arg-CH2–), 27.35 (Arg-CH2–). HRMS calcd for
29.0
C30H52N14O11 ([MþH]þ): 785.4013, found: 785.4008. [
a]
1.888
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2009.04.084.
D
(H2O, c 0.047). lmax¼250 nm, ¼12,000.
3

Y. Xu et al. / Tetrahedron 65 (2009) 5228–5239
5239
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