Solid-phase synthesis and biological evaluation of a uridinyl branched peptide urea library

Article abstract of DOI:10.1016/j.bmcl.2007.09.097

A 1000-member uridinyl branched peptide library was synthesized on PS-DES support using IRORI technology. High-throughput screening of this library for anti-tuberculosis activity identified several members with a MIC₉₀ value of 12.5 μg/mL.

Full text of DOI:10.1016/j.bmcl.2007.09.097

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6899–6904
Solid-phase synthesis and biological evaluation of
a uridinyl branched peptide urea library
Dianqing Sun,a Victoria Jones,b Elizabeth I. Carson,a Robin E. B. Lee,a
Michael S. Scherman,b Michael R. McNeilb and Richard E. Leea,*
aDepartment of Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163, USA
bDepartment of Microbiology, Colorado State University, Fort Collins, CO 80523, USA
Received 5 July 2007; revised 26 September 2007; accepted 28 September 2007
Available online 4 October 2007
Abstract—A 1000-member uridinyl branched peptide library was synthesized on PS-DES support using IRORI technology. High-
throughput screening of this library for anti-tuberculosis activity identified several members with a MIC90 value of 12.5 lg/mL.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Due to the emergence and spread of multidrug-resistant
HO
NHR
microbes such as multidrug-resistant Mycobacterium
tuberculosis, methicillin-resistant Staphylococcus aureus,
and penicillin-resistant Streptococcus pneumoniae, there
is an urgent need to develop new antibiotics to treat
these problematic pathogens.1 Mureidomycins (Fig. 1)
were first isolated from the culture filtrate of Streptomy-
ces flavidovirens in 1989,2 and these nucleoside peptidyl
antibiotics have notable activity in vitro against a vari-
ety of drug resistant pathogens with an acceptable selec-
tivity index. Mureidomycin C (R = Gly) is the most
active member within this chemical class with minimum
inhibitory concentration (MIC) values of 0.1–3.13 lg/
mL against various strains of Pseudomonas aeruginosa.3
Mechanistic studies have revealed that Mureidomycins
are inhibitors of the MraY enzyme, a transmembrane
translocase, which performs an essential role in bacterial
peptidoglycan biosynthesis and is currently not targeted
by any approved antibacterial treatments.4 In the litera-
ture, a number of studies have reported the synthesis
and evaluation of Mureidomycin and Pacidamycin ana-
logues that have garnered an understanding of the struc-
ture activity relationship of this class suggesting that
some chemical modifications to these molecules are pos-
sible.5 The major problem of this class of inhibitors is
O
O
NH
H3C
O
N
CH3
H
N
H
H
N
O
HOOC
OH
N
N
N
H
O
O
O
OH
SCH3
Mureidomycin A (R = H)
C (R = Gly)
O
NH2
R3
O
NH
HN
N
O
O
H
H
O
R2
N
N
N
H
OH
OH
O
R1
O
1
Figure 1. Mureidomycins A, C, and uridinyl branched peptide urea
library 1.
their large molecular weight and consequent poor bio-
availability. Mureidomycins thus represent an attractive
natural product scaffold for the development of novel
antibiotic agents, especially if more bioavailable ana-
logues could be developed.
The synthesis of chemical libraries derived around natu-
ral product templates is an attractive approach to dis-
covery of pharmacologically active compounds. In this
study we report the generation and evaluation of a sim-
plified uridinyl branched peptide urea library with struc-
Keywords: Mureidomycins; Uridine-based library; Solid-phase synthe-
sis; IRORI technology; Anti-tuberculosis activity; Tuberculosis;
Antibiotic.
*
Corresponding author. Tel.: +1 901 448 6018; fax: +1 901 448
6828; e-mail: relee@utmem.edu
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.097

6900
D. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6899–6904
tural similarity to the Mureidomycin class of antibiotics.
We recently reported the solid-phase synthesis of a thy-
midinyl/20-deoxyuridinyl Ugi library6 and a thymidinyl
dipeptide urea library7 for use as discovery libraries
and probes for sugar nucleotide utilizing enzymes. Bio-
logical screening of these libraries identified a few active
compounds against M. tuberculosis (MIC90 = 50 lg/
mL). This study focused on the solid-phase synthesis
and biological evaluation of an advanced, more complex
uridinyl branched peptide urea library 1 (Fig. 1). Com-
pared with the previous libraries, two important struc-
tural features were incorporated into this library. First,
the nucleoside scaffold is uridine derived rather than thy-
midine or 20-deoxyuridine. Since uridine templates are
more commonly present in naturally occurring nucleo-
side antibiotics including tunicamycins, liposidomycins,
capuramycins, etc.,8 a uridine scaffold may have a better
chance of being recognized by the target enzyme. Sec-
ond, a branched functionality was introduced to explore
greater structural diversity and to increase the potential
for a broader range of interactions with the target
enzyme.
O
O
O
N3
O
HN
O
O
OH
OH
O
N
O
N
H
H
Fmoc-Dpr(ivDde)-OH
Fmoc-Ser(N3)-OH
2
3
Figure 2. Building blocks evaluated for branched functionality
incorporation.
based on cost. Key starting material 3 (ꢀ100 g) was
prepared in a large scale in 3-step sequence from
Fmoc-Ser-OH using the protocol of Schmidt10 with a
minor modification: PPh3/CBr4/NaN3 was used to
replace PPh3/DEAD/HN3 for the azide introduction
step for safety purposes.
A 1000-member library with three sites of diversity
(R110 · R210 · R310) was synthesized using IRORI di-
rected sorting technology11 as outlined in Scheme 1.
The two sets of Fmoc protected amino acids (Fmoc-
AA1 or AA2-OH) and one set of isocyanates or chloro-
formates with diverse functional groups were selected as
building blocks for the synthesis and are listed in Figure
3. Solid-supported PS-DES-Ser(N3)-uridine 5 was pre-
pared in bulk in a 500 mL solid-phase peptide synthe-
sizer using a five-step synthesis of PS-DES resin
activation,12 resin loading, azide reduction, Fmoc-
Ser(N3)-OH coupling, and Fmoc removal using stan-
dard protocols.7 The only minor modification was the
Fmoc removal step, which was performed using 25%
4-methylpiperidine rather than piperidine due to new
restrictions on the use of piperidine.13 The freshly pre-
pared resin 5 was evenly distributed into 1000 MiniKans
containing Rf tags (60 mg, 0.087 mmol/Kan). The first
library step was then performed by Fmoc-AA1-OH cou-
pling using DIC-HOBt activation, in 10 reaction flasks
each containing 100 MiniKans.7 This was followed by
washing, pooling, and Fmoc deprotection on mass prior
to sorting into 10 reaction vessels. Capping with the sec-
ond diversity element using either isocyanates R2 {1–9}
or chloroformate R2 {10} was then performed to pro-
vide the corresponding solid supported urea or carba-
mate derivatives 6. Washing and pooling of MiniKans
was followed by azide reduction to activate the branch-
ing position. The final diversity step of Fmoc-AA2-OH
coupling was then performed. Fmoc deprotection and fi-
nal simultaneous cleavage of the linker and side chain
protecting groups by 10% TFA in DCM gave the tar-
geted library 1 (Fig. 4) as discrete compounds.
From a synthetic viewpoint, these modifications posed
new challenges for the synthesis of this library 1. First,
in contrast to the chemical structures of thymidine or
20-deoxyuridine, the starting nucleoside contains an
extra 20-hydroxy group. This hydroxyl could lead to
over-acylation byproducts and decreased loading capac-
ity of the resin due to bis-addition to the solid support.
Therefore, a parallel experiment was designed and car-
ried out to evaluate if 20-OH causes over-acylation and
compare the loading capacity of uridine, 20-deoxythymi-
dine, and 20-deoxyuridine. The loading capacity, deter-
mined by following an established protocol,7 was
found to be 52% for the uridine, slightly lower than that
obtained for thymidine and 20-deoxyuridine. The
decreased loading is attributable to increased steric
hindrance and some bis-loading of the diol. Evidence
for over-acylation of the free hydroxyl group was not
seen under the standard peptide and urea formation
condition used in the library synthesis.
The next major synthetic challenge involved the intro-
duction of the branched functionality. Two potential
strategies could be applied based on availability of start-
ing materials and compatibility with the silyl ether resin
linker. The first approach evaluated used an orthogo-
nally protected diaminoacid derivative, ivDde protected
1,3-diaminopropionic acid (Fmoc-Dpr(ivDde)-OH,9
2
in Fig. 2). The ivDde protecting group is stable to Fmoc
removal conditions of 20% piperidine and can be selec-
tively removed by 2% hydrazine in DMF allowing for
selective cleavage of either protecting group. The second
approach evaluated used a modified Fmoc protected
azido deoxyserine residue (Fmoc-Ser(N3)-OH, 3 in
Fig. 2). In this case the azido group is used to mask
the second amino functionality. Orthogonal azide reduc-
tion or Fmoc removal conditions could then be used to
selectively afford each primary amine. In test reactions
both methods yielded good results after final cleavage
based upon model compound synthesis. Fmoc-
Ser(N3)-OH was ultimately chosen for library synthesis
Library members bearing the same R3 substituent from
the last step of synthesis were archived in glass vials in
8 · 12 array format. A randomly selected row (12 sam-
ples) of each array was subsequently selected for reverse
phase HPLC and mass spectrometry analysis (total 120
samples, 12% of library size). Purity of products was
confirmed by HPLC with UV254 nm detection and MS.

D. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6899–6904
6901
O
N
O
NH
NH
Et
Si
N3
O
N
O
f-h
H
Et
a-b
c-e
O
O
N
N3
H
H2N
OH
O
OH
O
O
PS-DES
5
4
.
O
N
CF3CO2H
O
NH2
R3
O
NH
NH
N3
HN
O
N
O
O
H
O
H
i-l
H
O
H
O
R2
N
N
R2
N
N
N
N
H
H
OH
O
R1
O
O
R1
O
OH OH
O
1
6
Scheme 1. Synthesis of targeted library 1 on solid support. Reagents and conditions: (a) 1,3-dichloro-5,5-dimethylhydantoin (3 equiv), anhydrous
CH2Cl2, rt, 2 h; (b) 50-azido-50-deoxyuridine (3 equiv), imidazole (3.5 equiv), anhydrous DMF, rt, 4 h; (c) SnCl2/HSPh/NEt3 (1:4:5), rt, overnight; (d)
Fmoc-Ser(N3)-OH (5 equiv), HOBt (5 equiv), anhydrous CH2Cl2/DMF (1/1, v/v), DIC (5 equiv), rt, 5 h; (e) 4-methylpiperidine/DMF (25%, v/v), rt,
20 min.; (f) Fmoc-AA1-OH (3.5 equiv), HOBt (3.5 equiv), anhydrous CH2Cl2/DMF (1/1, v/v), DIC (3.5 equiv), rt, overnight; (g) 4-methylpiperidine/
DMF (25%, v/v), rt, 1 h; (h) R2NCO or i-butylOC(O)Cl/TEA (4 equiv), anhydrous CH2Cl2, 24 h; (i) SnCl2/HSPh/NEt3 (1:4:5), rt, overnight; (j)
Fmoc-AA2-OH (3.5 equiv), HOBt (3.5 equiv), anhydrous CH2Cl2/DMF (1/1, v/v), DIC (3.5 equiv), rt, overnight; (k) 4-methylpiperidine/DMF (25%,
v/v), rt, 1 h; (l) 10% TFA/CH2Cl2, rt, overnight. The point of attachment of 50-azido-50-deoxyuridine to the solid support could be via 20-OH or 30-
OH.
S
O
O
AA1 {1-10}
O
H
N
OH
Fmoc
OH
Fmoc
N
OH
Fmoc
N
H
Fmoc
OH
Fmoc
OH
N
H
N
H
H
O
O
O
O
{2}
{5}
{1}
{3}
{4}
O
O
N
O
HN
O
O
HN
O
Fmoc
OH
Fmoc
OH
Fmoc
OH
Fmoc
OH
N
H
Fmoc
OH
N
H
{7 }
N
H
N
H
N
H
O
O
O
O
O
{6}
R3 {1-10}
{8}
{9
}
{10
}
NCO MeO
NCO
NCO
O
NCO
NCO
NCO
NCO
{2}
{1}
{
3
}
{4}
{
5
}
{6}
O
O
O
NCO
Cl
NCO
O
{7 }
{9 }
{10}
{8}
AA2 {1-10}a
a
Figure 3. Building blocks for library 1 (10 · 10 · 10) synthesis. The same set of building blocks as AA1 {1–10} except for {2} Fmoc-Phe-OH was
used to replace Fmoc-b-Ala-OH and {9} Fmoc-Phe(NO2)–OH was used to replace Fmoc-Phe-OH.
Thirty percent of the samples analyzed had >80% HPLC
purity, 43% had HPLC purity ranging from 60% to 80%,
21% had 40–60% purity, and 6% gave an HPLC purity
of <40%. Among the 120 analyzed samples, 97% of
them provided the desired product by mass spectrome-
try. The overall yield was slightly disappointing, 8% by
gravimetric analysis. However, this yield was sufficient
to supply enough compound for all the required antimi-
crobial testing in multiple assays.
This library was screened for anti-tuberculosis activity.14
Ten initial hits were identified from primary screening

6902
D. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6899–6904
.
CF3CO2H
O
NH2
R3
O
NH
HN
N
O
O
H
H
O
R2
N
N
N
H
R1
OH
1
OH
O
O
NH2
R1 {
CH3
1
-10}=
OH
S
OH
NH
NH2
O
O
ß-Ala
{
1
}
{2
}
{3
}
{
4
}
{
5
}
{
6
}
{7
}
{
8
}
{
9
}
{10}
R2 {
1
-
10} =
H3CO
N
N
H
N
N
H
N
H
H
H
N
H
{
1
}
{
2
}
{
3
}
{
4
}
{5
}
{6}
O
O
N
H
O
N
H
N
H
O
{
7
}
{
8
}
{
9
}
{10}
NH2
R3 {
1-10} =
NO2
OH
NH
S
OH
NH2
O
O
CH3
}
{1
{
2
}
{
3
}
{4
}
{
5
}
{
6
}
{
7
}
{
8
}
{
9
}
{10}
Figure 4. Uridinyl branched peptide urea targeted library 1.
against M. tuberculosis (H37Rv). These 10 library mem-
bers were subsequently resynthesized by applying the
same solid-phase methodology, purified by preparative
RP-HPLC, and retested. Their characterization data
and anti-tuberculosis activity are shown in Table 1.
The structures of these compounds were assigned and
confirmed by mass spectrometry, 1H and 13C NMR,
for anti-tuberculosis activity. These 10 compounds were
also assessed for antimicrobial activity against Bacillus
anthracis (Sterne strain), Bacillus subtilis, Escherichia
coli (K-12), Enterococcus faecalis, and methicillin-sus-
ceptible Staphylococcus aureus (MSSA). No inhibitory
activity was found, this suggests that these compounds
have the potential to be developed into narrow spectrum
antibiotics targeting M. tuberculosis.
1
1H-13C HSQC, and H-1H COSY.15 The most active
compounds are 1 {9,1,10}, {8,5,9}, {8,1,10}, and
{10,5,10} with MIC value of 12.5 lg/mL. Hydrophobic
moieties (e.g., tryptophan at R3 position and n-hexyl or
4-methoxyphenyl at R2 position) appear to be required
In conclusion, a more complex uridinyl branched pep-
tide urea library was synthesized using IRORI directed
sorting technologies. High-throughput screening of this
Table 1. Characterization data and anti-tuberculosis activity of library members after resynthesis, purification, and retesting of primary hits
MS [M+Na]a (m/z)
HPLC purityb (%)
MIC90 (lg/mL)
c
tR (min)
Compound
1 {10,8,7}
1 {10,8,10}
1 {10,6,10}
1 {10,1,10}
1 {10,9,10}
1 {9,1,10}
1 {8,5,9}
813.4
885.5
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
96 (98)
4.67
4.94
5.09
5.22
4.83
5.23
4.79
5.04
4.99
4.98
>200
50
50
871.5
851.5
25
809.5
790.5 [M+1]
856.5
100
12.5
12.5
12.5
100
12.5
1 {8,1,10}
1 {4,6,9}
1 {10,5,10}
806.5 [M+1]
822.5
873.5
a Mass spectra were recorded on a Bruker Esquire LC–MS using ESI.
b
˚
Analytical RP-HPLC was conducted on a Shimadzu HPLC system with a Phenomenex C18 column (100 A, 3 lm, 4.6 · 50 mm), flow rate 1.0 mL/
min, and a gradient of solvent A (H2O 0.1% TFA) and solvent B (CH3CN): 0–2 min 100% A; 2–8 min 0–100% B (linear gradient). UV detection at
254 nm; figures in parentheses indicate HPLC purity detected at 218 nm.
c Retention time.

D. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6899–6904
6903
In the primary assay compounds were tested at 20, 10, and
5 lg/mL concentrations, based on recovered yield. The
resynthesized compounds were tested as a serial 1:1
dilution from 200 to 1.56 lg/mL for accurate MIC
determination. A hit in the primary screen assay was
determined to be compounds with MIC90 < 20 lg/mL.
Controls included wells containing no drug as a negative
control and isoniazid as a positive control (reproducible
MIC90 0.031 lg/mL).
library identified several library members with moderate
anti-tuberculosis activity (MIC: 12.5 lg/mL). These
compounds represented a good starting point for further
expansion and optimization, a more focused small
library is currently being designed and synthesized in
an effort to enhance this activity of this class. Studies
to determine if these compounds inhibit the MraY
pathway in M. tuberculosis remain to be performed.
These studies will be reported in due course.
15. Representative analytical data for library members after
resynthesis and purification by preparative HPLC.
-
Acknowledgments
CF3CO2
O
O
N
NH3
HN
N
NH
H
We thank National Institutes of Health Grant AI057836
for financial support. We thank Angela Buckman for
help with the large scale preparation of Fmoc-Ser(N3)-
OH and Jerrod Scarborough’s assistance with HPLC
analysis of the library.
O
O
H
N
H
H
O
N
N
N
H
OH OH
O
O
1{9,1,10}. 64.4 mg, 17.8% overall purified yield. 1H NMR,
500 MHz (DMSO-d6):d 11.34 (d, 1H, J = 1.7 Hz, N3H),
10.98 (d, J = 2.0 Hz, 1H, NH(Trp)), 8.58 (t, J = 5.7 Hz,
1H, NH), 8.14 (d, J = 8.5 Hz, 1H, NH), 8.05 (t,
J = 5.6 Hz, 1H, NH), 8.02 (d, J = 3.9 Hz, 3H, NH3þ),
7.70 (d, J = 7.8 Hz, 1H, CH(Trp)), 7.66 (d, J = 8.1 Hz, 1H,
C6H), 7.36 (d, J = 8.1 Hz, 1H, CH(Trp)), 7.26 (m, 2H,
Phe), 7.19 (m, 4H, 3CH(Phe) and CH(Trp)), 7.09 (dd,
J = 7.1 and 8.1 Hz, 1H, CH(Trp)), 7.00 (dd, J = 7.1 and
7.8 Hz, 1H, CH(Trp)), 6.18 (t, J = 5.4 Hz, 1H, NH(Urea)),
6.11 (d, J = 6.8 Hz, 1H, NH(Urea)), 5.74 (d, J = 5.4 Hz,
1H, H-10), 5.63 (dd, J = 2.0 and 8.1 Hz, 1H, C5H), 5.42 (br
s, 1H, OH), 5.18 (br s, 1H, OH), 4.47 (m, 1H, CH(Ser)),
4.24 (m, 1H, CH(Phe)), 4.06 (t, J = 5.4 Hz, 1H, H-20), 3.95
(m, 1H, CH(Trp)), 3.86 (m, 2H, H-30 and H-40), 3.20–3.58
References and notes
1. Antimicrobial Resistance. WHO Fact Sheet No. 194.
Health Communications, WHO. Geneva, 2002; <http://
www.who.int/inf-fs/en/fact194.html>.
2. Inukai, M.; Isono, F.; Takahashi, S.; Enokita, R.; Saka-
ida, Y.; Haneishi, T. J. Antibiot. 1989, 42, 662.
3. Isono, F.; Katayama, T.; Inukai, M.; Haneishi, T. J.
Antibiot. 1989, 42, 674.
4. Inukai, M.; Isono, F.; Takatsuki, A. Antimicrob. Agents
Chemother. 1993, 37, 980.
5. For papers related to Mureidomycins, see (a) Gentle, C.
A.; Bugg, T. D. H. J. Chem. Soc., Perkin Trans. 1 1999,
1279; (b) Gentle, C. A.; Harrison, S. A.; Inukai, M.;
Bugg, T. D. H. J. Chem. Soc., Perkin Trans. 1 1999,
1287; (c) Bozzoli, A.; Kazmierski, W.; Kennedy, G.;
Pasquarello, A.; Pecunioso, A. Bioorg. Med. Chem. Lett.
2000, 10, 2759; (d) Howard, N. I.; Bugg, T. D. H.
Bioorg. Med. Chem. 2003, 11, 3083; For papers related
to pacidamycins, see (e) Boojamra, C. G.; Lemoine, R.
(m, 5H, CH2-50, CH2(Ser), and CHa(Trp)), 3.02 (m, 2H,
0
CHa (Trp)and CHb(Phe)), 2.89 (m, 2H,0 NCH2(n-Hex)),
2.79 (dd, J = 9.5 and 13.9 Hz, 1H, CHb (Phe)), 1.22 (m,
8H(n-Hex)), 0.84 (t, J = 7.1 Hz, 3H, CH3(n-Hex)). 13C
NMR, 500 MHz (DMSO-d6):d 172.50, 169.72, 168.96,
163.06, 158.10, 150.71, 141.27, 137.84, 136.34, 129.18,
128.11, 127.01, 126.28, 124.96, 121.16, 118.42, 111.47,
106.87, 102.00, 88.32, 82.33, 72.52, 70.84, 55.13, 52.85,
52.55, 41.22, 40.49, 39.0 (overlapped with DMSO-d6
peak), 37.54, 31.02, 29.67, 27.33, 26.00, 22.07, 13.92. Mass
spectrum (ESI) m/z [M+1]+ 790.5. HPLC purity:100%,
tR = 5.23 min.
´
C.; Lee, J. C.; Leger, R.; Stein, K. A.; Vernier, N. G.;
Magon, A.; Lomovskaya, O.; Martin, P. K.; Chamber-
land, S.; Lee, M. D.; Hecker, S. J.; Lee, V. J. J. Am.
Chem. Soc. 2001, 123, 870; (f) Boojamra, C. G.;
Lemoine, R. C.; Blais, J.; Vernier, N. G.; Stein, K.
A.; Magon, A.; Chamberland, S.; Hecker, S. J.; Lee, V.
J. Bioorg. Med. Chem. Lett. 2003, 13, 3305.
O2N
6. Sun, D.; Lee, R. E. Tetrahedron Lett. 2005, 46, 8497.
7. Sun, D.; Lee, R. E. J. Comb. Chem. 2007, 9, 370.
8. For a recent review regarding naturally occurring nucle-
oside antibiotics, see Kimura, K.; Bugg, T. D. H. Nat.
Prod. Rep. 2003, 20, 252.
-
CF3CO2
O
O
N
NH3
NH
HN
O
O
H
N
H
H
O
N
N
N
9. Fmoc-Dpr(ivDde)-OH is available from Novabiochem,
($948/5 g).
H
MeO
OH OH
O
O
10. Schmidt, U.; Mundinger, K.; Biedl, B.; Haas, G.; Lau, R.
Synthesis 1992, 1201.
11. <http://www.irori.com>.
12. PS-DES resin (100–200 mesh, loading: 1.45 mmol/g) is
commercially available from Argonaut Technologies, now
a Biotage Company.
13. Hachmann, J.; Lebl, M. J. Comb. Chem. 2006, 8, 149.
14. Compounds were screened for MIC activity against M.
tuberculosis H37Rv using the microbroth dilution method
in Middlebrook 7H9 ADC media according to CLSI
guidelines. Compounds were dissolved and diluted in
DMSO. Four microliters of this solution was then added
to 196 lL of culture media containing 2 · 104 bacteria.
OH
1
1{8,5,9}.75.0 mg, 19.8% overall purified yield. H NMR,
500 MHz (DMSO-d6):d 11.34 (d, 1H, J = 1.7 Hz, N3H),
9.20 (br s, 1H, OH), 8.54 (t, J = 5.7 Hz, 1H, NH), 8.49 (s,
1H, NH(Urea)), 8.36 (d, J = 8.8 Hz, 1H, NH), 8.10 (m,
5H, CH, CH, andNH3þ), 8.02 (t, J = 5.7 Hz, 1H, NH),
7.66 (d, J = 8.1 Hz, 1H, C6H), 7.45 (d, J = 8.5 Hz, 2H),
7.13 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.3 Hz, 2H), 6.72 (d,
J = 8.8 Hz, 2H), 6.66 (d, J = 8.3 Hz, 2H), 6.17 (d,
J = 7.1 Hz, 1H, NH(Urea)), 5.74 (d, J = 5.4 Hz, 1H, H-
10), 5.64 (dd, J = 2.0 and 8.1 Hz, 1H, C5H), 5.45 (br s, 1H,

6904
D. Sun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6899–6904
OH), 5.18 (br s, 1H, OH), 4.52 (m, 1H, CH(Ser)), 4.36 (m,
13C NMR, 500 MHz (DMSO-d6): d 172.40, 169.68,
168.06, 163.04, 155.90, 155.15, 153.95, 150.69, 146.71,
143.06, 141.17, 133.11, 130.88, 130.10, 127.38, 123.46,
119.05, 115.01, 113.81, 101.99, 88.33, 82.28, 72.56, 70.89,
55.04, 54.79, 53.15, 51.96, 41.19, 40.56, 37.21, 36.53. Mass
spectrum (ESI) m/z [M+Na]+ 856.5. HPLC purity: 100%,
tR = 4.79 min.
1H, CH(Tyr)), 4.06 (t, J = 5.1 Hz, 1H, H-20), 4.02 (m, 1H,
CH(Phe(4-NO2)), 3.87 (m, 2H, H-30 and H-40), 3.68 (m,
1H, CHa(Ser)), 3.67 (s, 3H, CH3), 3.34 (m, overlapped
a0
with H2O peak, 2H, CH2-50), 3.16 (m, 02H, CH (Ser) and
CHb(Phe(4-NO2)), 2.94 (m, 2H, CHb (Phe(4-NO2)) and
0
CHc(Tyr)), 2.69 (dd, J = 8.8 and 13.9 Hz, 1H, CHc (Tyr)).