N-glycosylthioureido aglyco-ristocetins without platelet aggregation activity.
ABSTRACT The water-soluble N-methoxy-PEG-yl-, N-beta-D-glucopyranosyl- and N-beta-D-maltosylthioureido aglyco-ristocetin were prepared which, in contrast to ristocetin A, did not induce thrombocyte aggregation. The antibacterial activity of N-beta-D-maltosylthioureido aglyco-ristocetin A against MRSA was comparable to that of ristocetin A, while its activity against Enterococcus faecalis (VRE, TSE) is somewhat stronger when compared to those of vancomycin and ristocetin A.
- SourceAvailable from: Evelien Vanderlinden[Show abstract] [Hide abstract]
ABSTRACT: We report on a new anti-influenza virus agent, SA-19, a lipophilic glycopeptide derivative consisting of aglycoristocetin coupled to a phenylbenzyl-substituted cyclobutenedione. In Madin-Darby canine kidney cells infected with influenza A/H1N1, A/H3N2, or B virus, SA-19 displayed a 50% antivirally effective concentration of 0.60 μM and a selectivity index (ratio of cytotoxic versus antiviral concentration) of 112. SA-19 was 11-fold more potent than unsubstituted aglycoristocetin and was active in human and nonhuman cell lines. Virus yield at 72 h p.i. was reduced by 3.6 logs at 0.8 μM SA-19. In contrast to amantadine and oseltamivir, SA-19 did not select for resistance upon prolonged virus exposure. SA-19 was shown to inhibit an early postbinding step in virus replication. The compound had no effect on hemagglutinin (HA)-mediated membrane fusion in an HA-polykaryon assay and did not inhibit the low-pH-induced refolding of the HA in a tryptic digestion assay. However, a marked inhibitory effect on the transduction exerted by retroviral pseudoparticles carrying an HA or vesicular stomatitis virus glycoprotein (VSV-G) fusion protein was noted, suggesting that SA-19 targets a cellular factor with a role in influenza virus and VSV entry. Using confocal microscopy with antinucleoprotein staining, SA-19 was proven to completely prevent the influenza virus nuclear entry. This virus arrest was characterized by the formation of cytoplasmic aggregates. SA-19 appeared to disturb the endocytic uptake and trap the influenza virus in vesicles distinct from early, late, or recycling endosomes. The aglycoristocetin derivative SA-19 represents a new class of potent and broad-acting influenza virus inhibitors with potential clinical relevance.Journal of Virology 06/2012; 86(17):9416-31. · 5.08 Impact Factor
glucopyranosyl- and N-b-D-maltosylthioureido aglyco-
ristocetin were prepared which, in contrast to ristocetin A,
did not induce thrombocyte aggregation. The antibacterial
activity of N-b-D-maltosylthioureido aglyco-ristocetin A
against MRSA was comparable to that of ristocetin A,
while its activity against Enterococcus faecalis (VRE, TSE)
is somewhat stronger when compared to those of
vancomycin and ristocetin A.
The water-soluble N-methoxy-PEG-yl-, N-b-D-
synthesis, antibacterial activity
glycopeptide antibiotics, platelet aggregation,
Ristocetin A (1) was discovered by American researchers
 fifty years ago, but its glycopeptide structure was
elucidated much later . Similarly to vancomycin and
teicoplanin, 1 is also effective against MRSA , but
causes thrombocytopenia  making the antibiotic
inapplicable for therapeutic purposes. However, it was
shown later that in the blood of patients suffering from von
Willebrand’s disease 1 does not induce thrombocyte
aggregation [5, 6]. As a result, 1 is now used as a routine
laboratory agent to diagnose von Willebrand’s disease.
Further studies [7, 8] on the glycopeptide antibiotics
revealed that vancomycin and actinoidin, possessing similar
structure, do not exhibit platelet aggregation, consequently
the mechanism of the effect of the latter two antibiotics on
thrombocytes must be different from that of 1.
Structure-activity relationship studies have shown that
the a-L-rhamnopyranosyl moiety of the heterotetrasaccharide
side-chain  and the C-terminal methoxycarbonyl and
phenolic hydroxyl groups of the heptapeptide aglycone 
can be responsible for the agglutination effect of 1. In
contrast, no effect of the free amino groups on the platelet
aggregation properties was observed . The above
structural elements facilitate the binding of the antibiotic to
specific sequences of von Willebrand’s factor (vWF)
thereby inducing the interaction of vWF with platelet
glycoprotein 1b (GP1b) and initiating aggregation .
The dimerization ability of 1 may also play an important
role in this mechanism [9, 11].
Despite its significant antibacterial effect (Table 1),
due to the above-mentioned undesired haematological
complications 1 was not put into clinical practice.
The goal of our present work was to synthesize ristocetin
derivatives that retain the antibacterial activity but do not
induce platelet aggregation.
Results and Discussion
The glycopeptide antibiotics exert biological activity by
developing hydrogen bondings between the glycopeptide
aglycones and the L-Lys-D-Ala-D-Ala terminal monomer
fragment of the peptidoglycan located in the bacterial
N-Glycosylthioureido Aglyco-ristocetins without Platelet
Ferenc Sztaricskai, Gábor Pintér, Erzsébet Röth, Pál Herczegh, Szilvia Kardos,
Ferenc Rozgonyi, Zoltán Boda
Received: June 7, 2007 / Accepted: July 26, 2007
©Japan Antibiotics Research Association
J. Antibiot. 60(8): 529–533, 2007
THE JOURNAL OF
F. Sztaricskai (Corresponding author), G. Pintér, E. Röth, P.
Herczegh: Department of Pharmaceutial Chemistry, University of
Debrecen, H-4010 Debrecen, P.O.Box 70, Hungary,
S. Kardos, F. Rozgonyi: Institute of Medicinal Microbiology,
Semmelweis University, H-1445 Budapest, P.O.Box 370, Hungary
Z. Boda: IInd Department of Medicine, University of Debrecen,
H-4012 Debrecen, P.O.Box 58, Hungary
cell wall. This non-covalent interaction inhibits the
transglycosidation and transpeptidation processes and this
finally leads to termination of the cell-wall synthesis and
destruction of the bacteria.
Our studies have shown that aglyco-ristocetin (2),
prepared (Scheme 1) by means of deglycosylation  of
1, retains the antibacterial activity (Table 1) and this effect
is comparable  to that of vancomycin, teicoplanin and
linezolid used in clinical practice. However, 2 is not suitable
for thrombocyte-aggregation tests due to its extremely
limited solubility in water. To enhance water-solubility,
2 was first reacted with methoxypolyethyleneglycol
isothiocyanate (3) to furnish N-PEG-yl-thioureido aglyco-
ristocetin A (4) which was precipitated with ether and
purified by column chromatography.
The influence of the presence or absence and the mode
of attachment of the particular carbohydrate moieties in the
antibiotic molecules on the antimicrobial activity and on
the interaction with vWF are not fully demonstrated.
Deglycosylation of 1 leads not only to the loss of the
rhamnose unit, but to the removal of all of the O-glycosidic
bonds. Substitution of the N-terminal primary amino group
of 2 allows the synthesis of N-glycosyl thioureido
derivatives. For this purpose 2 was treated with 2,3,4,6-
tetra-O-acetyl-D-glucopyranosyl isothiocyanate (5) and
after chromatographic purification the partially acetylated
derivative (6a) of the target compound was isolated
with 59% yield (Scheme 1). Zemplén trans-esterification
 then afforded N-b-D-glucopyranosylthioureido aglyco-
ristocetin A (6). An analogous reaction of 2 and
(7) and subsequent O-deacetylation gave N-b-D-
maltosylthioureido aglyco-ristocetin A (8). The aglyco-
ristocetin derivatives 4, 6 and 8 possessed water-solubility
sufficient for studying both the antibacterial effect and the
thrombocyte aggregation properties.
Comparison of the antimicrobial activities of the
thioureido derivatives with 2 showed that introduction of
the N-methoxy-PEG-yl group to the molecule resulted in a
dramatical decrease in the antibiotic activity of 4, which
can not be completely explained by the more than doubled
molecular mass. At the same time, the activity of the N-b-
D-glucopyranosylthioureido derivative 6 was reduced only
to one-fourth, and extension of the side chain with the N-b-
D-maltosyl group 8 led to a significant increase in the
efficacy. 8 possesses a moderate, but definitive antibacterial
activity, its activity against VRE and TSE is higher than
those of vancomycin and 1 and as active towards MRSA as
Comparison of the antibacterial effects of the new antibiotic analogs
Enterococcus faecalis ATCC
51299 (VRE, TSE)
Enterococcus faecalis ATCC
29212 (VSE, TSE)
Staphylococcus aureus ATCC
Staphylococcus aureus ATCC
Bacillus subtilis ATCC 6633
24ND 168 0.5 0.54
123282 0.5 0.52
42 32841 0.51.5
0.52 128842 0.51
ATCC: American Type Culture Collection; VRE, TSE: Vancomycin resistant, Teicoplanin sensitive van B gene positive; VSE, TSE: Vancomycin
sensitive, Teicoplanin sensitive; MSSA: Methicillin-oxacillin sensitive; MRSA: Methicillin-oxacillin resistant, mec A gene positive; MRSE: Methicillin-
oxacillin resistant, mec A gene positive.
the parent antibiotic 1.
The enhanced solubility of the prepared new N-
substituted thioureido aglyco-ristocetin derivatives in water
and in the tris-buffer (pH ?7.4) allowed to study their
platelet aggregation properties. In contrast to 1 (Aggristin-
Kit®) used as control, neither of 4, 6 and 8 induced
aggregation of human thrombocytes in the concentration
range of 0.5 and 1.0?1.5mg/ml (Fig. 1). Aggregation of
Syntheses of N-substituted-thioureido aglyco-ristocetin derivatives.
PRP (platelet-rich plasma) in the blood of healthy patients
was inhibited by 6, but 4 and 8 did not show such an effect.
These results demonstrate that carbohydrates linked
through both O-glycosidic and N-glycosidic bonds to the
aglycone may also influence thrombocyte aggregation.
Although the carbohydrate components of the glycopeptide
antibiotics are not involved in the hydrogen bondings
between the aglycones and the peptidoglycan of the
bacterial cell-wall, but their location and mode of linkage
may influence the conformation of the molecule as a whole,
and thus development of the so-called binding pocket
and occasional dimerization of the molecule. Our findings
with 8, possessing weak but definitive antibiotic activity
and lack of thrombocyte aggreagtion, suggest that further
modifications of 2 by means of the methods of
carbohydrate chemistry may lead to the development of
additional valuable basic materials of new drugs.
2 was prepared  by the deglycosylation of 1, and 3 was
prepared from the appropriate monomethoxy-amino-PEG
by the reaction with thiophosgene . Specific optical
rotations were measured with a Perkin-Elmer 341
automatic polarimeter. The solutions were evaporated under
diminished pressure at 40°C. For NMR spectroscopy, a
Bruker 2005Y instrument operating at 200MHz and
50.3MHz frequencies, respectively, for the 1H and 13C
nuclei was used. Internal TMS was the reference material.
The UV spectra were obtained with a Jasco W550 UV-VIS
instrument, and the MALDI MS spectra were recorded with
a Bruker BIFLEX III mass spectrometer equipped with a
TOF analyzer. Thrombocyte aggregation was measured
using a Chromo-Ley Dual Channel Platelet Aggregometer,
Model 660. For thin layer chromatography Kieselgel 60
F254(Merck) layer was used and column chromatography
was performed on Silicagel 60 (0.063?0.2mm, Merck)
adsorbent and the following developing systems were
applied: (A) CH2Cl2-MeOH (9:1); (B) toluene-MeOH
(7:3); (C) toluene-MeOH (6:4); (D) toluene-MeOH
(1:1); (E) n-BuOH-AcOH-H2O (4:1:1). The spots were
visualized in UV light or by spraying with the Pauly
reagent. Investigation of the antibacterial activity of the
antibiotics and their derivatives was carried out by
measuring the MIC values as described earlier .
N-Methoxy-PEG-yl-thioureido Aglyco-ristocetin (4)
To a solution of 2 (0.085mmol) in DMSO (2.0ml) 3
(0.085mmol)  was added and the homogeneous
reaction mixture was kept at room temperature for 48
hours. The product 4 was precipitated with dry ether,
filtered off, washed with a small volume of ether and
the crude product (170mg) was purified by column
chromatography (A) to obtain 30.6% of pure 4. TLC: Rf
0.15 (A). UV: lmax282nm (MeOH). MS (MALDI) m/z
2326.2 (M?Na)?. Calcd. for C110H150O43N8S 2302.9.
N-b b-D-Glucopyranosylthioureido Aglyco-ristocetin (6)
To a solution of 2 (0.2mmol) in methanol (3.0ml) 5
(0.22mmol)  was added, the reaction mixture was kept
at room temperature for 20 hours and then concentrated
under diminished pressure. The residue was purified by
column chromatography (B) to obtain the acetylated
product 6a (58.3%). TLC: Rf 0.56 (C). [a]D
(c 0.2, MeOH). UV: lmax279nm (MeOH). MS (MALDI)
m/z 1585.78 (M?Na)?. Calcd. for C75H70O28N8S 1563.4.
The pH of a solution of 6a (0.1mmol) in dry methanol
(3.5ml) was adjusted to ?8 by the addition of 1M sodium
methoxide in methanol and the O-deacetylation  was
monitored by TLC (D) Rf 0.12. The product 6 (63.3%),
precipitated from the reaction mixture (12 hours, 20°C)
after concentration under diminished pressure, was filtered
off and dried. [a]D
279nm (MeOH). MS (MALDI) m/z 1417.8 (M?Na)?.
Calcd. for C67H62O24N8S 1395.3.
25??79.4 (c 0.25, MeOH). UV: lmax
properties of ristocetin A (1) and the N-b-D-maltosyl
derivative (8) with a Chrom-Ley Dual Channel Platelet
Aggregometer Model 660.
Investigation of the thrombocyte aggregation
Curve A: 1.5mg/ml Aggristin (commercial name of ristocetin
A). Curve B: 1.5mg/ml of 8 added to PRP (platelet rich plasma).
1H-NMR spectrum (carbohydrate portion, MeOD) d
ppm: 3.27, 3.40, 3.25, 3.26, 3.33, 3.59 (H6a, H6b).
13C-NMR spectrum (carbohydrate portion, MeOD) d
ppm: 77.7, 77.4, 72.7, 70.1, 77.9, 61.3 (C6).
N-b b-D-Maltosylthioureido Aglyco-ristocetin (8)
The acetylated product 8a (75%) was prepared from 2
(0.06mmol) and 7 (0.066mmol) as described above for the
preparation of 6. [a]D
280nm (MeOH). MS (MALDI) m/z 1873.8 (M?Na)?.
Calcd. for C87H86O36N8S 1851.7.
Zemplén O-deacetylation  of 8a furnished the target
8 (45%). TLC: Rf 0.34 (E). [a]D
UV: lmax280nm (MeOH). MS (MALDI) m/z 1580.1
(M?Na)?. Calcd. for C73H72O29N8S 1557.4.
1H-NMR spectrum (carbohydrate portion, MeOD) d
ppm: 3.85 (A4, A5), 3.88 (A3), 3.99 (A2), 4.02 (B4, B5), 4.12
(B2), 4.23 (B3), 4.17, 4.27 (A6, B6), 5.76 (A1, B1).
13C-NMR spectrum (carbohydrate portion, MeOD) d
ppm: 61.6 (A6, B6), 70.5 (A4, A5), 72.9 (A2), 73.0 (A3), 73.8
(B2), 77.0 (B5), 77.8 (B3), 78.1 (B4), 84.5 (B1), 100.8 (A1).
25??57.3 (c 0.11, MeOH). UV: lmax
25??55.0 (c 0.1, MeOH).
Research Found (Grant No.: OTKA TO46744, TO42512) for
financial support, Dr. Gyula Batta (Research Group of Antibiotics
of the Hungarian Academy of Sciences) and Dr. Sándor Kéki
(Department of Applied Chemistry, University of Debrecen) for
recording the NMR and the mass spectra. We are indebted to the
Gauze Institute of New Antibiotics (Moscow) for the sample of
ristocetin (ristomycin) A.
The authors thank the National Scientific
1.Grundy WE, Sinclair AC, Theriault RJ, Goldstein AW,
Rickler CJ, Warren HB, Oliver TI, Sylvester JC. Ristocetin,
microbiologic properties. Antibiotics Ann 687–692
Sztaricskai F, Harris CM, Neszmélyi A, Harris TM.
Structural studies of ristocetin A (ristomycin A).
Carbohydrate—aglycone linkages. J Am Chem Soc 102:
Neu HC. The crisis in antibiotic resistance. Science 257:
Gangarosa EJ, Johnson TR, Ramos HS. Ristocetin induced
thrombocytopenia: Site and mechanism of action. Arch
Intern Med 105: 83–89 (1960)
Howard MA, Firkin BG. Ristocetin a new tool in the
investigation of platelet aggregation. Thromb Diathesis
Haemorrh 26: 362–369 (1971)
6. Weis HJ, Rogers J, Brand H. Defective ristocetin—induced
platelet and its correction by factor VIII. J Clin Invest 52:
Boda Z, Solum NO, Sztaricskai F, Rák K. Study of platelet
agglutination induced by the antibiotics of the vancomycin
group: Ristocetin (ristomycin), actinoidin and vancomycin.
Thrombos Haemostas 42: 1164–1179 (1980)
Boda Z, Solum NO, Sztaricskai F, Rák K. Actinoidin: A new
inhibitor of ristocetin (and ristomycin)—induced platelet
agglutination. Thrombosis Research 17: 603–610 (1980)
Bardsley B, Williams DH, BaglinTP. Cleavage of rhamnose
from ristocetin A removes its ability to induce platelet
aggregation. Blood Coagulation and Fibrinolysis 9: 241–244
Berndt MC, Ward CM, Booth WJ, Castaldi PA, Mazurow
AV , Andrews RK. Identification of aspartic acid 514 through
glutamic acid 542 as glycoprotein 1b IX. complex receptor
recognition sequence on von Willebrand factor. Mechanism
and modulation of von Willebrand factor by ristocetin and
botrocetin. Biochemistry 31: 11144–11151 (1992)
Hoylaerts MF, Nuyts K, Peerlinck K, Deckmyn H, Vermylen
J. Promotion of binding of von Willebrand factor to platelet
glycoprotein 1b by dimers of ristocetin. Biochem J 306:
Wanner J, Tang D, McComas CC, Crowley BM, Jiang W,
Moss J, Boger DL. A new improved method for
deglycosidation of glycopeptide antibiotics exemplified with
vancomycin, ristocetin and ramoplanin. Bioorg Med Chem
Lett 13: 1169–1173 (2003)
Sztaricskai F, Batta Gy, Herczegh P, Balázs A, Jekö J, Röth
E, Szabó PT, Kardos SZ, Rozgonyi F, Boda Z. A new series
of glycopeptide antibiotics incorporating squaric acid
moiety. Synthesis, structural and antibacterial studies. J
Antibiot 59: 564–582 (2006)
Zemplén G, Gerecs Á, Hadácsi I. Saponification of
acetylated carbohydrates (in German). Chem Ber 69:
Boda Z, Sztaricskai F, Bognár R, Rák K, Zajka G, Daróczy
I. Method for preparation of ristomycin (ristocetin) A in
high quality and its reagent to the study of platelet
agglutination experiments. (in Hungarian) 3020/82
Hungarian Patent (June 28, 1984)
Mongondry P, Bonnans-Plaisance C, Jean M, Tassin JF.
Mild synthesis of amino-poly(ethylene glycol)s. Application
to steric stabilization of clays. Macromolecular Rapid
Commun 24: 1091–1100 (2003)
Hunsen M, Long DA, D’Ardenne CR, Smith AL. Mild one-
pot preparation of glycosyl bromides. Carbohydrate
Research 340: 2670–2674 (2005)
Kühne M, Györgydeák Z, Lindhorst TK. A simple method
for the preparation of glycosyl isothiocyanates. Synthesis 6: