N-Terminal guanidine derivatives of teicoplanin antibiotics strongly active against glycopeptide resistant Enterococcus faecium

Article abstract of DOI:10.1038/s41429-020-0313-6

Antibiotic resistance is one of the major challenges in healthcare of our time. To meet this challenge, we designed and prepared guanidine and lipophilic guanidine derivatives of the glycopeptide antibiotic teicoplanin to armed them with activity against

Full text of DOI:10.1038/s41429-020-0313-6

The Journal of Antibiotics
https://doi.org/10.1038/s41429-020-0313-6
ARTICLE
N-Terminal guanidine derivatives of teicoplanin antibiotics strongly
active against glycopeptide resistant Enterococcus faecium
Zsolt Szűcs1,2 Ilona Bereczki1 Erzsébet Rőth1 Márton Milánkovits3 Eszter Ostorházi3 Gyula Batta4
●
●
●
●
●
●
Lajos Nagy5 Zsuzsanna Dombrádi6 Anikó Borbás1 Pál Herczegh1
●
●
●
Received: 24 February 2020 / Revised: 26 March 2020 / Accepted: 15 April 2020
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2020
Abstract
Antibiotic resistance is one of the major challenges in healthcare of our time. To meet this challenge, we designed and
prepared guanidine and lipophilic guanidine derivatives of the glycopeptide antibiotic teicoplanin to armed them with
activity against the most threatening nosocomial bacteria, multiresistant enterococci. From teicoplanin and its
pseudoaglycone, a series of N-terminal guanidine derivatives have been prepared with free and amide C-terminal parts.
Six aliphatic and aromatic lipophilic carbodiimides were prepared and used for the synthesis of lipophilic guanidine
teicoplanin conjugates. All new N-terminal guanidine antibiotics showed high activity against a standard panel of Gram-
positive bacteria. Four selected derivatives displayed excellent antibacterial activity against a series of nosocomial VanA
Enterococcus faecium strains.
Introduction
Nevertheless, 80% of E. faecium isolates were vancomycin-
resistant in US hospitals [2–4]. According to a longitudinal
analysis rates of E. faecium in Europe increased in the time
interval of 2001–2014 from 1.4% (in 2001) to 4.3 %
(in 2014), while VRE rates among the same strain changed
from 4.7 to 20.3% [5]. VanA and VanB are the most frequent
phenotypes of E. faecium, VanA types being resistant
to vancomycin and teicoplanin [2]. Against this threat orita-
vancin, a semisynthetic glycopeptide antibiotic seems to
give some safety, showing low MIC values against VanA E.
faecium [5, 6].
In the framework of a systematic synthetic study on lipo-
philic modifications of teicoplanin and its (pseudo)aglycone
(s) [7–9], we recently reported on a series of derivatives with
simple lipophilic N-terminal substituents that possessed
comparable in vitro activity with that of oritavancin against
clinical isolates of VanA E. faecium [10, 11]. As a con-
tinuation of this study in the present paper, we describe the
synthesis of some N-terminal guanidine derivatives of teico-
planin and its pseudoaglycone.
Annually 25,000 people are killed in Europe by drug-resistant
bacteria [1]. Vancomycin-resistant enterococci (VRE) are the
leading cause of nosocomial infections [2]. The hospital
acquired infections are caused mostly by Enterococcus
faecalis and only 20% of them by Enterococcus faecium.
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-020-0313-6) contains supplementary
material, which is available to authorized users.
* Pál Herczegh
herczeghp@gmail.com
1
Department of Pharmaceutical Chemistry, University of Debrecen,
Debrecen Egyetem tér 1, H-4032, Hungary
2
Doctoral School of Pharmaceutical Sciences, University of
Debrecen, Debrecen, Hungary
3
Department of Medical Microbiology, Semmelweis University,
Budapest Nagyvárad tér 4, H-1089, Hungary
Guanidines are superbases, i.e. stronger bases than other
nitrogen compounds [12]. Therefore, their enhanced Cou-
lomb interactions as well as hydrogen bonds with proteins
can be expected. Several guanidine derivatives possess
antibacterial activity [13–16]. The mechanism of action of
glycopeptide antibiotics is based on their strong interaction
with bacterial peptidoglycan [17]. We hypothesized that
4
Department of Organic Chemistry, University of Debrecen,
Debrecen Egyetem tér 1, H-4032, Hungary
5
Department of Applied Chemistry, University of Debrecen,
Debrecen Egyetem tér 1, H-4032, Hungary
6
Department of Medical Microbiology, University of Debrecen,
Debrecen Egyetem tér 1, H-4032, Hungary

Z. Szűcs et al.
introduction of a guanidino substituent into the teicoplanin
molecule or into its derivatives will enhance their interac-
tions with the bacterial target D-alanine containing peptides
and as a consequence will improve the antibacterial activity.
Since lipophilic substituents of teicoplanin derivatives also
positively influence their bioactivity [7–11], various lipo-
philic guanidine moieties were also introduced into teico-
planin or its pseudoaglycone.
To obtain 2 we elaborated a novel, simple deglycosidation
method that involves treatment of 1 with concentrated
hydrochloric acid. This new method is much cheaper, easier
to carry out and considerably safer than the hydrogen-
fluoride mediated hydrolytic process reported by the
Boger group [18], which we have used in the past [7–10],
while the yield of teicoplanin pseudoaglycone 2 is similarly
excellent.
At first, a derivative of 2 with unsubstituted guanidino
group was prepared using the readily available pyr-
azolecarboxamidine reagent 3. The intermediate dicarbamate
derivative 4 was deprotected to give 5 (Scheme 1).
Results and discussion
Chemistry
One of the mildest ways of preparation of substituted
guanidine derivatives is the addition of primary amines onto
carbodiimides [19–21]. The preparation of derivatives car-
rying two lipophilic groups on the guanidine moiety
required the synthesis of N,N′-disubstituted carbodiimides.
For the introduction of guanidine moiety, the N-terminal
primary amino group of teicoplanin (1) or its pseudoagly-
cone 2 (T-ΨAgl, also known as teicoplanin A3-2) was used.
Scheme 1 Structure of starting
teicoplanin 1, teicoplanin
pseudoaglycone 2 (T-ΨAgl),
and transformation of
pseudoaglycone 2 into
guanidine derivatives 4 and 5.
Reagents and conditions:
a dimethylformamide (DMF),
N,N′-di-Boc-1H-pyrazole-1-
carboxamidine, Et3N, 40 °C,
overnight; b trifluoroacetic acid
(TFA), anisole, 25 °C, 1 h
O

N-Terminal guanidine derivatives of teicoplanin antibiotics strongly active against glycopeptide. . .
Carbodiimides with two electron-donating alkyl groups
display low reactivity to primary amines, but an aromatic
substituent can enhance their reactivity. Therefore, we
chose the phenyl group as one of the substituents of our
carbodiimides. Six aliphatic and aromatic lipophilic
amines 6a–f were reacted with phenyl isocyanate produ-
cing thioureas 7a–f in good yields. Subsequent treatment
of the latter compounds with methanesulfonyl chloride
and trimethylamine [22] resulted in carbodiimides 8a–f
(Scheme 2).
form of diphenylmethyl ester, yielding 9, which in the
reactions with carbodiimides 8a–d gave guanidines 13a–d.
The latter were deprotected by trifluoroacetic acid to pro-
vide final products 14a–d (Scheme 3). From teicoplanin (1),
which is a mixture of six different compounds (A2-1–A2-5
and A3-1, see Scheme 1), a six-component dimethylami-
nopropyl amide mixture of similar composition (11) was
obtained using PyBOP reagent, similarly to the dalba-
vancin molecule [23]. From 11 guanidino derivatives 16b,
16e, and 16f were prepared, respectively, each in the form
of inseparable mixture of six compounds. A diethylami-
nopropyl amide 10 of pseudoaglycone 2 was also obtained
from 1 by amidation and subsequent hydrolytic removal of
N-acylglucosamines and D-mannose. Reactions of 10 with
carbodiimides gave guanidine amides 15a–d (Scheme 3
and Table 1).
The synthetic steps of conjugation procedure of teico-
planin complex 1 and its pseudoaglycone 2 are summarized
in Scheme 3 and the structure of products are shown in
Table 1. The carboxylic group of 2 was protected in the
The structure of the new derivatives (see Table 1) was
proved by 2D NMR techniques (1H–1H COSY, 1H–1H
ROESY, 1H–13C HSQC, 1H–13C HMBC). The detailed
NMR assignation of the new derivatives as well as the
composition of 16b, 16e, and 16f can be found in the
supporting information.
Starting amine
6a R = n-hexyl
6b R = n-octyl
6c R = n-decyl
6d R = n-dodecyl
6e R = 4-phenylbenzyl
6f R = N-benzyl-piperidyl
Thiourea (Yield) Carbodiimide (Yield)
7a
7b
7c
7d
7e
7f
(65%)
(85%)
(90%)
(84%)
(92%)
(97%)
8a
8b
8c
8d
8e
8f
(86%)
(78%)
(40%)
(50%)
(45%)
(80%)
Antibacterial evaluation
The antibacterial activity of the new teicoplanin derivatives
was evaluated on a standard panel of eight Gram-positive
bacterial strains for preliminary results (Table 2). The
pseudoaglycone derivative 4 bearing a Boc protected
Scheme 2 Synthesis of lipophilic carbodiimide reagents 8a–f. Reagents
and conditions: a dichloromethane (DCM), phenyl isothiocyanate,
4-dimethylaminopyridine (DMAP), 25 °C, 1 h; b DCM, methanesulfonyl
chloride, Et3N, 25 °C, 30 min
Scheme 3 Synthesis routes to
lipophilic guanidine derivatives
of teicoplanin pseudoaglycone
(13–15) and teicoplanin (16).
Ester (9) and amide (10, 11)
structures formed on the C-
terminus are highlighted in
boxes. The structures of
lipophilic guanidine derivatives
13–16 are shown in Table 1.
Reagents and conditions: a c.
HCl, 0 °C to 25 °C, 4.5 h; b
DMF, diphenyldiazomethane, p-
toluenesulfonic acid, 25 °C, 16
h; c DMF, Et2N(CH2)3NH2,
PyBOP, 25 °C, 5 h; d DMF,
Me2N(CH2)3NH2, PyBOP, 25 °
C, 3 h; e DMF, carbodiimides,
25 °C, 3–4 h; f TFA, 25 °C, 1 h

Z. Szűcs et al.
Table 1 Structure of lipophilic guanidine derivatives of teicoplanin pseudoaglycon (13–15) and teicoplanin (16)
OR2
Cl
O
O
O
OH
O
HO
HO
O
Cl
O
O
NHAc
H
H
N
H
N
H
N
N
O
N
H
N
H
N
H
R4
HN
O
O
N
R3
O
HO
OR1
OH
O
OH
HO
R1
R2
R3
R4
n-hexyl
Compound#
13a
13b
13c
13d
14a
14b
14c
14d
15a
15b
15c
15d
16b
16e
16f
n-octyl
H
H
H
H
H
H
OCHPh2
n-decyl
n-dodecyl
n-hexyl
n-octyl
H
n-decyl
n-dodecyl
n-hexyl
n-octyl
H
N
N
n-decyl
n-dodecyl
n-octyl
H
α-D-mannose N-acyl-β-D-glucosamine
4-phenylbenzyl
N
N
N-benzyl-piperidyl
guanidine did not show any relevant activity, while its
deprotected analogue 5 was highly active against every
tested strain. This might very well be due to the capability
of the guanidine moiety to gain a positive charge, as well as
the ability to form extra hydrogen bonds as H-bond donor.
The former is considered to be significant in the early stages
of binding the D-Ala-D-Ala termini of cell wall precursors,
while the latter might help to counteract the loss of the
hydrogen bond due to the change of the terminal D-Ala to D-
Lac in the peptidoglycan precursors of glycopeptide resis-
tant enterococci.
Among the diphenylmethyl esters only the n-hexyl ana-
logue 13a seemed to have fairly good activity, all other
analogues (13b–d) were essentially inactive, indicating that
the structure and lipophilicity of substituents connecting to
the N-terminus—in this case the guanidino group—have a
strong influence on antibacterial activity. The latter is in
accordance with previous findings of many different
research groups [24–29], including our former results [7–
10, 30] in this field. Analogues with the free carboxyl group
(14a–d) gained significant activity against all strains,
including the VRE strains. Two of them, 14a and 14b

N-Terminal guanidine derivatives of teicoplanin antibiotics strongly active against glycopeptide. . .
Table 2 In vitro antibacterial activity (MIC) of guanidine derivatives of teicoplanins
B. subtilis
ATCC 6633 ATCC 29213
S. aureus MSSA S. aureus MRSA S. epidermidis S. epidermidis E. faecalis
E. faecalis
E. faecalis
15376 VanA
ATCC 33591
ATCC 35984
mecA
ATCC 29212 ATCC
51299 VanB
Teicoplanin
0.5
0.5
32
0.5
0.5
16
0.5
0.5
16
4.0
2.0
16
16
1.0
1.0
32
0.5
128
32
256
256
64
Vancomycin
4.0
16
4
5
0.8
4.0
128
128
128
6.4
1.6
4.0
4.0
2.0
4.0
1.0
8.0
0.8
1.0
0.5
0.4
1.0
64
0.4
2.0
32
0.4
2.0
4.0
64
0.4
0.8
4.0
32
0.8
2.0
16
0.4
4.0
16
0.8
2.0
16
13a
T-ΨAgl
13b
13c
13d
14a
14b
14c
14d
15a
15b
15c
15d
16b
16e
16f
diphenylmethyl
esters
256
128
0.8
0.2
2.0
8.0
4.0
4.0
2.0
8.0
0.8
2.0
0.5
128
128
0.4
0.4
4.0
4.0
1.0
2.0
2.0
8.0
0.8
2.0
1.0
64
128
256
0.5
0.2
1.0
32
64
32
16
128
0.2
0.2
4.0
2.0
32
64
0.2
0.05
2.0
0.8
1.0
1.0
4.0
2.0
0.4
1.0
0.5
0.2
0.05
8.0
0.8
0.5
2.0
0.5
1.0
0.4
2.0
0.5
0.4
0.8
4.0
8.0
16
T-ΨAgl free
C-terminus
64
64
32
32
T-ΨAgl amides
2.0
32
64
32
32
32
0.8
16
1.6
32
0.8
16
Teicoplanin amides
32
32
16
MIC minimum inhibitory concentration (µg ml−1), ATCC American Type Culture Collection, MSSA methicillin-sensitive Staphylococcus aureus,
MRSA methicillin-resistant Staphylococcus aureus, VSE vancomycin-sensitive enterococcus, mecA mecA gene expression in Staphylococcus,
vanA+ vanA gene positive, vanB+ vanB gene positive
bearing n-hexyl and n-octyl substituents on the guanidine
moiety were highly active even against VRE. The pseu-
doaglycone amides 15a–d although were as effective as
their carboxyl analogues against staphylococci, a significant
loss of activity was observed against enterococci. The
antibacterial properties of the three amide derivatives
obtained from the teicoplanin mixture (16b, 16e, and 16f)
were about the same as the pseudoaglycone amides, except
for the n-octyl analogue 16b, which was highly active
against every strain in the standard panel.
Four of the new derivatives 5, 14a, 14b, and 16b were
selected for in vitro evaluation against a collection of VanA
type enterococci isolated from patients at the Department
of Medical Microbiology, University of Debrecen. The
n-hexyl 14a and n-octyl 14b pseudoaglycone derivatives
with a free C-terminus displayed about the same activity
(Table 3), the MIC was below 0.5 µg ml−1 against most
strains, outperforming oritavancin in multiple cases. The
two isolates that seemed to be the most resistant to our
derivatives were E. faecium 11408 and E. faecium 17980,
which were still susceptible to oritavancin. The pseu-
doaglycone derivative 5 with the unsubstituted guanidine
group had similar activity against VRE strains as 14a and
14b, with the exception of the E. faecium 38415 strain,
which seemed to be resistant to this compound. The n-octyl
analogue prepared from teicoplanin mixture displayed
the same activity as 14a, the only resistant strain was E.
faecium 11408.
Conclusion
In summary, we have demonstrated a straightforward way
to transform the N-terminal amino group of teicoplanin or
its pseudoaglycone into unsubstituted or substituted guani-
dine groups. This modification led to derivatives with
enhanced in vitro antibacterial activity against glycopeptide
resistant Gram-positive bacteria including VanA type
enterococci. Four of the new compounds has shown equal
or higher activity than oritavancin against the 22 tested
nosocomial VRE strains in vitro (typically 0.2–0.4 μg ml−1).
Not considering the N-terminal guanidine group, these three
types of compounds have markedly different structures,
leading to completely distinct ionization behavior and
lipophilicity, hence water solubility. The fact that these
similar but yet very different antibiotics have practically
equally high activity, leaves us a wide range of opportu-
nities for structural adjustments to the existing derivatives
with the aim of tuning their in vivo behavior.
Experimental
General information
3-(Dimethylamino)-1-propylamine,
propylamine, pyrazolecarboxamidine (3) and N,N′-di-Boc-
1H-pyrazole-1-carboxamidine were purchased from Tokyo
3-(diethylamino)-1-

Z. Szűcs et al.
Table 3 In vitro antibacterial
activity (MIC) of selected
guanidine derivatives against
VanA enterococci
#
Strain
Source
TEI
VAN
ORI
14a
14b
16b
5
1
E. faecium 8663
E. faecium 22285
E. faecium 656
Bronchus
Urine
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
2
0.1
0.2
0.2
0.4
0.2
1.6
6.25
0.2
0.4
0.2
0.4
0.2
0.2
0.2
0.2
0.1
0.2
1.6
0.4
0.2
0.2
0.2
1
0.1
0.2
0.2
0.8
0.2
3.18
3.18
0.4
0.4
0.2
0.4
0.2
0.4
0.2
0.2
0.1
0.2
0.4
0.2
0.2
0.2
0.2
2
0.1
0.2
0.2
0.8
0.2
6.25
0.8
0.2
0.4
0.2
0.2
0.2
0.2
0.2
0.2
0.1
0.2
0.4
0.8
0.2
0.2
0.2
1
0.2
0.4
0.2
0.4
0.2
2
2
3
Wound
Drain
2
4
E. faecium 3452
E. faecium 4753
E. faecium 11408
E. faecalis 17980
E. faecium 24581
E. faecium 25192
E. faecium 29007
E. faecium 29810
E. faecium 29838
E. faecium 30458
E. faecium 31482
E. faecium 32445
E. faecium 35936
E. faecium 38276
E. faecium 38415
E. faecium 38522
E. faecium 39063
E. faecium 39759
E. faecium 42491
1
5
Decubitus
Drain
1
6
<0.25
2
12.5
6.25
7
Urine
8
Wound
Haemoculture
Urine
0.5
0.5
0.25
0.5
0.5
0.25
0.25
0.5
0.25
0.25
2
0.2
0.2
0.2
0.4
0.2
0.4
0.2
0.2
0.1
0.2
6.25
1.6
0.2
0.8
0.2
3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Wound
Urine
Cannula
Urine
Cannula
Urine
Urine
Wound
Decubitus
Wound
Drain
1
0.5
0.25
0.25
Urine
No. of MIC values above the breakpoint for teicoplanin (2 μg ml−1
No. of MIC values above the breakpoint for vancomycin (4 μg ml−1
)
)
1
0
1
3
MIC minimum inhibitory concentration (µg ml−1), TEI teicoplanin, VAN vancomycin, ORI oritavancin
Chemical Industry Co., Ltd. Oritavancin was purchased
from Xi’an Kerui Biotechnology Co., Ltd. (Xian, Shaanxi,
China) and checked by MALDI-TOF MS, 1D and 2D NMR
experiments. The vancomycin hydrochloride standard used
for the antibacterial evaluations was a gift from TEVA
Pharmaceutical Industries Ltd. (Debrecen, Hungary) and
teicoplanin was purchased from Shaanxi Sciphar Bio-
technology Co., Ltd (Xi’an, Shaanxi, China). Teicoplanin
for synthetic purposes was purchased from Xi’an Sgonek
Biological Technology Co., Ltd. (Weiyang Qu, Xian Shi,
Shaanxi Sheng, China).
ESI-HRMS spectra were recorded by a microTOF-Q type
QqTOFMS mass spectrometer (Bruker) and a maXis II
UHR ESI-QTOF MS instrument (Bruker) in positive ion
mode using MeOH as solvent. In the case of maXis II UHR
ESI-QTOF MS the following parameters were applied for
the electrospray ion source: capillary voltage: 3.5 kV; end-
plate offset: 500 V; nebulizer pressure: 0.8 bar; dry gas
temperature: 200 °C and dry gas flow rate: 4.5 l min−1
.
Constant background correction was applied for each
spectrum, the background was recorded before each sample
by injecting the blank sample matrix (solvent). Na-formate
or tuning mix ESI-TOF (Agilent) calibrant was injected
after each sample, which enabled internal calibration during
data evaluation. Mass spectra were recorded by otofControl
version 4.1 (build: 3.5, Bruker) and processed by Compass
DataAnalysis version 4.4 (build: 200.55.2969). For analy-
tical RP-HPLC a Waters 2695 Separations Module (Waters
Corp., Milford, USA) was used. The separations were car-
ried out on a VDSpher PUR 100 C18-M-SE, 5 μm, 150 ×
4.6 mm column (Batch# VD173001) at an injection volume
of 10 μl, using a flow rate of 1.0 ml min−1 with a Waters
2996 DADset at 254 nm and a Bruker MicroTOF-Q
type Qq-TOF MS instrument (Bruker Daltonik, Bremen,
Germany) as detectors. The following system was used for
The antibacterial evaluations were carried out as it was
described in our previous publication [8]. TLC was per-
formed on Kieselgel 60 F254 (Merck) with detection either
by immersing into ammonium molybdate-sulfuric acid
solution followed by heating or by using Pauly’s reagent for
detection. Flash column chromatography was performed
using Silica gel 60 (Merck 0.040–0.063 mm). The 1H NMR
(500 and 400 MHz), 13C NMR (125 and 100 MHz) and 2D
NMR spectra were recorded with a Bruker DRX-400 or a
Bruker Avance II 500 spectrometer at 298 K or 310 K.
1
Chemical shifts are referenced to Me4Si (0.00 ppm for H)
and to the solvent residual signals. The spectra were
evaluated using MestReNova and TopSpin softwares.

N-Terminal guanidine derivatives of teicoplanin antibiotics strongly active against glycopeptide. . .
the elutions: Solvent A: Water:MeCN 9:1 + 0.0025%v/v
TFA, Solvent B: MeCN. Gradient: 20% B from 0 to 20 min,
from 20% B to 80% B from 20 to 40 min, 80% B from 40 to
50 min, from 80% B to 20% B from 50 to 51 min. Solvent
A: Water:MeCN 9:1 + 0.0025%v/v TFA, Solvent B:
MeCN. The MicroTOF-Q mass spectrometer was equipped
with an electrospray ion source. The mass spectrometer was
operated in positive ion mode with a capillary voltage of
3.5 kV, an endplate offset of −500 V, nebulizer pressure of
1.8 bar, and N2 as drying gas with a flow rate of 9.0 l min−1
at 200 °C. The mass spectra were recorded by means of a
digitizer at a sampling rate of 2 GHz. The mass spectra were
calibrated externally using the exact masses of clusters
[(NaTFA)n + TFA]− from the solution of sodium tri-
fluoroacetate (NaTFA). The spectra were evaluated with the
DataAnalysis 3.4 software from Bruker.
by adding 2 N NaOH solution. The obtained white pre-
cipitate was filtered off and washed with water (pH 5.5).
After normal phase column chromatography, using MeCN:
H2O gradient elution, 2 was obtained in 76% yield. The
characterization data of 2 were identical to those previously
reported in the literature [7].
Compound 4
Teicoplanin A3-2 pseudoaglycone 2 (200 mg, 0.14 mmol)
was dissolved in dry DMF (3 ml) and triethylamine (78 μl,
0.56 mmol, 4.0 equiv.) was added, followed by N,N′-di-
Boc-1H-pyrazole-1-carboxamidine (86.9 mg, 0.28 mmol,
2.0 equiv.). The reaction mixture was stirred at 40 °C for
24 h. After cooling down to room temperature, diethyl ether
was added and the resulting white precipitate was filtered
off and washed with additional diethyl ether. The crude
product was purified by flash column chromatography
eluting with MeCN:H2O 97:3 → 95:5. Yield: 103 mg (44%)
off-white powder. ESI-HRMS: m/z 1687.3951 [M–H +
2Na]+; calcd: 1687.3976 (C77H75Cl2N10O27Na2+).
Synthesis
General procedure A for the synthesis of carbodiimides
(Scheme 2)
Step a: To the corresponding amine dissolved in dry
DCM, 1.0 equiv. of phenyl isothiocyanate was added,
then the reaction mixture was stirred for 2 h at room
temperature. The mixture was evaporated and the crude
product was purified by flash column chromatography
using i-hexanes:acetone mixtures as eluent. Step b: The
obtained thiocarbamide intermediate was dissolved in dry
DCM, then a catalytic amount of DMAP, 3.0 equiv. of
TEA and 2.0 equiv. of MsCl were added while cooling in
an ice bath. The mixture was allowed to warm to room
temperature. After 30 min silica gel was added and the
mixture was evaporated, then the crude product was
purified by flash column chromatography using i-hexanes:
ethyl acetate mixtures as eluent.
Compound 5
Compound 4 (80 mg, 49 μmol) and anisole (10.6 μl, 2.0
equiv.) was dissolved in TFA (1 ml) and the mixture was
stirred for 1 h at room temperature. The crude product
was precipitated by adding diethyl ether. The precipitate
was filtered off, washed, and dried. The crude product was
purified by flash column chromatography eluting with
MeCN:H2O = 9:1. Yield: 44 mg (63%) off-white powder.
ESI-HRMS: m/z 1465.3094 [M + Na]+; calcd: 1465.3102
(C67H60Cl2N10O23Na+).
Synthesis of carbodiimides (Scheme 1)
N-Hexyl-N′-phenylcarbodiimide (8a)
General procedure B for the deprotection of teicoplanin
pseudoaglycone DPM esters (step f in Scheme 3)
See General procedure A. Reactants for Step a: n-hexylamine
(2.64 ml, 20 mmol), phenyl isothiocyanate (2.7 g, 2.4 ml).
Eluent: i-hexanes:acetone = 8:2. Yield of thiocarbamide
intermediate 7a: 3.08 g (65%). Step b: thiocarbamide
(1010 mg, 5 mmol), DMAP (25 mg), trimethylamine (2.1 ml),
methanesulfonyl chloride (774 μl). Eluent: i-hexanes →
i-hexanes:EtOAc 98:2 → 97:3 → 96:4. Yield: 870 mg (56%,
The corresponding teicoplanin pseudoaglycone DPM ester
was dissolved in dry TFA (1–2 ml) at room temperature and
stirred for 1 h, then the mixture was co-evaporated with
toluene (15 ml) two times. The crude product was dissolved
in a MeCN:H2O 1:1 mixture, silica gel was added, the
mixture was evaporated and the product was purified by
flash column chromatography.
1
for two steps) yellow syrup. H NMR (400 MHz, CDCl3) δ
7.28 (m, 2H), 7.12–7.05 (m, 3H), 3.40 (t, J = 6.8 Hz, 2H),
1.67 (tt, J = 7.0 Hz, 2H), 1.46–1.37 (m, 2H), 1.31 (m, 4H),
0.89 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
140.90 (N = C = N), 129.44 (2C, 2 × CHAr), 124.64, 123.58
(2C, 2 × CHAr), 46.98 (N-CH2), 31.47, 31.42, 26.59, 22.66,
14.11 (CH3). ESI-HRMS: m/z 235.1809 [M + CH3OH + H]+;
calcd: 235.1805 (C13H18N2 + CH3OH + H+).
Compound 2
After the treatment of 1 with concentrated hydrochloric acid
(cteicoplanin = 100 mg ml−1) for about 4.5 h at room tem-
perature, the pH of the reaction mixture was set to pH 5.5

Z. Szűcs et al.
N-Octyl-N′-phenylcarbodiimide (8b)
136.14 (Ar-Cq), 129.44, 124.64, 123.59 (5C, 5 × ArCH),
46.98 (N-CH2), 32.06, 31.52, 29.77, 29.69, 29.65,
29.49, 29.25, 26.93, 22.83 (10C, 10 × CH2), 14.25 (CH3).
ESI-HRMS: m/z 319.2753 [M + CH3OH + H]+; calcd:
319.2744 (C19H30N2 + CH3OH + H+).
See General procedure A. Reactants for Step a: n-octylamine
(1.29 g, 1.65 ml, 10 mmol), phenyl isothiocyanate (1.35 g,
1.2 ml). Eluent: i-hexanes:acetone = 8:2. Yield of inter-
mediate 7b: 2.24 g (85%). Step b: thiocarbamide (1.32 g,
5 mmol), DMAP (25 mg), trimethylamine (2.09 ml), metha-
nesulfonyl chloride (774 μl). Eluent: i-hexanes → i-hexanes:
EtOAc 98:2 → 97:3 → 96:4. Yield: 900 mg (66%, for two
steps) yellow syrup. 1H NMR (360 MHz, CDCl3) δ 7.32–7.23
(m, 2H), 7.14–7.03 (m, 3H), 3.40 (t, J = 6.8 Hz, 2H), 1.67 (tt,
J = 6.8 Hz, 2H), 1.41 (tt, J = 15.0, 6.6 Hz, 2H), 1.35–1.20 (m,
8H), 0.87 (t, J = 7.1 Hz, 3H). 13C NMR (91 MHz, CDCl3) δ
140.91 (N=C=N), 136.14 (Ar-Cq), 129.43, 124.63, 123.58
(5C, 5 × ArCH), 46.97 (N-CH2), 31.88, 31.51, 29.29,
26.91, 22.76 (8C, 8 × CH2), 14.20 (CH3). ESI-HRMS: m/z
263.2118 [M + CH3OH + H]+; calcd: 263.2118 (C15H22N2 +
CH3OH + H+).
N-Phenyl-N′-4-phenylbenzyl carbodiimide (8e)
See General procedure A. Reactants for Step a: 4-
phenylbenzylamine (275 mg, 1.5 mmol), phenyl iso-
thiocyanate (180 μl). Eluent: i-hexanes:acetone = 8:2 →
7:3. Yield of intermediate 7f: 440 mg (92%). Step b:
thiocarbamide (420 mg, 1.32 mmol), DMAP (3 mg), tri-
methylamine (552 μl), mesyl chloride (204 μl). Eluent: i-
hexanes → i-hexanes:EtOAc 98:2 → 97:3 → 96:4. Yield:
1
170 mg (42%, for two steps) yellowish solid. H NMR
(360 MHz, CDCl3) δ 7.63–7.52 (m, 4H), 7.48–7.38 (m,
4H), 7.37–7.30 (m, 1H), 7.28–7.20 (m, 2 H), 7.12–7.05
(m, 1H), 7.04–6.97 (m, 2H), 4.59 (s, 2H). 13C NMR (91
MHz, CDCl3) δ 140.88, 140.74, 140.07, 137.41, 136.98
(5C, 5 × Cq), 129.45, 128.91, 127.95, 127.63, 127.52,
127.20, 125.01, 123.80 (14C, 14 × Ar-CH), 50.34 (N-
CH2). ESI-HRMS: m/z 317.1662 [M + CH3OH + H]+;
calcd: 317.1648 (C20H16N2 + CH3OH + H+).
N-Decyl-N′-phenylcarbodiimide (8c)
See General procedure A. Reactants for Step a: n-decy-
lamine (1573 μl, 8 mmol), phenyl isothiocyanate (955 μl).
Eluent: i-hexanes:acetone = 8:2. Yield of thiocarbamide
intermediate 7c: 2.1 g (90%). Step b: thiocarbamide
(1.00 g, 3.42 mmol), DMAP (20 mg), trimethylamine
(1.43 ml), mesyl chloride (530 μl). Eluent: i-hexanes → i-
hexanes:EtOAc 98:2 → 97:3 → 96:4. Yield: 350 mg (36%,
N-(1-Benzyl-4-piperidyl)-N′-phenylcarbodiimide (8f)
See General procedure A. Reactants for Step a: 4-amino-1-
benzylpiperidine (611 μl, 3 mmol), phenyl isothiocyanate
(358 μl). Eluent: i-hexanes:acetone = 7:3 → 6:4 + 0,1%V/V
Et3N. Yield of intermediate 7g: 950 mg (97%). Step b: thio-
carbamide (650 mg, 2 mmol), DMAP (10 mg), trimethyla-
mine (836 μl), methanesulfonyl chloride (309 μl). Eluent:
i-hexanes → i-hexanes:EtOAc 9:1 → 8:2 + 0,1%V/V Et3N.
1
for two steps) yellow syrup. H NMR (360 MHz, CDCl3)
δ 7.32–7.23 (m, 2H), 7.14–7.03 (m, 3H), 3.40 (t, J = 6.8
Hz, 2H), 1.67 (tt, J = 6.9 Hz, 2H), 1.41 (tt, J = 6.9 Hz,
2H), 1.27 (d, J = 9.4 Hz, 12H), 0.88 (t, J = 6.7 Hz, 3v).
13C NMR (91 MHz, CDCl3) δ 140.92 (N=C=N), 136.13
(Ar-Cq), 129.43, 124.63, 123.58 (5C, 5 × ArCH),
46.98 (N-CH2), 32.01, 31.52, 29.43, 29.24, 26.92,
22.81 (8C, 8 × CH2), 14.24 (CH3). ESI-HRMS: m/z
291.2437 [M + CH3OH + H]+; calcd: 291.2431 (C17H26N2 +
CH3OH + H+).
1
Yield: 470 mg (78%, for two steps) pale yellow solid. H
NMR (360 MHz, CDCl3) δ 7.45–7.34 (m, 2H), 7.32–7.22 (m,
6H), 7.20–7.14 (m, 2H), 4.39–4.25 (m, 1H), 3.46 (s, 2H),
2.74 (d, J = 12.1 Hz, 2H), 2.19–2.08 (m, 2H), 2.08–1.99 (m,
2H), 1.41 (dq, J = 11.1, 3.9 Hz, 2H). 13C NMR (91 MHz,
CDCl3) δ 138.31, 136.18 (2C, 2 × Ar-Cq), 130.22 (2C, 2 × Ar-
CH), 129.10 (2C, 2 × Ar-CH), 128.24 (2C, 2 × Ar-CH),
127.19, 127.07 (2C, 2 × Ar-CH), 125.06 (2C, 2 × Ar-CH),
63.01 (Cq-CH2), 52.09 (N-CH), 52.05 (2C, 2 × N-CH2), 31.77
(2C, 2 × N-CH-CH2).
N-Dodecyl-N′-phenylcarbodiimide (8d)
See General procedure A. Reactants for Step a: n-dodecy-
lamine (1112 mg, 1380 μl, 6 mmol), phenyl isothiocyanate
(811 mg, 718 μl). Eluent: i-hexanes:acetone = 8:2. Yield
of intermediate 7d: 1.33 g (69%). Step b: thiocarbamide
(1.33 g, 4.15 mmol), DMAP (20 mg), trimethylamine
(1.74 ml), methanesulfonyl chloride (642 μl). Eluent: i-
hexanes → i-hexanes:EtOAc 98:2 → 97:3 → 96:4. Yield:
Esters (Scheme 3)
Teicoplanin pseudoaglycone diphenylmethyl ester (9)
1
690 mg (40%, for two steps) yellow syrup. H NMR (360
MHz, CDCl3) δ 7.33–7.23 (m, 2H), 7.13–7.05 (m, 3H),
3.40 (t, J = 6.8 Hz, 2H), 1.67 (tt, J = 6.9 Hz, 2H), 1.46–1.35
(m, 2H), 1.27 (d, J = 10.0 Hz, 17H), 0.88 (t, J = 6.7 Hz,
3H). 13C NMR (91 MHz, CDCl3) δ 140.92 (N=C=N),
Teicoplanin A3-2 (1.3 g, 0.93 mmol) was dissolved in DMF
(8.0 ml) followed by the addition of p-toluenesulfonic
acid (176 mg, 1.02 mmol, 1.1 equiv.) and freshly prepared
diphenyldiazomethane (198 mg, 1.02 mmol, 1.1 equiv.).

N-Terminal guanidine derivatives of teicoplanin antibiotics strongly active against glycopeptide. . .
After stirring at room temperature overnight, ether was
added, the precipitated solid was filtered off and washed
with additional ether, then redissolved in MeCN:H2O 1:1
mixture, silica gel was added, the mixture was evaporated
and the product was purified by flash column chromato-
graphy. Eluent: MeCN:H2O = 95:5 → 90:10 → 85:15.
Yield: 920 mg (63%) white powder. The product was used
in the following steps without NMR characterization. ESI-
HRMS: m/z 1589.3664 [M + Na+]; calcd: 1589.3672
(C79H68Cl2N8O23Na+).
information (Table S1). ESI-HRMS: m/z 1853.6269 [M +
H]+; calcd: 1853.6256 (C98H98Cl2N10O23+).
Compound 14a
13a (37 mg, 20.9 μmol) was deprotected according to
General procedure B. Eluent: MeCN:H2O = 95:5 →
90:10 → 85:15. Yield: 28 mg (84%) white powder. NMR
data and spectra can be found in the supporting information
(Table S1). ESI-HRMS: m/z 1625.4360 [M + Na]+; calcd:
1625.4354 (C79H76Cl2N10O23Na+).
Compound 13a
Compound 14b
Diphenylmethyl ester 9 (100 mg, 63.8 μmol) was dissolved
in DMF (1.0 ml) and 8a (16 mg, 1.2 equiv.) in DMF
(1.0 ml) was added dropwise slowly. After 1 h of stirring at
room temperature ether (75 ml) was added, and the pre-
cipitated solid was filtered off and washed with additional
ether. The crude product was dissolved in methanol, silica
gel was added, the mixture was evaporated and the product
was purified by flash column chromatography. Eluent:
MeCN:H2O = 95:5 → 93:7. Yield: 47 mg (42%) white
powder. NMR data and spectra can be found in the sup-
porting information (Table S1). ESI-HRMS: m/z 1769.5303
[M + H]+; calcd: 1769.5317 (C92H87Cl2N10O23+).
13b (81 mg, 45 μmol) was deprotected according to General
procedure B. Eluent: MeCN:H2O = 95:5 → 90:10 → 85:15.
Yield: 33 mg (45%) white powder. NMR data and spectra
can be found in the supporting information (Table S1).
ESI-HRMS: m/z 1631.4857 [M + H]+; calcd: 1631.4848
(C81H81Cl2N10O23+).
Compound 14c
13c (85 mg, 46.5 μmol) was deprotected according to
General procedure B. Eluent: MeCN:H2O = 95:5 →
90:10 → 85:15. Yield: 38 mg (49%) white powder. NMR
data and spectra can be found in the supporting infor-
mation (Table S1). ESI-HRMS: m/z 1659.5162 [M + H]+;
calcd: 1659.5161 (C83H85Cl2N10O23+).
Compound 13b
The reaction of teicoplanin A3-2 diphenylmethyl ester 9
(195 mg, 0.125 mmol) in DMF (1.5 ml) and 8b (35 mg) was
carried out as described for 13a. Purification of 13b: DCM:
MeOH = 9:1 → 8:2. Yield: 99 mg (44%) white powder. NMR
data and spectra can be found in the supporting information
(Table S1). ESI-HRMS: m/z 1819.5469 [M + Na]+; calcd:
1819.5450 (C94H90Cl2N10O23Na+).
Compound 14d
13d (62 mg, 33.4 μmol) was deprotected according to
General procedure B. Eluent: Tol:MeOH = 1:1. Yield: 36
mg (63%) white powder. NMR data and spectra can be
found in the supporting information (Table S1). ESI-
HRMS: m/z 1709.5296 [M + Na]+; calcd: 1709.5293
(C85H88Cl2N10O23Na+).
Compound 13c
The reaction of teicoplanin A3-2 diphenylmethyl ester 9
(195 mg, 0.125 mmol) in DMF (1.5 ml) and 8c (38 mg) was
carried out as described for 13a. Purification of 13c:
DCM:MeOH = 90:10 → 85:15 → 80:20. Yield: 135 mg
(59%) white powder. NMR data and spectra can be found in
the supporting information (Table S1). ESI-HRMS: m/z
1825.5965 [M + H]+; calcd: 1825.5943 (C96H95Cl2N10O23+).
AMIDES (Scheme 3)
Compound 15a
Synthesis of diethylaminopropyl amide of teicoplanin A3-2
pseudoaglycone (10) [31]: teicoplanin (1.2 g, ~0.64 mmol)
was suspended in DMF (8.0 ml) followed by the addition
of N,N-diethyl-1,3-propanediamine (252 μl, 1.6 mmol, 2.5
equiv.). After 10 min of stirring at room temperature,
PyBOP® (333 mg, 1.0 equiv.) was added, then every half
hour additional amine (50 μl, 0.32 mmol, 0.5 equiv.) was
added for four times. By this time the mixture became
homogenous. After 2 h an additional portion of PyBOP®
Compound 13d
The reaction of teicoplanin A3-2 diphenylmethyl ester 9
(157 mg, 0.1 mmol) in DMF (1.0 ml) and 8d (35 mg) was
carried out as described for 13a. Purification of 13d: DCM:
MeOH = 9:1 → 8:2. Yield: 72 mg (38%) white powder.
NMR data and spectra can be found in the supporting

Z. Szűcs et al.
(166 mg, 0.32 mmol, 0.5 equiv.) was added, followed by
another 3 h of stirring, after which diethyl ether (200 ml)
was added to the mixture. The precipitated solid was filtered
off and washed with ether, then dried in vacuo. The crude
product was dissolved in c. HCl (5.0 ml) while cooling in an
ice bath, then stirred for 4.5 h. The pH of the reaction
mixture was set between 8 and 9 by adding 2 M NaOH
while cooling in an ice bath, and stirring vigorously. The
precipitated solid was filtered off and washed with a small
amount of dilute NaOH solution pH = 8–9. The crude
product was dissolved in a MeCN:H2O 1:1 mixture, silica
gel was added, the mixture was evaporated and the product
was purified by flash column chromatography. Eluent:
MeCN:H2O = 85:15 → 80:20 → 75:25 → 70:30 → 65:35
(+0.2% V/V AcOH). Yield: 464 mg (50% for two steps)
white powder. Synthesis of 15a: teicoplanin A3-2 diethy-
laminopropyl amide (92 mg, 60.8 μmol) was dissolved in
DMF (1.0 ml) and Et3N (17 μl, 121.5 μmol, 2.0 equiv.)
was added. Then, N-phenyl-N′-hexylcarbodiimide (15 mg,
73 μmol, 1.2 equiv.) in DMF (1.0 ml) was added dropwise
in 8–10 aliquots in the course of about 2 h. After another 2 h
of stirring at room temperature diethyl ether (75 ml) was
added, and the precipitated solid was filtered off and washed
with additional diethyl ether. The crude product was
dissolved in a MeCN:H2O 1:1 mixture, silica gel was
added, the mixture was evaporated and the product
was purified by flash column chromatography. Eluent:
MeCN:H2O = 90:10 → 85:15 → 80:20 → 75:25 (+0.2%V/
V AcOH). Yield: 30 mg (29%) white powder. NMR data
and spectra can be found in the supporting information
(Table S1). ESI-HRMS: m/z 1715.5894 [M + H]+; calcd:
1715.5899 (C86H93Cl2N12O22+).
Yield: 79 mg (71%). NMR data and spectra can be found in
the supporting information (Table S1). ESI-HRMS: m/z
1771.6527 [M + H]+; calcd: 1771.6525 (C90H101Cl2N12O22+).
Compound 15d
Teicoplanin A3-2 pseudoaglycone diethylaminopropyl amide
(140 mg, 92.5 μmol) was dissolved in DMF (1.5 ml). The
reaction and isolation was carried out as described for 15a.
Reactants: Et3N (26 μl), N-phenyl-N′-dodecylcarbodiimide
(32 mg,0.11 mmol). Eluent: MeCN:H2O = 90:10 → 85:15 →
80:20 → 75:25 (+0.1%V/V AcOH). Yield: 78 mg (47%)
white powder. NMR data and spectra can be found in the
supporting information (Table S1). ESI-HRMS: m/z
1799.6839 [M + H]+; calcd: 1799.6838 (C92H105Cl2N12O22+).
Compound 16b
Synthesis of dimethylaminopropyl amide of teicoplanin
(11) [31]: teicoplanin (600 mg, ~0.32 mmol) was sus-
pended in DMF (5.0 ml) followed by the addition of
N,N-dimethyl-1,3-propanediamine (100 μl, 0.8 mmol, 2.5
equiv.). After 10 min of stirring at room temperature
PyBOP® (166 mg, 1.0 equiv.) was added, then every half
hour additional amine (20 μl) was added for four times.
After the reaction mixture became homogenous, another
portion of PyBOP® (83 mg, 0.16 mmol, 0.5 equiv.) was
added. After 1 h of stirring ether:ethyl acetate = 1:1
mixture (100 ml) was added to precipitate the product,
which was filtered off and washed further with ether. The
crude product was dissolved in a MeCN:H2O 1:1 mixture,
silica gel was added, the mixture was evaporated and the
product was purified by flash column chromatography.
Eluent: MeCN:H2O = 80:20 → 75:25 → 70:30 (+0.1%V/
V AcOH). Yield: 362 mg (58%). Synthesis and isolation
of the guanidine derivative: see 15a. Reactants: 115 mg
(58 μmol) teicoplanin dimethylaminopropyl amide, Et3N
(2.0 equiv., 116 μmol, 16 μl), N-phenyl-N′-octylcarbodii-
mide (1.2 equiv., 70 μmol, 16 mg). Eluent: MeCN:H2O =
80:20 → 75:25 (+0.1%V/V AcOH). Yield: 55 mg (43 %)
white powder. NMR data and spectra can be found in the
supporting information (Table S1). ESI-HRMS: m/z
2194.8557 [M + H]+; calcd base peak: 2194.8495 (com-
ponents A2-2, 3) (C108H132Cl2N13O32+).
Compound 15b
Teicoplanin A3-2 diethylaminopropyl amide (92 mg,
60.8 μmol) was dissolved in DMF (1.0 ml). The reaction and
isolation was carried out as described for 15a. Reactants:
Et3N (17 μl, 121.5 μmol), N-phenyl-N′-octylcarbodiimide
(17 mg,73 μmol). Eluent: MeCN:H2O = 90:10 → 85:15 →
80:20 → 75:25 (+0.2%V/V AcOH). Yield: 48 mg (46%)
white powder. NMR data and spectra can be found in the
supporting information (Table S1). ESI-HRMS: m/z
1743.6226 [M + H]+; calcd: 1743.6212 (C88H97Cl2N12O22+).
Compound 15c
Compound 16e
Teicoplanin A3-2 pseudoaglycone diethylaminopropyl
amide (95 mg, 62.7 μmol) was dissolved in DMF (1.5 ml).
The reaction and isolation was carried out as described
for 15a. Reactants: Et3N (17.4 μl, 125 μmol), N-phenyl-
N′-decylcarbodiimide (20 mg, 75.4 μmol). Eluent: MeCN:
H2O = 90:10 → 85:15 → 80:20 → 75:25 (+0.2%V/V AcOH).
See the synthesis and isolation of 16b above. Reactants:
teicoplanin dimethylaminopropyl amide (115 mg, 58 μmol),
Et3N (16 μl, 116 μmol, 2.0 equiv.), N-phenyl-N′-4-
phenylbenzyl carbodiimide (20 mg, 70 μmol, 1.2 equiv.).
Eluent: MeCN:H2O = 80:20 → 75:25 (+0.1%V/V AcOH).
Yield: 68 mg (52%) white powder. NMR data and spectra

N-Terminal guanidine derivatives of teicoplanin antibiotics strongly active against glycopeptide. . .
can be found in the supporting information (Table S1).
ESI-HRMS: m/z 1124.9060 [M + 2H]2+; calcd base peak:
1124.9050 (components A2-2,3) (C113H127Cl2N13O322+).
6. Sweeney D, et al. Comparative in vitro activity of oritavancin and
other agents against vancomycin-susceptible and -resistant enter-
ococci. J Antimicrob Chemother. 2017;72:622–4.
7. Pintér G, et al. Diazo transfer-click reaction route to
new, lipophilic teicoplanin and ristocetin aglycon derivatives
with high antibacterial and anti-influenza virus activity: an
aggregation and receptor binding study. J Med Chem. 2009;52:
6053–61.
Compound 16f
See the synthesis and isolation of 16b above. Reactants:
teicoplanin dimethylaminopropyl amide (115 mg, 58 μmol),
Et3N (16 μl, 116 μmol, 2.0 equiv.), N-phenyl-N′-(1-benzyl-
4-piperidyl) carbodiimide (25 mg, 0.087 mmol, 1.5 equiv.).
After 3 h sufficient conversion was achieved (checked by
Cellulose F TLC—eluent: n-PrOH: c. NH4OH = 7:3). ether:
ethyl acetate = 1:1 mixture (100 ml) was added to pre-
cipitate the product, which was filtered off and washed with
ether. The crude product was dissolved in MeCN:H2O:
acetone 1:1:1 mixture (1.0 ml), and applied to a column
loaded with Sephadex® LH-20 gel in the same solvent
mixture. The obtained product was purified further by flash
column chromatography. Eluent: MeCN:H2O = 80:20 →
75:25 → 70:30 (+0.1%V/V AcOH). Yield: 35 mg (27%).
NMR data and spectra can be found in the supporting
information (Table S1). ESI-HRMS: m/z 1128.4303 [M +
2H]2+; calcd base peak: 1128.5078 (components A2-2,3)
(C112H132Cl2N14O322+).
8. Szűcs Z, et al. Lipophilic teicoplanin pseudoaglycon derivatives
are active against vancomycin- and teicoplanin-resistant enter-
ococci. J Antibiot. 2017;70:664–70.
9. Szűcs Z, et al. Synthesis and biological evaluation of lipophilic
teicoplanin pseudoaglycon derivatives containing a substituted
triazole function. J Antibiot. 2017;70:152–7.
10. Szűcs Z, et al. New semisynthetic teicoplanin derivatives have
comparable in vitro activity to that of oritavancin against clinical
isolates of VRE. J Antibiot. 2019;72:524–34.
11. Vimberg V, et al. Fluorescence assay to predict activity of the
glycopeptide antibiotics. J Antibiot. 2019;72:114–7.
12. Ishikawa T, editor. Superbases for organic synthesis. New York:
Wiley; 2009.
13. Zamperini C, et al. Identification, synthesis and biological activity
of alkyl-guanidine oligomers as potent antibacterial agents. Sci
Rep. 2017;7:8251–63.
14. Pasero C, et al. Alkyl-guanidine compounds as potent broad-
spectrum antibacterial agents: chemical library extension and
biological characterization. J Med Chem. 2018;61:9162–76.
15. Kuppusamy R, et al. Guanidine functionalized anthranilamides as
effective antibacterials with biofilm disruption activity. Org Bio-
mol Chem. 2018;16:5877–88.
16. Teng P, et al. Novel bis-cyclic guanidines as potent membrane-
active antibacterial agents with therapeutic potential. Chem Com-
mun. 2017;53:11948–51.
17. Binda E, Marinelli F, Marcone GL. Old and new glycopeptide
antibiotics: action and resistance. Antibiotics. 2014;3:572–94.
18. Wanner J, et al. A new and improved method for deglycosida-
tion of glycopeptide antibiotics exemplified with vancomycin,
ristocetin, and ramoplanin. Bioorg Med Chem Lett. 2003;13:
1169–73.
Acknowledgements This work was supported by the European
Regional Development Fund under the projects GINOP-2.3.2-15-
2016-00044 and GINOP-2.3.3-15-2016-00004 and by the European
Social Fund under the project EFOP-3.6.3-VEKOP-16-2017-00009.
This research was also funded by the National Research, Development
and Innovation Office of Hungary (K119509).
Compliance with ethical standards
19. Suhs T, König B. Synthesis of functionalized guanidino amino
acids. Chem Eur J. 2006;12:8150–7.
Conflict of interest The authors declare that they have no conflict of
interest.
20. Hosamani B, Narendra N, Prabhu G, Sureshbabu VV. Synthesis
of N-urethane protected amino alkyl (S-methyl)-isothiouronium
compounds and carbodiimide tethered peptidomimetics: an
application for guanidino and substituted guanidino peptido-
mimetics synthesis. RSC Adv. 2014;4:48920–30.
21. Chen J, Pattarawarapan M, Zhang AJ, Burgess K. Solution- and
solid-phase syntheses of substituted guanidinocarboxylic acids.
J Comb Chem. 2000;2:276–81.
22. Fell JB, Coppola GM. A mild and efficient preparation of car-
bodiimides. Synth Commun. 1995;25:43–7.
23. Lin SW, Carver PL, DePestel DD. Dalbavancin: a new option
for the treatment of gram-positive infections. Ann Pharmacother.
2006;40:449–60.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
1. Laxminarayan R. Antibiotic effectiveness: balancing conservation
against innovation. Science. 2014;345:1299–301.
2. Cattoir V, Leclercq R. Twenty-five years of shared life with
vancomycin-resistant enterococci: is it time to divorce? J Anti-
microb Chemother. 2013;68:731–42.
3. Cattoir V, Giard J-C. Antibiotic resistance in Enterococcus
faecium clinical isolates. Expert Rev Anti Infect Ther. 2014;
12:239–48.
4. Arias CA, Murray BE. Emergence and management of drug-
resistant enterococcal infections. Expert Rev Anti Infect Ther.
2008;6:637–55.
24. Nagarajan R. Structure-activity relationships of vancomycin-type
glycopeptide antibiotics. J Antibiot. 1993;46:1181–95.
25. Pavlov AY, et al. Modification of glycopeptide antibiotic
eremomycin by the action of alkyl halides and study on anti-
bacterial activity of the compounds obtained. J Antibiot. 1994;
47:225–32.
26. Malabarba A, et al. N15-alkyl and N15,N15-dialkyl derivatives of
teicoplanin antibiotics. J Antibiot. 1990;43:1107–21.
27. Sarkar P, Yarlagadda V, Ghosh C, Haldar J. A review on cell wall
synthesis inhibitors with an emphasis on glycopeptide antibiotics.
Med Chem Commun. 2017;8:516–33.
5. Mendes RE, et al. Longitudinal (2001-14) analysis of enterococci
and VRE causing invasive infections in European and US hos-
pitals, including a contemporary (2010-13) analysis of oritavancin
in vitro potency. J Antimicrob Chemother. 2016;71:3453–9.

Z. Szűcs et al.
28. Yarlagadda V, Akkapeddi P, Manjunath GB, Haldar J. Membrane
active vancomycin analogues: a strategy to combat bacterial
resistance. J Med Chem. 2014;57:4558–68.
29. Printsevskaya SS, et al. Structure-activity relationship studies of a
series of antiviral and antibacterial aglycon derivatives of the
glycopeptide antibiotics vancomycin, eremomycin, and dechlor-
oeremomycin. J Med Chem. 2005;48:3885–90.
30. Csávás M, et al. Synthesis and antibacterial evaluation
of some teicoplanin pseudoaglycon derivatives containing
alkyl- and arylthiosubstituted maleimides. J Antibiot. 2015;68:
579–85.
31. Malabarba A, et al. Synthesis and biological properties of N63-
carboxamides of teicoplanin antibiotics. Structure-activity rela-
tionships. J Med Chem. 1989;32:2450–60.