Synthesis and antibacterial activities of novel tyrocidine A glycosylated derivatives towards multidrug-resistant pathogens

Article abstract of DOI:10.1002/psc.2774

Glycosylation can have a multifaceted impact on the properties and functions of peptides and plays a critical role in interacting with or binding to the target molecules. Herein, based on the previously reported method for macrocyclic glycopeptide synthesis, two series of tyrocidine A glycosylated derivatives (1a-f and 2a-f) were synthesized and evaluated for their antibacterial activities to further study the structure and activity relationships (SAR). Biological studies showed that the synthetic glycosylated derivatives had good antibacterial activities towards methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. SAR studies based on various glycans and linkages were used to enhance the biochemical profile, resulting in the identification of several potent antibiotics, such as 1f, with a great improved therapeutic index than tyrocidine A.

Full text of DOI:10.1002/psc.2774

Special Issue Article
Received: 6 January 2015
Revised: 27 February 2015
Accepted: 2 March 2015
Published online in Wiley Online Library: 21 April 2015
(wileyonlinelibrary.com) DOI 10.1002/psc.2774
Synthesis and antibacterial activities of
novel tyrocidine A glycosylated
derivatives towards multidrug-
resistant pathogens‡
Yan Zou,§ Qingjie Zhao,§ Chunmei Zhang, Liang Wang, Wenjuan Li, Xiang Li,
Qiuye Wu* and Honggang Hu*
Glycosylation can have a multifaceted impact on the properties and functions of peptides and plays a critical role in interacting
with or binding to the target molecules. Herein, based on the previously reported method for macrocyclic glycopeptide synthesis,
two series of tyrocidine A glycosylated derivatives (1a–f and 2a–f) were synthesized and evaluated for their antibacterial activities
to further study the structure and activity relationships (SAR). Biological studies showed that the synthetic glycosylated
derivatives had good antibacterial activities towards methicillin-resistant Staphylococcus aureus and vancomycin-resistant
Enterococcus. SAR studies based on various glycans and linkages were used to enhance the biochemical profile, resulting in the
identification of several potent antibiotics, such as 1f, with a great improved therapeutic index than tyrocidine A. Copyright ©
2015 European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: tyrocidine A; N-glycosylated peptide; solid-phase peptide synthesis; antibacterial activity; antibiotic resistance
biological effects on multidrug-resistant pathogens [methicillin-
resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
Introduction
Antibiotic resistance has been a growing public health crisis and
has emerged as one of the preeminent public health concerns of
the 21st century [1,2]. Nowadays, some drug-resistant organisms
like multidrug-resistant pathogens have acquired resistance to
almost all of antibiotics that are widely used in clinic, making drugs
such as vancomycin, daptomycin, and linezolid less valid for
clinical treatment of infection, which were previously reserved for
highly resistant bacterial strains, our only and last line of defense
from the bacteria [3,4]. Therefore, new antibacterial molecules are
urgently needed to combat antibiotic resistance, which has led to
an increasing demand of new synthetic strategies that can afford
new types of antibiotics [5].
In recent years, great efforts have been made to structurally
modify naturally occurring macrocyclic peptide antibiotics such as
vancomycin, daptomycin, and telavancin [6–8]. One of such types
of antibiotics is tyrocidine A (1, Figure 1), a cyclic decapeptide
isolated from Bacillus subtilis, which showed strong antibacterial
activities against a number of drug-resistant pathogens [9,10].
Tyrocidine A represents an appealing lead compound in the
development of novel antibacterial agents, despite its strong side
effects [11–15]. Our previous work has reported an efficient method
to provide glycosylated derivatives of tyrocidine A [16]. Preliminary
biological results showed that the linkage between tyrocidine A
and glycans was crucial for retaining the antibacterial activities.
Therefore, to further explore the influence of different glycans
and linkages on antibacterial activity, in the present study, two
series of novel tyrocidine A glycosylated derivatives (1a–f and
2a–f, Figure 2) were designed, synthesized, and evaluated for their
Enterococcus (VRE)].
Chemistry
Our research plan was to investigate the influence of different
glycans on the antibacterial activities as well as alternative linkage
between glycan and cyclic peptide scaffolds. In achieving this
aim, as shown in Figure 3, our first step was to prepare fully
protected glycosylated asparagines derivatives 3a–3f and 4a–4f
as key building blocks following the reported methods (Scheme 1)
[16]. Moreover, compared with the previous work, acetyl was
chosen as another protective group of hydroxyls in 4b and 4c to
make the synthetic route more efficient [17].
With these building blocks in hand, the next procedure was to
construct the linear glycopeptides by solid-phase peptide synthesis
*
Correspondence to: Qiuye Wu and Honggang Hu, and Department of Organic
Chemistry, School of Pharmacy, Second Military Medical University, Shanghai
200433, China. E-mail: wuqy6439@sohu.com; huhonggang_fox@msn.com
§
These authors contributed equally to this work.
‡
Special issue of contributions presented at the 13th Chinese International Peptide
Symposium, Peptides: Treasure of Chemistry and Biology, June 30 to July 4, 2014 in
Datong, Shanxi, China.
Department of Organic Chemistry, School of Pharmacy, Second Military Medical
University, Shanghai 200433, China
J. Pept. Sci. 2015; 21: 586–592
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

NOVEL TYROCIDINE A GLYCOSYLATED DERIVATIVE
with Fmoc-Phe-OH in the presence of DIPEA. Next, the glycopep-
tides were assembled via sequential introduction of Fmoc-Pro-
OH, Fmoc-D-Phe-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Val-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, building blocks 3a–4f, and Fmoc-
D-Phe-OH using 20% piperidine as Fmoc-removed reagents and HCTU
(N,N,N',N'-Tetramethyl-O-(6-chloro-1H-benzotriazol-1-yl)uronium
hexafluorophosphate)/DIPEA in coupling reactions [18–23].
Subsequently, the loaded resins were treated with AcOH/TFE/
dichloromethane (DCM) (1 : 1 : 8) to give the linear glycopeptides
9a–f and 10a–f [16]. The released glycopeptides were directly
used in the next step without further purification. 1-Hydroxybenzotriazole
(HOBt), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluoro-
phosphate (PyBOP), and N,N-diisopropylethylamine were chosen as
the coupling reagents for cyclization. Both HPLC and MS results
indicated that the cyclization reactions were clean and produced
only the cyclic products 11a–f and 12a–f. The protecting groups of
12b–c were removed with diluted methanolic CH3ONa solution
and then treated with TFA, DCM, and Et3SiH (2:8:1, 5ml) to afford
the target compounds 2b–c [21]. Compounds 1a–f, 2a, and 2d–f
Figure 1. The structure of tyrocidine A.
and then unite their C-terminus and N-terminus via chemical
condensation to obtain cyclic glycopeptides. As shown in Scheme 2,
the first Phe residue was anchored to 2-chlorotrityl chloride resin
Figure 2. Structure of the target compounds 1a–f and 2a–f.
Figure 3. Structure of fully protected glycosylated asparagine derivatives 3a–4f.
J. Pept. Sci. 2015; 21: 586–592
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

ZOU ET AL.
Scheme 1. General synthetic route for building blocks 3a–4f, reagents and conditions: (a) BF3.Et2O, DCM, H(OCH2CH2)nN3 and (b) CH3ONa-CH3OH; then,
NaH, BnBr, DMF; (c) Pd-CaCO3, H2; then, DCC, HOBt, Fmoc-Asp-OtBu, DCM; (d) 25% TFA, DCM.
Scheme 2. Synthetic route for target compounds, reagents and conditions: (a) HOAc, TFE, DCM (1 : 1 : 8), RT; (b) PyBOP, HOBt, N,N-diisopropylethylamine,
DCM; (c) CH3ONa-CH3OH; (d) Pd–C, H2; and (e) TFA, Et3SiH, DCM (2 : 1 : 8). SPPS, solid-phase peptide synthesis.
were obtained by treating 11a–f, 12a, and 12d–f with palladium-
catalyzed hydrogenation and TFA/DCM/Et3SiH successively [16].
The crude peptides were purified by RP-HPLC to afford 1a–f and
2a–f in good purities and overall yield 8–14%. All the final products
were fully characterized by NMR and high resolution (HR)-quadru-
pole (Q)-TOF-MS.
added into a 96-well plate. Fresh human red blood cells were
diluted 100-fold in phosphate-buffered saline (10 mM Na2HPO4,
1.8 mM KH2PO4, 137mM NaCl, and 2.7 mM KCl, pH 7.4), and the
diluted red blood sample was transferred into each well and mixed
with the peptide solution (final volume of 50 μl). The plate was
incubated overnight at room temperature and visually inspected
for cell lysis. MHC was defined as the lowest concentration required
to lyse half of the red blood cells.
Biological Evaluation
In our previous work, compound 1a has been successfully prepared
and just tested for its antibacterial activity towards B. subtilis [16].
Herein, the antibacterial activities of tyrocidine A, 1a, and new
synthetic glycosylated derivatives 1b–f and 2a–f were further
evaluated using MRSA and VRE. Their hemolytic activities, defined
as minimal hemolytic concentrations (MHCs), were also
determined. Most of the tyrocidine A glycosylated derivatives were
active against all bacterial species, with MIC values ranging from
6.25 to 25 μg/ml (Table 1). A published method was adapted for
MHC determination [24,25]. The cyclic peptide stock solutions were
serially diluted (twofold) in H2O, and the diluted solutions were
Results and Discussion
The tyrocidine A glycosyl analogues were synthesized using
standard Fmoc-solid-phase peptide synthesis (Scheme 2). In this
work, we tried two different methods to prepare glycosyl
asparagines derivatives. The first one was that acetyl groups were
replaced by benzyl to protect the hydroxyl groups of sugars, and
it could be easily deprotected by catalytic hydrogenation. The
second one was that the acetyl groups were directly used to
protect hydroxyl groups and deprotected by diluted
MeONa/MeOH after cyclization of liner peptide without affecting
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Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015; 21: 586–592

NOVEL TYROCIDINE A GLYCOSYLATED DERIVATIVE
Table 1. In vitro antibiotic activity towards MRSA and VRE of the target
compounds
the title compounds showed higher antibacterial activity and
improved therapeutic indexes than tyrocidine A. Fully acetylated
glycosylated amino acid building blocks were successfully used to
prepare title compounds 2b and 2c, which made the total synthetic
process more efficient. The present method is cost-efficient and can
be used for synthesizing other glycopeptides and cyclic glycopep-
tides. Compound 1f was identified as the most potent compound
and had been used in the further biological study.
Compound
MIC
(μg/ml)
MHC
(μg/ml)
Therapeutic index
(MHC/MIC)
MRSA
VRE
MRSA
VRE
Tyrocidine A
25
25
6.25
25
0.25
1
0.25
1
1a
1b
1c
1d
1e
1f
25
25
25
25
6.25
6.25
12.5
12.5
25
0.25
0.5
0.5
0.5
2
0.25
0.5
0.5
0.5
2
12.5
25
12.5
25
Experimental Section
25
25
General Experimental Methods
12.5
25
12.5
25
2a
2b
2c
2d
2e
2f
25
1
1
Purification of crude peptides was carried out by using HPLC
(LC-6A, Shimadzu, Kyoto, Japan) with dual wavelength (215 and
254 nm). 1H NMR and 13C-NMR spectra were recorded at 300
and 600 MHz with the chemical shifts reported in parts per million
(δ) downfield from tetramethylsilane (TMS). Coupling constants
(J) are reported in hertz (Hz). ESI-MS and HR-Q-TOF-MS were
measured with a Finnigan LCQ Deca XP Max mass spectrometer
(Thermo Electron Corporation, Beverly, Massachusetts, USA) and
an Agilent 6538 UHD Accurate Mass Q-TOF LC/MS mass
spectrometer (Agilent Technologies, Santa Clara, California, USA),
respectively. Thin layer chromatography (TLC) was performed on
silica gel plates detected by charring with phosphomolybdic acid
in EtOH or 5% H2SO4 in EtOH. Commercial anhydrous solvents
and reagents were used without further purification. Glycosyl as-
paragines derivatives 3a–3f and 4a–4f were synthesized by our re-
ported procedure [16,21].
12.5
25
12.5
12.5
25
12.5
6.25
12.5
12.5
6.25
1
1
0.25
1
0.5
0.5
1
12.5
12.5
25
12.5
12.5
1
0.25
0.5
MRSA, methicillin-resistant Staphylococcus aureus; VRE, vancomycin-
resistant Enterococcus; MHC, minimal hemolytic concentration.
other groups. In our present work, glycosyl asparagines derivatives
3a–3e, 4a, 4d, and 4e were synthesized by the first procedure,
while intermediates 4b–c were prepared by the second method.
All glycosyl asparagines derivatives were obtained successfully.
There were five steps in the first procedure, while there were only
three steps in the later procedure that was more efficient. In the
synthesis of linear peptide, we chose HCTU/DIPEA as coupling
reagents instead of HBTU/HOBt/DIPEA. A single coupling reaction
using HCTU without HOBt and a 30-min reaction time is enough
for most amino acids [23].
Synthesis of the Title Compounds 1a–f and 2a–f (General
Procedure)
After a mixture of trityl resin 5 (100mg, active Cl, and 0.12 mmol),
Fmoc-Phe-ONa (100mg), DIPEA (0.2 ml), and DCM was shaken on
a vortex mixer at RT for 3 h, the resin was filtered off and washed
several times with MeOH and DCM. The Phe-linked resin was then
loaded onto the automatic peptide synthesizer for the construction
of full length peptide/glycopeptides. The protocols employed were
deprotection of Fmoc with 20% piperidine in N-methyl-2-pyr-
rolidone (NMP) and peptide coupling using 10 eq. of amino acid
and 10 eq. of HCTU/DIPEA. All deprotection and coupling reactions
were set to perform for 2 h at RT. Amino acids Fmoc-Pro-OH, Fmoc-
D-Phe-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Val-OH, Fmoc-
Tyr(tBu)-OH, and Fmoc-Gln(Trt)-OH were sequentially installed to
construct an octapeptide on the resin. Glycosyl amino acids 3a–f
and 4a–f (2eq.) were, respectively, mixed with DCC and HOBt in
DCM, and the mixture was shaken with a vortex mixer at RT over-
night. After the resin was washed sequentially with NMP, MeOH,
and DCM, it was treated with 20% piperidine in NMP at RT for
2 h. The resin was washed again with NMP, MeOH, and DCM
and was finally subjected to coupling with Fmoc-D-Phe-OH
(5eq.) and de-Fmoc by the same methods to form the desired
glycopeptides on resin (7a–f and 8a–f).
As shown in Table 1, the antibacterial activities of compounds
(1c, 1f, 2b, 2d, and 2e) were higher than those of the other com-
pounds. The MHC value of compounds 1a, 1d–f, 2a–b, and 2d–e
were higher than that of tyrocidine A. Compared with tyrocidine
A, which had an MHC/MIC ratio of 0.25, most of the analogues
had substantially improved therapeutic indexes (MHC/MIC in the
range of 0.25–2). Compound 1f showed the best overall properties.
The MIC value of compound 1f against MRSA and VRE was twice
lower than that of tyrocidine A. To test the importance of the
linkage between tyrocidine A and glycan, compounds 2a–f were
synthesized with a longer linkage compared with compounds
1a–f. The MIC value of compounds 2b and 2d–e was lower than
that of compounds 1b and 1d–e, against MRSA and VRE in vitro.
The MHC/MIC ratio of compounds 2b and 2e was higher than that
of compounds 1b and 1e, respectively. But compound 1f that
contained α-mannose had an MHC/MIC ratio of 2 that was eight
times higher than that of tyrocidine A and compound 2f. These
results suggested that a short linkage (compound 1f) could be
beneficial for derivatives with α-glycans, while a long linkage
(compounds 2b and 2d–e) could be favorable for compounds with
β-glycans. Finally, compound 1f was identified as the most effective
compound, and further biological study is in progress.
To remove the linear peptides 7a–f and 8a–f from the resin, the
peptide-loaded resin was treated with a mixture of HOAc and TFE in
DCM (1 : 1 : 8, 15ml) for 1 h at RT. The resin was filtered off and was
washed with DCM and DCM/MeOH mixture. The washings were
combined and condensed in vacuum to give crude 9a–f and
10a–f. 9a, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2707.3; found,
2707.4. 9b, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2707.3; found,
2707.6. 9c, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2155.1; found,
Conclusion
In summary, 12 macrocyclic glycopeptides with different linkages
were synthesized through a practical and versatile method to
further explore the structure and activity relationships. Most of
J. Pept. Sci. 2015; 21: 586–592
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
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ZOU ET AL.
2154.8. 9d, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2275.1; found,
2275.5. 9e, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2275.1; found,
2274.9. 9f, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2275.1; found,
2275.1. 10a, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2751.3; found,
2751.7. 10b, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2751.3; found,
2751.4. 10c, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2199.1; found,
2199.2. 10d, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2319.1; found,
2319.2. 10e, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2319.1; found,
2319.0. 10f, MALDI-TOF-MS: calcd for [M+ Na]+ m/z, 2319.1; found,
2319.3.
For peptide/glycopeptide cyclization, after PyBOP (3 eq.), HOBt
(3eq.), and DIPEA (6eq.) were dissolved in DCM (200ml) in a
round-bottom flask, 9a–f and 10a–f dissolved in DCM (0.5 mg/ml)
was dropped into the solution in a period of 2 h. After the addition
was finished, the mixture was stirred for another 10h. Then, DCM
was removed, and the resulting residue was briefly purified by
Sephadex LH-20 (Energy Chemical, Shanghai, China) to afford
11a–f and 12a–f. 11a, MALDI-TOF-MS: calcd for [M+ Na]+ m/z,
2689.3; found, 2689.5. 11b, MALDI-TOF-MS: calcd for [M+ Na]+
m/z, 2689.3; found, 2689.7. 11c, MALDI-TOF-MS: calcd for [M+
Na]+ m/z, 2137.1; found, 2136.8. 11d, MALDI-TOF-MS: calcd for [M
+ Na]+ m/z, 2257.1; found, 2257.5. 11e, MALDI-TOF-MS: calcd for
[M+ Na]+ m/z, 2257.1; found, 2256.9. 11f, MALDI-TOF-MS: calcd
for [M+ Na]+ m/z, 2257.1; found, 2257.1, 12a, MALDI-TOF-MS: calcd
for [M+ Na]+ m/z, 2733.3; found, 2733.6. 12b, MALDI-TOF-MS: calcd
for [M+ Na]+ m/z, 2733.3; found, 2733.1. 12c, MALDI-TOF-MS: calcd
for [M+ Na]+ m/z, 2181.1; found, 2180.9. 12d, MALDI-TOF-MS: calcd
for [M+ Na]+ m/z, 2301.1; found, 2301.3. 12e, MALDI-TOF-MS: calcd
for [M+ Na]+ m/z, 2301.1; found, 2301.1. 12f, MALDI-TOF-MS: calcd
for [M+ Na]+ m/z, 2301.1; found, 2301.4.
3.71 (m, 1H), 3.66 (m, 2H), 3.61 (t, 1H, J= 8.7 Hz), 3.58–3.54 (m, 3H),
3.48–3.42 (m, 3H), 3.39–3.28 (m, 7H), 3.21 (d, 2H, J = 11.4 Hz), 3.12
(t, 2H, J = 12.6 Hz), 2.86 (m, 1H), 2.25 (m, 1H), 2.37–2.24 (m, 3H),
2.04 (m, 1H), 1.96–1.83 (m, 8H), 1.58 (m, 1H), 1.51 (m, 1H), 1.35
(m, 1H), 1.24 (d, 6H, J= 15.0 Hz), 1.17 (d, 4H, J = 6.0 Hz), 1.15 (d, 4H,
J= 6.0 Hz), 0.52 (m, 1H). 13C-NMR (150 MHz, CD3OH, TMS): δ
176.42, 172.20, 172.08, 171.90, 171.60, 171.49, 171.13, 171.05 (2C),
170.97, 170.56, 169.93, 155.37, 137.25, 137.13, 135.04, 129.06 (3C),
128.82 (2C), 128.25 (2C), 127.80 (2C), 127.60 (2C), 127.43 (2C),
127.17, 126.71, 126.06 (2C), 125.94, 114.48 (2C), 103.25, 102.08,
78.75, 75.33, 74.51, 74.46, 72.98 (2C), 70.60, 68.43, 67.12, 60.74,
59.98, 59.56, 58.03, 56.78, 54.97, 54.30, 53.27, 50.83, 50.56, 49.47,
45.78, 41.25, 39.73, 39.39, 38.44, 36.70, 36.30, 35.53, 31.70, 30.89,
29.84, 25.07, 24.49 (2C), 22.51, 22.06 (2C), 21.57, 21.18 (2C), 17.65
(2C). HR-Q-TOF-MS calcd for C80H112N13O24+, (M+H)+, 1638.7938,
found, 1638.7969.
1b, yield 19.5 mg, 9.9%. 1H NMR (600 MHz, CD3OD, TMS): δ 9.39
(s, 1H), 9.29 (s, 1H), 8.90 (d, 1H, J = 8.4Hz), 8.60 (s, 1H), 8.56 (s, 1H),
7.87 (s, 1H), 7.52 (d, 1H, J = 9.6Hz), 7.44 (s, 2H), 7.34–7.16 (m, 15H),
6.80 (d, 2H, J = 6.0 Hz), 6.48 (d, 2H, J = 6.0Hz), 5.86 (s, 1H), 5.48
(d, 1H, J = 6.0Hz), 5.10 (s, 1H), 5.01 (s, 1H), 4.88 (s, 1H), 4.65
(m, 2H), 4.52 (m, 1H), 4.20 (d, 1H, J = 7.8 Hz), 4.15–4.09 (m, 2H),
4.35 (m, 1H), 3.84 (m, 1H), 3.77 (m, 1H), 3.74–3.61 (m, 7H), 3.55
(t, 2H, J= 9.0Hz), 3.48 (m, 1H), 3.43–3.34 (m, 3H), 3.32–3.20
(m, 6H), 3.19–3.13 (m, 2H), 3.12–3.21 (m, 3H), 2.82 (m, 1H), 2.42
(m, 1H), 2.34–2.18 (m, 3H), 2.02 (m, 1H), 1.96–1.77 (m, 6H), 1.72
(m, 2H), 1.54 (m, 1H), 1.47 (m, 1H), 1.31 (m, 1H), 1.19 (d, 6H,
J= 13.2 Hz), 1.12 (d, 4H, J = 5.4 Hz), 1.09 (d, 4H, J = 4.8 Hz), 0.98
(m, 1H), 0.47 (m, 1H). HR-Q-TOF-MS calcd for C80H112N13O24+, (M
+H)+, 1638.7938, found, 1638.7945.
For the deprotection of 11a–f and 12a and 12d–f, firstly, they
were mixed with 10% Pd–C (10 mg) in CH3OH (10 ml), and the
mixture was stirred under a H2 atmosphere at RT for 24 h. After
the catalyst was filtered off, the solution was condensed in a
vacuum. The residue was then dissolved in a mixture of TFA,
DCM, and Et3SiH (2: 8 : 1, 5 ml). After the mixture was stirred at RT
for 2 h, the solvent was removed in vacuum, and the residue was
co-evaporated with toluene for a few times. The crude product
was dissolved in water (5 ml) and extracted with ether (3× 3 ml)
and was then purified by HPLC (conditions: Supelco Discovery
C18, 250× 10 mm, suitable ratio of acetonitrile-0.1%TFA in water-
0.1%TFA, 2 ml/min) to give 1a–f, 2a, and 2d–f.
For the deprotection of 12b–c, firstly, they were mixed with 50 ml
of 0.05 M NaOMe in methanol and stirred at RT for 3 h, and it was
neutralized to pH 6–7 using Amberlyst H+ resin. The solution was
condensed in a vacuum. The residue was then dissolved in a
mixture of TFA, DCM, and Et3SiH (2: 8 : 1, 5 ml). After the mixture
was stirred at RT for 2 h, the solvent was removed in vacuum, and
the residue was co-evaporated with toluene for a few times. The
crude product was dissolved in water (5ml) and extracted with
ether (3× 3 ml) and was then purified by HPLC (conditions: Supelco
Discovery C18, 250 ×10mm, suitable ratio of acetonitrile-0.1%TFA in
water-0.1%TFA, 2 ml/min) to give 2b–c. The characterization data of
the title compounds are given later.
1c, yield 14.1 mg, 8.1%. 1H NMR (600MHz, CD3OD, TMS): δ 9.39
(s, 1H), 9.28 (s, 1H), 8.89 (d, 1H, J= 8.4Hz) 8.60 (s, 1H), 7.80 (d, 1H,
J= 7.2 Hz), 7.50 (d, 1H, J= 8.4Hz), 7.39 (d, 2H, J = 6.6Hz), 7.30–7.12
(m, 15H), 6.75 (d, 2H, J = 7.8Hz), 6.42 (d, 2H, J= 7.8Hz), 5.82 (s, 1H),
5.47 (s, 1H), 5.00 (s, 1H), 4.95 (s, 1H), 4.77 (s, 1H), 4.71 (s, 1H), 4.60
(m, 2H), 4.50 (m, 1H), 4.15 (d, 1H), 4.07 (d, 2H), 4.14 (d, 1H,
J= 7.2 Hz), 4.11–4.05 (m, 2H), 3.97–3.88 (m, 1H), 3.86–3.72 (m, 1H),
3.62 (m, 1H), 3.55 (m, 2H), 3.52–3.44 (m, 1H), 3.44–3.33 (m, 3H),
3.30–3.18 (m, 7H), 3.18–2.86 (m, 6H), 2.80 (m, 1H), 2.40 (t, 1H,
J= 12.9 Hz), 2.36–2.10 (m, 4H), 1.98 (m, 1H), 1.93–1.65 (m, 9H), 1.53
(m, 1H), 1.43 (s, 1H), 1.32 (m, 1H), 1.15 (dd, 6H, J = 15.3 Hz), 1.07
(m, 9H), 0.44 (s, 1H). HR-Q-TOF-MS calcd for C73H100N13O18+, (M
+H)+, 1446.7304, found, 1446.7283.
1d, yield 16.6 mg, 9.4%. 1H NMR (600 MHz, CD3OD, TMS): δ 9.35
(s, 1H), 9.25 (s, 1H), 8.90 (d, 1H, J = 8.4Hz), 8.60 (s, 1H), 7.82 (s, 1H),
7.52 (d, 1H, J = 9.0 Hz), 7.40–7.12 (m, 17H), 6.79 (d, 2H, J = 7.8Hz),
6.43 (d, 2H, J = 7.2 Hz), 5.84 (s, 1H), 5.47 (d, 2H, J = 5.4Hz), 5.00
(s, 1H), 4.72 (s, 1H), 4.67–4.55 (m, 2H), 4.50 (m, 1H), 4.20 (d, 1H,
J= 7.8 Hz), 4.15 (d, 1H, J = 7.2 Hz), 4.08 (t, 1H, J = 5.4 Hz), 3.92
(m, 1H), 3.78–3.63 (m, 3H), 3.55 (dd, 1H, J= 12.0, 5.4Hz), 3.48
(s, 1H), 3.40–3.30 (d, 2H, J = 10.2 Hz), 3.29–3.20 (m, 5H), 3.18–3.11
(m, 4H), 3.06 (t, 1H, J = 13.2 Hz), 2.95 (s, 2H), 2.83–2.76 (m, 1H), 2.40
(t, 1H, J= 6.9Hz), 2.32–2.13 (m, 4H), 2.20 (m, 1H), 1.92–1.75
(m, 7H), 1.70 (m, 2H), 1.52 (m, 1H), 1.45 (m, 1H), 1.30 (s, 1H), 1.16
(t, 6H, J = 7.5Hz), 1.12–0.98 (dd, 8H, J = 16.8Hz), 0.95 (m, 1H), 0.43
(s, 1H). HR-Q-TOF-MS calcd for C74H102N13O19+, (M+H)+, 1476.7409,
found, 1476.7405.
1a, the NMR data was consistent with our report [16]: yield
1
27.3 mg, 13.9%. H NMR (600MHz, CD3OD, TMS): δ 9.46 (s, 1H),
9.36 (s, 1H), 9.16 (s, 1H), 8.95 (s, 2H), 8.66 (s, 1H), 7.93 (s, 1H), 7.56
(d, 1H, J =9.0 Hz), 7.49 (s, 2H), 7.34–7.22 (m, 15H), 6.84 (d, 2H,
J = 7.8Hz), 6.51 (d, 2H, J = 7.2Hz), 5.93 (s, 1H), 5.54 (s, 1H), 5.06
(s, 1H), 5.00 (s, 1H), 4.86 (s, 1H), 4.68 (m, 2H), 4.56 (s, 1H), 4.37
(d, 1H, J = 7.8Hz), 4.33 (d, 1H, J= 6.6Hz), 4.19 (m, 2H), 4.03 (m, 1H),
3.88 (d, 1H, J = 2.4Hz), 3.85 (d, 1H, J = 7.8Hz), 3.82–3.76 (m, 4H),
1e, yield 19.2 mg, 10.8%. 1H NMR (600 MHz, CD3OD, TMS): δ 9.40
(s, 1H), 9.26 (s, 1H), 8.88 (d, 1H, J= 9.0Hz), 8.57 (s, 1H), 7.85 (d, 1H,
J= 7.2 Hz), 7.51 (d, 1H, J = 9.0 Hz), 7.39 (d, 2H), 7.32–7.10 (m, 15H),
6.78 (d, 2H, J = 7.8 Hz), 6.45 (d, 2H, J = 7.8Hz), 5.85 (s, 1H), 5.48
(s, 1H), 5.00 (s, 1H), 4.93 (s, 1H), 4.74 (s, 1H), 4.67–4.57 (m, 2H), 4.50
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Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015; 21: 586–592

NOVEL TYROCIDINE A GLYCOSYLATED DERIVATIVE
(m, 1H), 4.19 (d, 1H, J = 7.2 Hz), 4.14 (d, 1H, J= 7.2Hz), 4.09 (t, 1H,
J= 5.4Hz), 3.92 (m, 1H), 3.81 (d, 1H), 3.78–3.68 (m, 2H), 3.66 (m,
2H), 3.53 (dd, 1H, J= 9.9 Hz), 3.49–3.40 (m, 3H), 3.35 (m, 2H), 3.30–
3.18 (m, 4H), 3.15 (d, 1H, J = 11.4 Hz), 3.06 (t, 3H, J = 13.2Hz), 2.81
(m, 1H), 2.41 (t, 1H, J = 12.9 Hz), 2.30–2.17 (m, 3H), 2.05–1.96
(m, 1H), 1.93–1.65 (m, 9H), 1.52 (d, 1H, J = 6.0 Hz), 1.45 (s, 1H),
1.36–1.25 (m, 1H), 1.17 (t, 6H, J = 6.6Hz), 1.13–1.02 (dd, 9H, J = 17.1
Hz), 0.43 (s, 1H). HR-Q-TOF-MS calcd for C74H102N13O19+, (M+H)+,
1476.7409, found, 1476.7421.
2.40–2.31 (m, 1H), 2.31–2.14 (m, 4H), 2.10 (s, 2H), 2.00 (s, 3H),
1.86 (s, 5H), 1.73 (s, 3H), 1.53 (s, 2H), 1.38 (d, J = 28.4 Hz, 8H),
1.22–1.07 (m, 12H), 0.97 (t, J= 6.7 Hz, 3H), 0.51 (s, 1H). HR-Q-
TOF-MS calcd for C75H105N13O19, (M+2H)/2+, 745.8819, found,
745.8825.
2d, yield 19.8 mg, 10.8%. 1H NMR: (600 MHz, CD3OD, TMS) δ: 0.42
(1H, s) 0.89–0.98 (6H, m) 1.09–1.12 (2H, dd, J =18Hz) 1.13–1.14 (6H,
t, J= 1.8Hz), 1.28 (2H, m), 1.44 (1H, m), 1.49 (1H, m), 1.65 (2H, m),
1.78 (6H, m), 1.97 (1H, m), 2.15 (4H, m), 2.39–2.41 (1H, t,
J = 13.2Hz), 2.79 (1H, m) 2.90 (2H, s) 3.05–3.06 (1H, t, J = 9 Hz),
3.21–3.22 (1H, d, J = 7.8Hz), 3.19 (10H, m), 3.36–3.37 (3H, d,
J = 9 Hz), 3.44 (8H, m) 3.69–3.70 (1H, d, J = 6 Hz), 3.882–3.886
(1H, d, J = 2.4Hz), 4.06–4.07 (1H, t, J = 5.4Hz), 4.14–4.15 (1H,
d, J = 8.4 Hz), 4.24–4.25 (1H, d, J = 7.8Hz), 4.47 (1H, m), 4.57
(2H, m), 4.69 (1H, s), 4.97 (2H, s), 5.45–5.46 (1H, d, J= 6 Hz),
5.81–5.83 (1H, t, J = 10.4 Hz), 6.43–6.44 (2H, d, J = 7.2 Hz),
6.77–6.78 (2H, d, J = 7.8Hz), 7.17 (14H, m), 7.50–7.51 (2H, d,
J = 9.6Hz), 7.80–7.82 (1H, d, J = 8.4Hz), 8.60 (1H, s), 8.88–8.89
(2H, d, J = 8.4Hz) 9.34 (1H, s). 13C NMR (150MHz, CD3OD,
TMS) δ: 172.32, 172.18, 171.96, 171.52, 171.44, 171.02, 170.33,
170.07, 155.38, 137.14, 137.05, 135.00, 129.05, 128.83, 128.27,
127.81, 127.56, 127.46, 127.15, 126.70, 126.00, 125.86, 114.46,
102.52, 76.21, 76.13, 73.22,69.72, 69.23, 68.45, 67.84, 60.88,
59.67, 57.92, 56.78, 54.97, 54.26, 53.45, 52.99, 50.48, 49.31,
45.84, 41.25, 39.63, 38.98, 38.62, 36.98, 36.61, 35.36, 31.65,
29.85, 28.03, 25.02, 24.48, 22.54, 22.04. HR-Q-TOF-MS calcd for
C76H107N13O20, (M+2H)/2+, 760.8872, found, 760.8881.
1f, yield 17.9 mg, 10.1%. 1H NMR (600 MHz, CD3OD, TMS): δ 9.40
(m, 3H), 8.90 (m, 2H), 8.64 (s, 1H), 7.86 (s, 1H), 7.55 (d, 1H,
J= 9.0Hz), 7.41 (m, 2H), 7.35–7.14 (m, 14H), 6.80 (d, 2H, J = 7.8Hz),
6.46 (d, 2H, J = 6.6 Hz), 5.88 (s, 1H), 5.50 (d, 1H, J = 4.8Hz),
5.13–4.98 (m, 2H), 4.82–4.71 (s, 1H), 4.70–4.57 (m, 3H), 4.56–4.47
(t, 1H, J = 4.8Hz), 4.17 (d, 1H, J= 7.2Hz), 4.12 (s, 1H), 3.84 (s, 1H),
3.71 (m, 3H), 3.65–3.55 (m, 5H), 3.50 (t, 1H, J = 10.8 Hz), 3.42–3.34
(m, 2H), 3.32–3.22 (m, 4H), 3.17 (m, 1H), 3.10–2.92 (m, 3H), 2.81
(m, 1H), 2.41 (t, 1H, J = 12.6 Hz), 2.34–2.18 (m, 4H), 2.08–1.97
(m, 1H), 1.94–1.78 (m, 6H), 1.78–1.66 (m, 3H), 1.53 (m, 1H), 1.46
(m, 1H), 1.38–1.26 (m, 1H), 1.22–1.16 (m, 6H), 1.14–1.02 (m, 8H),
+
0.5–0.4 (d, 1H, J =3.0 Hz). HR-Q-TOF-MS calcd for C74H102N13O19
,
(M+H)+, 1476.7409, found, 1476.7408.
2a, yield 21.4 mg, 10.6%. 1H NMR (600 MHz, DMSO) δ 7.86
(d, J = 8.0 Hz, 1H), 7.56 (d, J= 9.0Hz, 1H), 7.42 (s, 2H), 7.38–7.17
(m, 20H), 6.84 (d, J = 7.6Hz, 2H), 6.53 (s, 2H), 5.90 (s, 1H), 5.53
(s, 1H), 5.41 (s, 1H), 5.02 (s, 2H), 4.78 (s, 2H), 4.72–4.60 (m, 4H), 4.54
(s, 2H), 4.39 (d, J = 8.4Hz, 1H), 4.21 (d, J =8.2 Hz, 1H), 4.13
(t, J =5.8 Hz, 1H), 3.28 (ddd, J= 42.5, 29.0, 10.2 Hz, 10H), 3.14–3.00
(m, 4H), 2.88 (s, 2H), 2.46 (d, J= 13.1 Hz, 2H), 1.58 (dd, J = 35.6,
28.2 Hz, 6H), 1.43–1.36 (m, 10H), 1.28 (s, 9H), 1.24–1.07 (m, 17H),
0.96 (t, J = 7.0Hz, 6H), 0.50 (s, 1H). HR-Q-TOF-MS calcd for
C82H115N13O25, (M+2H)/2+, 841.9136, found, 841.9131.
2e, yield 17.9 mg, 9.8%. 1H NMR:(600 MHz, CD3OD, TMS) δ: 0.46
(1H, s), 0.90–1.00 (8H, m), 1.08–1.09 (2H, dd, J = 6 Hz), 1.15–1.16
(6H, t, J = 7.8 Hz), 1.30 (3H, m), 1.42 (1H, m), 1.43 (1H, m), 1.68
(3H, m), 1.81 (8H, m), 1.99 (1H, m), 2.18 (4H, m), 2.40 (1H, t,
J = 13.2Hz), 2.83 (1H, m), 2.96 (1H, t, J = 13.2 Hz), 3.07 (1H, t,
J = 12.6Hz), 3.15–3.16 (1H, d, J = 10.8 Hz), 3.25 (4H, m) 3,36 (4H, m),
3.50 (10H, m), 3.77 (2H, m), 3.88 (2H, m), 4.08–4.09 (1H, t,
J = 5.4Hz), 4.16–4.18 (1H, d, J= 7.8 Hz), 4.23–4.24 (1H, d, J = 7.8 Hz),
4.50 (1H, m), 4.52 (3H, m), 4.71 (1H, s), 4.93 (3H, m), 5.49–5.50
(1H, d, J = 4.2Hz), 5.84–5.86 (1H, t, J = 4.2 Hz), 6.45–6.46 (2H, d,
J = 7.8Hz), 6.79–6.80 (2H, d, J = 7.8Hz), 7.15 (16H, m), 7.52–7.53
(2H, d, J = 8.4 Hz), 7.41–7.43 (2H, d, J = 7.2Hz), 7.83–7.85 (1H, d,
J = 8.4Hz), 8.62 (1H, s) 8.73 (1H, s) 8.92–8.93 (2H, d, J = 9.0 Hz),
9.26–9.34 (3H, t, J = 7.8 Hz). HR-Q-TOF-MS calcd for C76H107N13O20,
(M+2H)/2+, 760.8872, found, 760.8879.
2b, yield 27.6 mg, 13.7%. 1H NMR (600MHz, CD3OD, TMS) δ: 0.42
(1H, s), 0.90–1.08 (6H, m), 1.10–1.11 (2H, dd, J = 6 Hz), 1.292–1.299
(6H, t, J = 4.2Hz), 1.29 (2H, m), 1.43 (1H, m), 1.51 (1H, m), 1.66
(3H, m), 1.75 (6H, m), 1.99 (1H, m), 2.16 (4H, m), 2.41 (1H, t,
J= 12.6 Hz), 2.76 (1H, m), 2.92 (1H, m), 3.05–3.07 (1H, t, J = 9.6Hz),
3.15–3.16 (1H, d, J = 6.6 Hz), 3.37–3.38 (1H, d, J = 1.8 Hz), 3.45 (14H,
m), 3.83 (4H, m), 4.08–4.09 (1H, t, J = 6 Hz), 4.15–4.16 (1H, d,
J= 7.8 Hz), 4.25–4.26 (1H, d, J = 7.8 Hz), 4.48 (1H, m), 4.58 (2H, m),
4.71 (1H, s), 4.95 (1H, m), 5.18–5.19 (1H, d, J = 4.8Hz), 5.48–5.49
(1H, d, J = 4.8Hz), 5.84–5.86 (1H, t, J = 10.2Hz), 6.44–6.45 (2H, d,
J= 4.2 Hz), 6.78–6.79 (2H, d, J = 7.8Hz), 6.79 (18H, m), 7.370–7.379
(2H, d, J = 5.4 Hz), 7.52–7.53 (2H, d, J= 9 Hz), 7.79 (1H, m), 8.61 (1H,
m), 8.74 (1H, s), 8.88–8.96 (2H, d, J = 4.8Hz), 9.42–9.44 (3H, t,
J= 6 Hz). 13C NMR (150MHz, CD3OD, TMS) δ: 178.30, 174.18,
173.99, 173.47, 173.38, 173.32, 172.92, 172.30, 171.94, 157.24,
139.01, 138.84, 136.87, 130.94, 130.72, 130.66, 129.69, 129.45,
129.35, 129.03, 128.81, 128.58, 127.90, 127.76, 116.34, 104.30,
102.86, 81.16, 77.85, 76.63, 75.07, 74.80, 74.66, 74.10, 71.52, 71.08,
70.36, 69.78, 62.77, 62.19, 61.53, 59.83, 58.64, 56.83, 56.23,
54.96, 52.34, 51.20, 47.71, 43.12, 41.54, 40.97, 40.84, 40.46,
38.89, 38.51, 37.29, 33.54, 32.79, 31.71, 29.89, 26.90, 26.36,
24.40, 23.94, 23.40, 23.05, 19.51, 19.40. HR-Q-TOF-MS calcd for
C82H115N13O25, (M+2H)/2+, 841.9136, found, 841.9129.
2f, yield 23.9mg, 13.1%. 1H NMR: (600MHz, CD3OD, TMS) δ: 0.41
(1H,s), 0.85–1.00 (8H, m), 1.07–1.08 (2H, dd, J = 6 Hz), 1.13–1.14
(6H, t, J = 6.6 Hz), 1.28 (3H, m), 1.44 (1H, s), 1.51 (1H, m), 1.69–1.83
(10H, m), 1.93 (1H, m), 2.21–2.22 (4H, d, J = 7.8Hz), 2.39–2.41
(1H, t, J = 12.6Hz), 2.76 (1H, m), 2.95 (4H, m), 3.13–3.15 (1H, d,
J = 10.8Hz), 3.25 (4H, m), 3.44 (10H, m), 3.70 (3H, m), 3.78 (2H, m),
4.08 (1H, s), 4.14–4.15 (1H, d, J = 7.2 Hz), 4.50 (1H, s), 4.60 (3H, m),
4.72–4.73 (2H, d, J= 7.2Hz), 4.93 (1H, s), 5.00 (1H, s), 5.48 (1H, s),
5.86 (1H, s), 6.42–6.43 (2H, d, J = 7.8Hz), 6.76–6.77 (2H, d,
J = 7.8Hz), 7.13 (16H, m), 7.37–7.38 (2H, d, J = 6 Hz), 7.50–7.52
(2H, d, J= 15.6 Hz), 7.84 (1H, s), 8.60 (1H, s), 8.88–8.89 (2H, d,
J = 9.0Hz), 9.27–9.35 (2H, m). 13C NMR (150 MHz, CD3OD, TMS) δ:
176.51, 172.27, 172.11, 172.06, 171.61, 171.46, 171.04, 170.49,
170.04, 155.38, 137.15, 137.00, 135.01, 129.06, 128.86, 128.80,
128.26, 127.60, 127.47, 127.14, 126.71, 126.07, 125.89, 114.47,
99.60, 72.66, 70.67, 70.25, 69.39, 68.81, 66.71, 65.33, 61.01, 59.65,
58.00, 56.79, 54.97, 54.37, 53.12, 50.91, 50.51, 49.36, 45.83, 41.27,
39.83, 39.08, 38.55, 37.08, 36.29, 35.36, 31.67, 30.89, 29.82, 27.98,
25.00, 24.49, 22.54. HR-Q-TOF-MS calcd for C76H107N13O20, (M+2H)/2+,
760.8872, found, 760.8870.
2c, yield 16.2 mg, 9.1%. 1H NMR (600MHz, DMSO) δ 7.57
(d, J = 8.9Hz, 1H), 7.44–7.17 (m, 16H), 6.90 (s, 2H), 6.59 (s, 2H), 5.86
(s, 1H), 5.51 (s, 1H), 5.41 (s, 1H), 4.76 (s, 1H), 4.70 (d, J = 11.1 Hz,
1H), 4.62 (s, 3H), 4.54 (s, 1H), 4.39 (d, J = 8.3 Hz, 1H), 4.20 (d,
J= 7.8Hz, 1H), 4.12 (s, 1H), 3.88 (s, 1H), 3.85–3.75 (m, 2H),
3.73–3.60 (m, 3H), 3.52 (s, 1H), 3.47–3.42 (m, 2H), 3.33–3.17
(m, 7H), 3.06 (dd, J =45.4, 32.4Hz, 4H), 2.90 (s, 1H), 2.48 (s, 1H),
J. Pept. Sci. 2015; 21: 586–592
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

ZOU ET AL.
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We are grateful to the National Natural Science Foundation of
China (no. 21402235 and 20902109), State Project for Essential
Drug Research and Development (2012ZX09502-001-005 and
2012ZX09J12108-01), and Instrumental Analysis Center of our
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