Synthesis of fluorescent ristocetin aglycon derivatives with remarkable antibacterial and antiviral activities

Article abstract of DOI:10.1016/j.ejmech.2012.10.030

Isoindole and benzoisoindole derivatives of ristocetin aglycon have been prepared by reaction with o-phthalaldehyde or naphthalene-2,3-dialdehyde and various thiols. The new compounds exhibited potent antibacterial and anti-influenza virus activity. The c

Full text of DOI:10.1016/j.ejmech.2012.10.030

European Journal of Medicinal Chemistry 58 (2012) 361e367
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of fluorescent ristocetin aglycon derivatives with remarkable
antibacterial and antiviral activities
,
a
a
a
b
a
c
Attila Sipos a , Gábor Máté , Erzsébet Roth , Anikó Borbás , Gyula Batta , Ilona Bereczki , Sándor Kéki ,
*
}
István Jóna d, Eszter Ostorházi e, Ferenc Rozgonyi e, Evelien Vanderlinden f, Lieve Naesens f,
Pál Herczegh a
,
*
a Department of Pharmaceutical Chemistry, Medical and Health Science Centre, University of Debrecen, Egyetem tér 1, H-4010 Debrecen, Hungary
b Department of Organic Chemistry, University of Debrecen, Egyetem tér 1, H-4010 Debrecen, Hungary
c Department of Applied Chemistry, University of Debrecen, Egyetem tér 1, H-4010 Debrecen, Hungary
d Department of Physiology, Medical and Health Science Centre, University of Debrecen, P. O. Box 22, Debrecen 4012, Hungary
e Microbiology Laboratory, Department of Dermatology, Venerology and Dermatooncology, Semmelweis University, Mária u. 41, H-1085 Budapest, Hungary
f Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Isoindole and benzoisoindole derivatives of ristocetin aglycon have been prepared by reaction with
o-phthalaldehyde or naphthalene-2,3-dialdehyde and various thiols. The new compounds exhibited
potent antibacterial and anti-influenza virus activity. The cluster forming and fluorescent properties of
the aglycon derivatives were also studied.
Received 15 May 2012
Received in revised form
15 October 2012
Accepted 17 October 2012
Available online 25 October 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Dedicated to the memory of Prof. Ferenc
Sztaricskai who passed away on 23/03/2012.
Keywords:
Glycopeptide
Antiviral
Antibacterial
Aggregation
Multivalence
Fluorescence
1. Introduction
D-mannoses, D-glucose, D-arabinose, L-rhamnose and L-ristosamine)
attached to the aglycon. In spite of its good antibacterial activity
against Gram-positive strains, including methicillin-resistant S.
aureus (MRSA) [5], this antibiotic has not been used in therapy, due
to it’s unwanted side-effect to cause aggregation of blood platelets
[6]. Bardsley and co-workers recognized that the removal of the
Glycopeptide antibiotics vancomycin and teicoplanin are used
for treating serious Gram-positive bacterial infections that are
resistant to other antibiotics [1,2]. Due to the emergence and spread
of glycopeptide-resistant enterococci (GRE) and glycopeptide
intermediate-resistant Staphylococcus aureus (GISA), as well as
teicoplanin-resistant Staphylococcus haemolyticus [3], there is an
urgent need for new antibiotics active against resistant bacteria.
Intensive research in this field was performed over the past 20
years [2].
L-rhamnose moiety from ristocetin A eliminates this aggregating
property [7]. In the framework of studies on synthetic modifications
of aglycons of glycopeptide antibiotics, we anticipated that agly-
coristocetin (2) could be a promising compound for obtaining new
semisynthetic derivatives lacking the platelet aggregation property.
Since the important work of Nagarajan et al. [8], who was the
first to report on lipophilic N-alkyl derivatives of glycopeptide
antibiotics with considerable activity against vancomycin-resistant
bacteria, the concept of inserting lipophilic substituents led to
several active antibiotics and the subclass of lipoglycopeptides (e.g.
oritavancin, telavancin and dalbavancin) [9].
Ristocetin A (1) is a glycopeptide-type antibiotic produced by
Nocardia lurida [4]. This molecule contains six sugar moieties (two
* Corresponding authors. Tel.: þ36 52 512 900x22895; fax: þ36 52 512 914.
E-mail addresses: sipos.attila@pharm.unideb.hu (A. Sipos), herczeghp@
gmail.com (P. Herczegh).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.030

362
A. Sipos et al. / European Journal of Medicinal Chemistry 58 (2012) 361e367
HO
HO
O
HO
O
OH
O
O
O
OH
HO
OH
O
OH
OH
CH
O
O
HO
OH
3
HO
O
O
O
CH
OH
3
H N
O
2
O
O
O
H
H
H
N
H
H
N
NH
H
O
2
N
H
N
H
N
H
H
HN
O
O
H COOC
3
HO
O
OH
CH
OH
3
HO
O
OH
OH
O
1
OH
OH
OH
O
O
O
HF / anisole
-18 °C
HO
H
OH
61 %
O
O
H
H
N
H
H
N
2 h
NH
H
O
2
N
H
N
H
N
H
H
HN
O
O
H COOC
3
HO
O
OH
CH
OH
3
HO
OH
2
Scheme 1. Synthesis of aglycoristocetin (2).
In the past few years, we have synthesized a series of new
The formation of isoindole and benzoisoindole derivative in
three-component reactions has been utilized extensively for fluo-
rescent labelling of target molecules. The fluorogenic reaction
involves the heteroring closing of o-phthalaldehyde (or its benzo-
logs) with primary amine and a thiol (Scheme 2) and known for its
relative robustness [16].
The investigation of the mechanism of reaction attracted
considerable attention and it is widely agreed now that the multi-
step reaction [17] yields more stable isoindoles with bulky and
lipophilic thiols. As it is usually recommended, reactions and isola-
tion steps were performed with protection from light and products
were stored in dark and at 0 ꢀC. Under these conditions we have not
observed considerable decomposition during our investigations.
In addition to the previous consideration on the applied thiol
reagent, the available structureeactivity relationships were also
reviewed to construct the more interesting aglycoristocetin deriv-
atives. On one hand, it is known that the more lipophilic moieties
inserted in the proximity of the N-terminal resulted high antibac-
terial activities as it is discussed in references [11e13]. On the other
hand, some S-glycosides were also applied in order to form products
aglycoristocetin derivatives possessing not only high antibacterial
activity but, in several cases, robust anti-influenza virus activity
was noted [10e13]. On the basis of NMR and dynamic light
scattering measurements, the antibacterial activity of some of our
glycopeptide aglycon derivatives can be attributed to their
multivalent interaction of their self-assembled aggregates with the
bacterial cell wall peptidoglycan [13]. In order to study their
mechanism of action in a cellular context (e.g. in influenza
virus-infected cells), we were interested in synthesizing fluorescent
aglycoristocetin derivatives. One of the well known methods
for fluorescent tagging of amino acids is the preparation of their
isoindole derivatives. Here we report the synthesis and physico-
chemical and biological evaluation of a series of such aglycor-
istocetin isoindole derivatives. In order to synthesize fluorescent
aglycoristocetin derivatives with potentially interesting pharma-
cological properties, the widely used three-component isoindole-
formation reaction [14] was chosen for the derivatization of the
N-terminal of this glycopeptide.
2. Results and discussion
CHO
2.1. Chemistry
R
NH2 +
R' SH
+
N
R
CHO
The synthesis of the aimed compounds was started with the
preparation of ristocetin aglycon (2) from the parent antibiotics
using anhydrous hydrogen fluoride treatment (Scheme 1) accord-
ing to Boger et al. [15].
S
R'
Scheme 2. General formation of 1,2-disubstituted isoindoles.

A. Sipos et al. / European Journal of Medicinal Chemistry 58 (2012) 361e367
363
Table 1
Formation of isoindole and benzoisoindole derivatives of aglycoristocetin 2.
Product
Starting dialdehyde
Thiol reagent (ReSH)
Formed isoindole moiety (R0Ne)a
Yields (%)
log Pb
3
57
5.48
4
52
1.43
5
6
7
50
33
26
4.26
6.44
7.18
8
9
19
38
5.62
7.67
(continued on next page)

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A. Sipos et al. / European Journal of Medicinal Chemistry 58 (2012) 361e367
Table 1 (continued )
Product
Starting dialdehyde
Thiol reagent (ReSH)
Formed isoindole moiety (R0Ne)a
Yields (%)
19
log Pb
10
5.70
a
General procedure: 0.11 mmol dialdehyde, 0.11 mmol thiol in 3 mL MeOH (protected from light, under argon) stirred for 20 min at 0 ꢀC. 0.05 mmol 2 in 3 mL MeOH added
and stirred for 2 h at RT.
b
log P values were calculated with ChemSketch logP add-on [18].
with a chemical profile tending to the parent antibiotic 1. In Table 1
the structure of products and yields were concluded.
The structure of products 3e10 was confirmed by 2D NMR
techniques, IR spectroscopy and MALDI-ToF measurements.
concentrations of our products 3e10, in comparison to as well as
three antiviral reference agents, are summarized in Table 3.
With the exception of compound 9, all the new aglycoristocetin
derivatives showed considerable anti-influenza A virus activity.
Compounds 6 and 7 were effective against influenza B virus as well.
Several of our new compounds exerted their antiviral effect at
remarkably lower concentrations than the clinically used reference
agents. From a comparison of the antiviral EC50 values for
compounds 3e10 with those of unsubstituted aglycoristocetin 2, it
is evident that the newly formed 1,2-(benzo)isoindole moiety has
a positive impact on anti-influenza virus activity. However, the
(benzo)isoindole derivatized glycopeptides also had moderate
2.2. Antibacterial studies
The antibacterial activity of our products 3e10 was tested
against a panel of strains of Gram-positive bacterial species
(Table 2) using broth microdilution method as previously described
[10]. Infections caused by some of these strains within the species
are known to be susceptible to glycopeptide antibiotic therapy;
however, this panel of bacteria includes such strains of the species
too that are mainly responsible for nosocomial infections and
resistant to glycopeptide agents.
Most of our derivatives were more active against this panel of
bacteria than the reference glycopeptide antibiotics. Interestingly,
the hardly accessible biofilm-forming Staphylococcus epidermidis
was found to be more sensitive to derivative 3, 6 and 9 than to the
reference antibiotics. In other words, these products can easily
penetrate the outmost biofilm layer and inhibit peptidoglycan
synthesis. Importantly, in case of Enterococcus faecalis possessing
the vanA resistance gene compounds 3e10 exhibited remarkably
lower inhibitory concentrations when compared to vancomycin
and teicoplanin, two antibiotics currently used in the clinic. A
similar observation was made with E. faecalis containing the vanB
resistance gene, since compounds 3e10 were again much more
active than vancomycin and derivatives 8 and 9 were as effective as
the original teicoplanin. It can be concluded that lipophilicity of
side-chains attached to the N-terminal of aglycon 2 was found to be
favourable in the inhibition of tested strains in agreement with the
results for compounds 7e9.
cytotoxicity, since their MCC values were in the range of 6e20
mM. In
contrast, the parent aglycon 2 was not toxic at 100 M (the highest
m
concentration tested). This cytotoxicity was even more pronounced
for derivatives 3 and 10, which were the most active antiviral
compounds in this series.
2.4. Fluorescent activity
An important reason for designing this set of new glycopeptides
was to obtain derivatives with fluorescent activity. This property,
together with high antibacterial activity, should offer an excellent
opportunity for the study of physiological functions of bacteria at
single-cell level [19]. In Table 4 the excitation and emission maxima
of compounds 3e10 are shown, together with reference data for
isoindole-labelled amino acids.
Our studies were started with the recording of UVevis spectra in
methanol, which confirmed that the aglycoristocetin 2 had no
absorption at or above 300 nm, while the characteristic absorption
band was found to be in the region of 328e334 nm for the
isoindole-derivatives and 340e341 nm for the benzoisoindole-
derivatives. These data served as starting points for recording the
fluorescent spectra of compounds 3e10. Isoindole derivatives are
often used for fluorescent-tagging of amino acids, therefore
a comparison was made with some typical literature data [20]. The
excitation maxima of our compounds 3e8 (for representative
spectra see Fig. 1 Panel A) are very similar to these published data,
2.3. Antiviral activity
It was reported by our groups that some lipophilic derivatives of
aglycoristocetin are potently active against influenza A and B
viruses [10e13]. The anti-influenza virus activity and cytotoxic
Table 2
Antibacterial activity of reference and newly synthesized glycopeptides 3e10.
Bacterium strains
MIC (mg/mL)
Teicoplanin
Vancomycin
Ristocetin
3
4
5
6
7
8
9
10
B. subtilis ATCC 6633
0.5
0.5
0.5
4
16
1
0.5
0.5
0.5
2
4
1
16
128
32
2
128
4
8
2
1
1
4
2
8
8
2
1
1
4
1
1
1
2
8
4
4
4
4
4
4
4
2
1
1
0.5
1
2
4
1
2
1
0.5
2
1
0.5
1
2
0.5
1
1
0.5
0.5
0.5
1
2
2
2
4
2
2
2
2
S. aureus MSSA ATCC 29213
S. aureus MRSA ATCC 33591
S epidermidis ATCC 35984 biofilm
S. epidermidis mecAþ
2
0.5
0.5
0.5
0.5
1
E. faecalis ATCC 29212
0.5
0.5
0.5
E. faecalis ATCC 15376 vanAþ
256
0.5
256
128
16
256
E. faecalis ATCC 51299 vanBþ
MIC: Minimum Inhibitory Concentration, ATCC: American Type Culture Collection, MSSA: Methicillin Sensitive Staphylococcus aureus, MRSA: Methicillin Resistant Staphy-
lococcus aureus, Biofilm: biofilm forming strain, vanAþ: vanA gene positive, vanBþ: vanB gene positive.

A. Sipos et al. / European Journal of Medicinal Chemistry 58 (2012) 361e367
365
Table 3
Anti-influenza virus activity and cytotoxicity of novel and reference compounds.
3. Conclusions
Introduction of the isoindole and benzoisoindole moieties into
the N-terminal position of the aglycoristocetin molecule resulted in
some compounds with pronounced antibacterial activity against
a panel of bacteria including biofilm-forming and resistant strains.
We assume the self-assembly of the new compounds and the
arising multivalent interaction of the aggregates with the repeating
unit of the bacterial cell wall peptidoglycan to be a key-element in
the mechanism of the antibacterial activity. The calculated log P
values (Table 1) confirm the strong lipophilic effect of the newly
formed moieties which directly contributes to aggregation. For
some derivates the cluster formation has been proved by dynamic
light scattering measurements. It is noteworthy, however, that
some ristocetin aglycon derivatives having lipophilic side chains in
the N-terminal position, prepared recently in this laboratory,
turned out to be completely inactive against bacteria [13]. There-
fore, the high antibacterial activity of the present series of ristocetin
aglycon derivatives demonstrates the important contribution of the
(benzo)isoindole moiety to the mechanism of action, indepen-
dently of its lipophilic character. In addition, it does not seem to be
direct correlation between the measure of lipophilicity (log P) and
the biological activities of compounds 3e10. On the basis of the
presented antibacterial test results the acetyl-protected glycosyl
units (derivatives 3 and 5) are the less efficient thiyl substituents.
Similarly to our previous results [10e13] the lipophilic
modification of aglycoristocetin resulted in antibiotic derivatives
with remarkable anti-influenza virus activity. Incorporation of
isoindoles into the aglycoristocetin calls for high inhibitory activity
against the tested influenza A strains almost in all cases (including
glycosyl-thiyl substituted derivatives 3e5), and additionally two
compounds (6 and 7) with strongly lipophilic character displayed
outstanding anti-influenza B virus activity as well. Taking, however,
the significantly different antiviral properties of the structurally
resembling 6 and 9, it is hard to draw further conclusions for the
structureeactivity relationships.
Compounds
EC50
(
m
M)
MCC (mM)
Influenza
A/PR/8/34
Influenza
A/HK/7/87
Influenza
B/HK/5/72
3
4
5
6
7
8
9
10
0.16
0.27
1.8
0.67
1.4
0.30
>100
0.08
2.3
0.072
2.3
1.8
0.68
4.0
1.8
>100
0.36
6.8
1.8
8.9
>100
>100
>100
1.4
ꢂ4
20
20
6.4
20
20
8.9
>100
>100
>100
>100
34
20
ꢂ0.8
>100
>100
ꢂ20
>500
Aglycoristocetin
Zanamivir
Ribavirin
Amantadine
0.3
6.8
7.6
8.9
>500
2.3
The experiments were performed in Madin Darby canine kidney cells. See reference
[22] for a detailed description of the antiviral evaluation procedure.
EC50: 50% effective concentration, or concentration producing 50% inhibition of
virus-induced cytopathic effect, as determined by visual scoring of the CPE. MCC:
Minimum compound concentration that causes
alteration of normal cell morphology.
a microscopically detectable
however the emission results show a considerable hypsochromic
shift. The reason for this could be the difference in the secondary
interactions between solvent and ligand molecules or between the
glycopeptide part and the isoindole moiety. There is also a signifi-
cant difference with the isoindole derivatives attached to carbo-
hydrate substituents and heterocycles. The excitation spectra of
benzoisoindole compounds 9e10 (for representative spectra see
Fig. 1 Panel B) have typically a band with fine structure and, as
a consequence of the extended
excitation and emission maxima have a bathochromic shift in
comparison to the isoindole congeners.
p-electron structure, both the
2.5. Aggregation study
Because of the promising antiviral and antibacterial activity of
our (benzo)isoindole derivatives, further synthetic and biological
studies are necessary on similar substrates to expand this field of
knowledge and establish sound conclusions. Thiswork is inprogress.
We examined the aggregation of compounds 6 and 9 in aqueous
solutions containing small amount of DMSO for proper solubility,
using dynamic light scattering measurements. This experiment
showed the presence of aggregates with an effective diameter of
more than 100 nm in monomodal distribution (Fig. 2).
4. Experimental
These results pointed to a slight difference between 2-S-phenyl-
isoindole derivative
6 and 2-S-phenyl-benzoisoindole 9, as
4.1. General information
compound 6 showed size distribution maximum at 160 nm while
for compound 9 a 180 nm distribution maximum was observed. The
aggregation-forming ability of the products is considered a direct
consequence of the insertion of the new lipophilic moiety as it is
confirmed by the log P values presented in Table 1. In view of current
progress in this field of research, multivalence is an important tool
in the generation of more potent anti-infective agents [13].
The starting materials and solvents were purchased from
commercial sources (SigmaeAldrich or Fluka) and used as received.
1H and 13C NMR spectra were recorded at 500 and 125 MHz,
respectively, with a Bruker DRX II 500 spectrometer at 300 K, using
MeOH-d4 as solvents and TMS (tetramethylsilane) as internal
standard. Signal assignment was aided by 2D HSQC, COSY and
TOCSY techniques of which exact parameters were performed in
accordance of previously published methods [21]. The assignment
of the substituted (benzo)isoindole moieties were performed on the
basis of previously reported full assignation of compound 2 [21].
Mass spectra were recorded with Bruker Biflex-III MALDI TOF mass
spectrometer. High-resolution mass spectral measurements were
performed with a Bruker micrOTOF-Q instrument in the ESI mode.
IR spectra were recorded on PerkineElmer 283 B spectrometer.
The dynamic light scattering measurements were performed on
Brookhaven Light Scattering device equipped with a BI-9000 digital
correlator. Fluorescence measurements were performed in 1 ꢁ1 cm
cuvettes in a SPEX Fluoromax single photon counting spec-
trometer (Jobin-Yvon, Spex Industries, Edison, NJ, U.S.A.). Data were
collected at a rate of 1 Hz and stored digitally for later analysis. For
Table 4
Excitation (ex) and emission (em) wavelength maxima for compounds 3e10.
Compounds
lex (nm)
lem (nm)
3
4
5
6
7
8
9
10
333
334
334
337
338
370
397
406
394
423
424
417
434
430
454
340; 359; 375; 394a
341; 361; 380a
337
Reference data for amino acids
with isoindolesb
a
Fine structure with multiple maxima.
Data obtained from Ref. [20].
b

366
A. Sipos et al. / European Journal of Medicinal Chemistry 58 (2012) 361e367
Fig. 1. The excitation and emission spectra of compounds 4 (Panel A) and 9 (Panel B).
purification of the products flash chromatography on Merck silica
gel (Kieselgel 60), 0.040e0.063 mm (70e230 mesh) was used. Thin
layer chromatography (TLC) was performed on Kieselgel 60 F254
(Merck). Spots were visualized by irradiation under UV lamp, and/or
by spraying with an ammonium-molibdenate/sulfuric acid solution
and heating, or using Pauly-reagent. Evaporations were carried out
under diminished pressure at 35e40 ꢀC (bath temperature).
Investigation of the antibacterial activity of the antibiotics and their
derivatives was carried out by measuring the MIC values as
described earlier [21]. The cell-culture based assays to determine
the inhibitory effect on influenza virus replication against, have
been published elsewhere [22].
2.15 (4 ꢁ OAc), 3.81 (COOCH3), 4.10, 4.48, 4.87, 5.35, 5.40, 5.45, 5.81
(glycopeptides chiral CH), 4.85, 4.88, 5.13, 5.22, 5.42 (carbohydrate
hydrogens), 7.32, 7.36, 7.45, 7.46, 7.48 (isoindole moiety). 13C NMR
(MeOH-d4, 500 MHz) d: 19.7 (OAc), 23.3 (OAc), 52.4 (COOCH3), 68.0,
70.0, 72.7, 74.3 (carbohydrate carbons), 125.6, 131.3, 137.8, 138.1,
138.5 (isoindole moiety), 170.5, 171.0, 171.4, 171.6, 171.9, 172.2
(peptide carbonyls), 173.9 (COOCH3).
4.2.2. Product 4
Off-white powder. TLC (DCM:MeOH ¼ 8:2): 0.22. MALDI-TOF
MS: [M þ Na]þ ¼ 1490.3 m/z, calculated for C74H65N7O24SNa
1490.4; HRMS (ESI) m/z found: 1468.3861 [M þ H]þ, calculated:
1468.3874 [M þ H]þ; nmax (KBr disc) 3418, 2928, 1654, 1516, 1210;
4.2. General procedure
1H NMR (MeOH-d4, 500 MHz)
d: 3.38, 3.41, 3.48, 3.64, 3.75
(carbohydrate hydrogens), 3.81 (COOCH3), 4.14, 4.51, 4.88, 5.33,
5.40, 5.48, 5.80 (glycopeptides chiral CH), 3.87 (COOCH3), 7.30, 7.33,
Dialdehyde (0.11 mmol) and thiol reagent (0.11 mmol) were
dissolved in 3.0 ml of methanol in a flask protected from light and
under argon atmosphere. The resulting solution was stirred at 0 ꢀC.
A solution of 58 mg aglycoristocetin 2 (0.05 mmol) in 3.0 mL of
methanol was added to this with constant argon gas flow and
stirred for further 2 h at 0 ꢀC. The reaction was followed by TLC with
UV and Pauly detection. The product mixture was carefully
evaporated under diminished pressure and purified with column
chromatography. Yields were reported in Table 1.
7.40, 7.41, 7.48 (isoindole moiety). 13C NMR (MeOH-d4, 500 MHz)
d:
53.1 (COOCH3), 58.0, 63.2, 71.4, 73.1, 82.3 (carbohydrate C), 124.7,
137.8, 138.1, 138.2, 138.4 (isoindole moiety), 170.7, 171.3, 171.4, 171.6,
171.9, 172.4 (peptide carbonyls), 173.4 (COOCH3).
4.2.3. Product 5
Off-white powder. TLC (DCM:MeOH ¼ 9:1): 0.25. MALDI-TOF
MS: [M þ Na]þ ¼ 1657.6 m/z, calculated for C82H74N8O27SNa
1657.7; HRMS (ESI) m/z found: 1635.4462 [M þ H]þ, calculated:
1635.4457 [M þ H]þ; nmax (KBr disc) 3435, 1739, 1649, 1516, 1210;
4.2.1. Product 3
Off-white powder. TLC (DCM:MeOH ¼ 95:5): 0.40. MALDI-TOF
MS: [M þ Na]þ ¼ 1658.4 m/z, calculated for C82H73N7O28SNa
1658.4; HRMS (ESI) m/z found: 1636.4258 [M þ H]þ, calculated:
1636.4297 [M þ H]þ; nmax (KBr disc) 3394, 2925, 2854, 1749, 1662,
1H NMR (MeOH-d4, 500 MHz)
(COOCH3), 3.81 (COOCH3), 4.11, 4.51, 4.79, 5.32, 5.45, 5.41, 5.85
(glycopeptides chiral CH), 4.97, 5.09, 5.15, 5.38 (carbohydrate
hydrogens), 7.29, 7.39, 7.44, 7.47, 7.49 (isoindole moiety). 13C NMR
d
: 1.91, 2.04, 2.08, 2.15 (4 ꢁ OAc), 3.88
1595, 1513, 1213; 1H NMR (MeOH-d4, 500 MHz)
d: 1.91, 2.04, 2.08,
(MeOH-d4, 500 MHz) d: 19.0 (OAc), 23.4 (OAc), 53.7 (COOCH3), 68.4,
Fig. 2. Intensity (Irel%)-size distribution (d) results for compounds 6 (Panel A) and 9 (Panel B).

A. Sipos et al. / European Journal of Medicinal Chemistry 58 (2012) 361e367
367
72.7 (carbohydrate C), 124.8, 138.0, 138.1, 138.5, 138.8 (isoindole
moiety), 170.6, 171.0, 171.3, 171.4, 172.1, 172.4 (peptide carbonyls),
173.2 (COOCH3).
7.43, 7.53, 8.04, 8.28 (benzoisoindole moiety), 7.97, 8.36 (pyrimidyl
moiety). 13C NMR (MeOH-d4, 500 MHz)
: 53.7 (COOCH3), 131.6,
147.8,171.8 (pyrimidyl moiety),114.2,126.5,130.6,130.8,131.2,131.7
(benzoisoindole moiety), 170.5, 171.0, 171.2, 171.4, 172.0, 172.3
(peptide carbonyls), 173.8 (COOCH3).
d
4.2.4. Product 6
Off-white powder. TLC (DCM:MeOH ¼ 9:1): 0.20. MALDI-TOF
MS: [M þ Na]þ ¼ 1404.5 m/z. calculated for C74H59N7O19SNa
1404.7; HRMS (ESI) m/z found: 1382.3658 [M þ H]þ, calculated:
1382.3659 [M þ H]þ; nmax (KBr disc) 3420, 2925, 2853, 1662, 1513,
Acknowledgements
The work is supported by the TÁMOP 4.2.1/B-09/1/KONV-2010-
0007 project. The project is co-financed by the European Union and
the European Social Fund. The work was also supported by the
Hungarian Research Fund OTKA through Grants K 79126, T-46186,
K 101850, OTKA-NKTH CK 77515, and NK 68578. L.N. and E.V.
acknowledge the technical assistance from Leentje Persoons and
Wim van Dam, and the financial support from the Flemish Fonds
voor Wetenschappelijk Onderzoek (FWO No. 9.0188.07) and the
Geconcerteerde Onderzoeksacties (GOA/10/014).
1209; 1H NMR (MeOH-d4, 500 MHz)
d: 3.81 (COOCH3), 3.81
(COOCH3), 4.12, 4.50, 4.84, 5.40, 5.44, 5.47, 5.80 (glycopeptides
chiral CH), 6.95 (PheSe), 7.18, 7.49, 7.50, 7.51, 7.55 (isoindole
moiety). 13C NMR (MeOH-d4, 500 MHz)
d: 53.4 (COOCH3), 124.9,
137.4, 137.5, 137.6, 137.8 (isoindole moiety), 170.1, 170.9, 171.2, 171.6,
172.1, 172.3 (peptide carbonyls), 173.8 (COOCH3).
4.2.5. Product 7
Off-white powder. TLC (CH2Cl2:MeOH ¼ 9:1): 0.11. MALDI-TOF
MS: [M þ H]þ ¼ 1439.2 m/z, calculated for C75H60N8O19S2 1439.3;
HRMS (ESI) m/z found: 1439.3327 [M þ H]þ, calculated: 1439.3332
[M þ H]þ; nmax (KBr disc) 3420, 2927, 1659, 1575, 1208; 1H NMR
Appendix A. Supplementary information
Supplementary data related to this article can be found at doi:
10.1016/j.ejmech.2012.10.030.
(MeOH-d4, 500 MHz) d: 3.84 (COOCH3), 3.81 (COOCH3), 4.13, 4.53,
4.80, 5.39, 5.40, 5.45, 5.78 (glycopeptides chiral CH), 7.79, 7.82, 7.83,
7.96 (isoindole moiety), 7.96, 8.01 (benzothiazole moiety). 13C NMR
(MeOH-d4, 125 MHz)
d
: 53.4 (COOCH3), 121.2, 126.6, 132.8, 150.9,
References
163.1 (benzothiazole moiety), 124.5, 137.9, 138.2, 138.4 (isoindole
moiety), 170.6, 171.1, 171.2, 171.3, 171.9, 172.3 (peptide carbonyls),
173.4 (COOCH3).
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4.2.6. Product 8
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Off-white powder. TLC (CH2Cl2:MeOH ¼ 8:2): 0.67. MALDI-TOF
MS: [M þ Na]þ ¼ 1406.4 m/z, calculated for C72H57N9O19SNa
1406.4; HRMS (ESI) m/z found: 1384.3560 [M þ H]þ, calculated:
1384.3564 [M þ H]þ; nmax (KBr disc) 3391, 2926, 1661, 1513, 1207;
1H NMR (MeOH-d4, 500 MHz)
d: 3.86 (COOCH3), 3.81 (COOCH3),
4.12, 4.49, 4.85, 5.35, 5.40, 5.46, 5.80 (glycopeptides chiral CH), 7.82,
7.86, 8.02 (isoindole moiety), 8.03, 8.57, 8.60 (pyrimidyl moiety). 13
NMR (MeOH-d4, 125 MHz) : 53.4 (COOCH3), 126.1, 138.7, 146.8,
C
d
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172.4 (pyrimidyl moiety), 125.6, 138.1, 138.6, 138.7 (isoindole
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}
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4.2.7. Product 9
}
}
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Pale brown powder. TLC (Tol:MeOH ¼ 7:3): 0.30. MALDI-TOF
MS: [M þ Na]þ ¼ 1454.5 m/z, calculated for C78H61N7O19SNa
1454.4; HRMS (ESI) m/z found: 1432.3820 [M þ H]þ, calculated:
1432.3816 [M þ H]þ; nmax (KBr disc) 3387, 2932, 1657, 1510, 1207;
}
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1H NMR (MeOH-d4, 500 MHz)
d: 3.81 (COOCH3), 4.14, 4.48, 4.90,
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}
5.33, 5.39, 5.45, 5.79 (glycopeptides chiral CH), 6.98, 7.04 (Ph-S),
F. Sztaricskai, E. Roth, E. Ostorházi, F. Rozgonyi, L. Naesens, P. Herczegh, J. Med.
7.17, 7.18, 7.20, 7.43, 8.35 (benzoisoindole moiety). 13C NMR
Chem. 52 (2009) 6053e6061.
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(MeOH-d4, 500 MHz) d: 53.4 (COOCH3), 122.6, 122.8, 124.0, 124.2,
125.0, 126.4, 126.7 (benzoisoindole moiety), 135.9, 136.6 (PheS),
170.4, 171.0, 171.3, 171.4, 171.9, 172.4 (peptide carbonyls), 173.6
(COOCH3).
b P. Zuman, Chem. Rev. 104 (2004) 3217e3238.
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4.2.8. Product 10
Pale brown powder. TLC (CH2Cl2:MeOH ¼ 9:1): 0.23. MALDI-TOF
MS: [M þ K]þ ¼ 1472.4 m/z, calculated for C76H59N9O19SK 1472.5;
HRMS (ESI) m/z found: 1434.3729 [M þ H]þ, calculated: 1434.3721
[M þ H]þ; nmax (KBr disc) 3384, 2925, 2856, 1664, 1508, 1206; 1H
}
}
[21] F. Sztaricskai, G. Batta, P. Herczegh, A. Balázs, J. Jeko, E. Roth, P.T. Szabó,
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NMR (MeOH-d4, 125 MHz) d: 3.88 (COOCH3), 3.81 (COOCH3), 4.13,
4.50, 4.89, 5.37, 5.40, 5.43, 5.78 (glycopeptides chiral CH), 7.36, 7.42,