New conjugates of muramyl dipeptide and nor-muramyl dipeptide linked to tuftsin and retro-tuftsin derivatives significantly influence their biological activity

Article abstract of DOI:10.1016/s1734-1140(12)70749-1

The synthesis and biological activity of new conjugates of muramyl dipeptide (MDP) and nor-muramyl dipeptide (nor-MDP) with tuftsin and retro-tuftsin derivatives containing isopeptide bond between e-amino group of lysine and carboxyl group of simple amino

Full text of DOI:10.1016/s1734-1140(12)70749-1

Short communication
New conjugates of muramyl dipeptide
and nor-muramyl dipeptide linked to tuftsin
and retro-tuftsin derivatives significantly
influence their biological activity
1
2
2
Krystyna Dzierzbicka , Anna Wardowska , Ma³gorzata Rogalska ,
2
Piotr Trzonkowski
Department of Organic Chemistry, Gdansk University of Technology, G. Narutowicza 11/12, PL 80-233 Gdañsk,
Poland
Department of Clinical Immunology and Transplantology, Medical University of Gdansk, Dêbinki 7,
PL 80-210 Gdañsk, Poland
Correspondence: Krystyna Dzierzbicka, e-mail: kd@chem.pg.gda.pl
Abstract:
The synthesis and biological activity of new conjugates of muramyl dipeptide (MDP) and nor-muramyl dipeptide (nor-MDP) with
tuftsin and retro-tuftsin derivatives containing isopeptide bond between e-amino group of lysine and carboxyl group of simple amino
acids such as Ala, Gly and Val are presented. We presumed, based on the cytokine profile, that the examined conjugates of tuftsin and
MDPwere capable of activating antibacterial mechanisms by switching on Th1 immune response. The most active were compounds
11, 14 and 19–23.
Key words:
muramyl dipeptide, nor-muramyl dipeptide, tuftsin derivatives, synthesis, immunomodulator
Introduction
20]. We have previously reported the synthesis of the
conjugates of MDP and nor-muramyl dipeptide (nor-
MDP) with tuftsin [2, 3, 6, 18, 19]. The most propi-
tious compounds were then further examined in vivo.
The elaborated animal model of experimentally in-
duced sepsis allowed us to screen them reliably as po-
tential therapeutic agents [18]. Importantly, tested al-
ready compounds were found as efficient stimulators
of innate immunity, which resulted in slowing down
Muramyl dipeptide (MurNAc-L-Ala-D-isoGln; MDP)
1 (Fig. 1), a component of peptidoglycan, stimulates
various functions of macrophages and increases non-
specific resistance of the host against numerous mi-
croorganisms [1, 4, 7, 15–17]. Another immunomodu-
lator, tuftsin, is a natural tetrapeptide, H-Thr-Lys-
Pro-Arg-OH (TKPR) 2, present in the peripheral of experimental sepsis. Hence, the examined already
blood of humans and other mammals, where it stimu- conjugates are reckoned rather as adjuvants than sepa-
lates monocytes, macrophages, and neutrophils [14, rate therapeutic agents [18].
217

Fig. 1. Structure of muramyl dipeptide (MDP) 1 and tuftsin (TKPR) 2
Scheme 1. Synthesis of MDP or nor-MDP with tuftsin 10–18 and retro-tuftsin 19–23
218

Conjugates of MDP and nor-MDP
In this study, we present the synthesis of new set of out on a MALDI MS (a Biflex III MALDI-TOF spec-
the conjugates 10–23 (Scheme 1) together with the trometer, Bruker Daltonics, Germany).
screening for their immunomodulatory activity. Novel
conjugates contain MDP or nor-MDP with tuftsin and
General procedure for the preparation
of compounds 10–23
retro-tuftsin derivatives with isopeptide bond. Intro-
duction of the additional residue at e-amino group of
lysine by -NHCO- formation made the isopeptide
bond stronger than peptide bond in central chain. This
modification increased chemical resistance and activ-
ity of the conjugates as compared to tuftsin [9–11,
13]. We hope that it will also increase the half-time of
the compounds and their bioavailability, which need
to be verified in structure-activity relationship studies.
To a stirred solution of MDP 3a or nor-MDP 3b de-
rivatives (0.1108 mmol) in anhydrous DMF (1 ml)
cooled to –15°C, NMM (0.1108 mmol) and isobutyl
chloroformate (0.1108 mmol) were added, followed
after 15 min by the addition of cooled solution of Thr-
Lys(Z-Y)-Pro-Arg(NO )-OMe trifluoroacetate 6a,b or
Arg(NO )-Pro-Lys(Z-Y)-Thr-OMe trifluoroacetate 7a–c
(0.1293 mmol) and NMM (0.1293 mmol) in anhy-
drous DMF (0.5 ml) and the stirring was continued
for 4 h at –15°C and then for 24 h at room tempera-
ture. After evaporation of the solvent, the reaction
mixture was purified using radial chromatography
and preparative TLC in solvent CHCl -MeOH (4:1,
v/v) or CHCl -MeOH (9:1, v/v) to obtain compounds
8a–i and 9a–e. The final products 10–23 were hydro-
genolyzed in 50% methanolic acetic acid containing
palladium black, purified with preparative TLC in
solvent n-BuOH-AcOH-H O (4:2:2, v/v/v), and ly-
ophilized. Below we present analytical characteristics
of the examined compounds:
Materials and Methods
1-Benzyl-MDP 3a and 1-benzyl-nor-MDP 3b de-
scribed in previous papers [5, 8, 12] were used for the
synthesis of conjugates with tuftsin derivatives (H-
Thr-Lys(Y)-Pro-Arg-OMe, Y = Ala, Val) and retro-
tuftsin derivatives (H-Arg-Pro-Lys(Y)-Thr-OMe, Y =
Ala, Gly, Val) (Scheme 1). Acylation of the Thr or
Arg amino group of partially protected pentapeptides
by MDP or nor-MDP was performed using the mixed
anhydride method with isobutyl chloroformate and
N-methylmorpholine (NMM) in dry dimethylforma-
mide (DMF). The protected conjugates were isolated
and purified by preparative TLC. The protected pen-
tapeptides (Boc-Thr-Lys(Z-Y)-Pro-Arg(NO )-OMe,
Y = Ala, Val) 4a,b or (Boc-Arg(NO )-Pro-Lys(Z-Y)-
Thr-OMe, Y = Ala, Gly, Val) 5a-c were synthesized
by the conventional chemical procedure also using
mixed anhydride method, isolated by column chroma-
tography and purified with preparative TLC on silica
Nor-Mur(NAc)-Ala-D-Glu(Thr-Lys(Ala)-Pro-Arg-Ome)-
NH 10. Yield 42%; amino acid analysis (6 M, 110°C,
20 h): Ala, Arg, Glu, Lys, Pro, Thr; MALDI-TOF calcd.
for C H N O , 1046.1, found 1046.9 (M+H) .
Nor-Mur(NAc)-Pro-D-Glu(Arg-Pro-Lys(Ala)-Thr-OMe)-
NH 19. Yield 30%; amino acid analysis (6 M, 110°C,
20 h): Arg, Glu, Lys, Pro, Thr, Val; MALDI-TOF calcd.
for C H N O , 1072.2, found 1073.1 (M+H) .
General procedure for viability tests
gel. Finally, the Boc group was removed from the Peripheral blood mononuclear cells (PBMC) were ob-
peptides by the treatment with trifluoroacetic acid tained from buffy coats by standard ficol gradient
(TFA) to transform them to trifluoroacetates 6a,b and centrifugation and separated to peripheral blood leu-
7a–c, which were subsequently used for the synthesis kocytes (PBL) and monocytes. All subsets were then
of conjugates with MDP or nor-MDP. The final prod- disposed (4 × 10 cells per well) in RPMI 1640 me-
ucts 8a–i and 9a–e were hydrogenolyzed in 50% dium containing 10% fetal calf serum and incubated
methanolic acetic acid containing palladium black, for 24 h in an atmosphere of 5% CO at 37°C in tripli-
purified with preparative TLC and lyophilized to ob- cates with the following final concentration of the ex-
tain hygroscopic solids. Qualitative amino acid analy- amined conjugates: 0 (control), 0.0001, 0.001, 0.01, and
ses of the hydrolyzates of the compounds were per- 0.1 mg/ml. The readout of the test was performed with
formed by TLC. Detection by: UV and ninhydrin. The colorimetric MTT assay. After incubation, 3-(4,5-
purity of the conjugates was confirmed with H-NMR dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bro-
and MS. The mass spectrometry analysis was carried mide (MTT) was added to the wells in the final con-
219

160
140
120
100
80
Fig. 2. The viability tests – the effect of
the chosen conjugates is presented.
Results of MTT assay are presented as
the percentage of the viability obtained
in control cultures (without tested com-
pounds, control culture = 100%). A – Vi-
ability of PBMC. Conjugate ‘19’ is cir-
cled as it was significantly different from
native compounds. B – Viability of PBL.
The circled conjugates are those sig-
nificantly different from native com-
pounds. C – Viability of monocytes. As-
terisk (*) marks significant difference
between the conjugate and tuftsin and
the cross (#) marks significant differ-
ence between the conjugate and MDP.
The results throughout the figure are
presented as medians (rectangles in-
side the boxes), 25–75% percentiles
(the boxes) and minimum-maximum
(error bars outside the boxes)
A
*, #
0.1 mg/ml
0.01 mg/ml
0.001 mg/ml
0.0001 mg/ml
,#
*
140
120
100
80
,#
*
B
0.1 mg/ml
0.01 mg/ml
0.001 mg/ml
0.0001 mg/ml
60
40
140
120
100
80
C
60
0.1 mg/ml
0.01 mg/ml
0.001 mg/ml
0.0001 mg/ml
40
220

Conjugates of MDP and nor-MDP
centration of 1 mg/ml and the plates were incubated immune cells influenced the results. These activities
for additional 4 h. Then, the reaction was stopped by were then further confirmed with cytokine release
addition of 100 μl of isopropanol. Optical density was assay [18]. We have assesed the influence of the syn-
read at 570 nm on the automated plate reader (FL600, thesized compounds on the viability of white blood
Bio-Tec, Japan). The results were then adjusted to cells: either heterogeneous population of PBMC or PBL
control cultures (cultures with 0 mg/ml of the conju- and monocytes. In the next step, we analyzed the cyto-
gates), where the viability of control was treated as kine profile secreted by immune cells when stimulated
100%.
with the examined compounds. Three cytokines, two
proinflammatory (IL-6, TNFa), as well as anti-
inflammatory (IL-10), were detected.
Cytokine profile
Viability tests
Separate set of the cultures of PBMC, PBL and mono-
cytes was performed in order to assess conjugate-
stimulated secretion of the cytokines in culture super-
natants. The concentration of the examined com-
poundes used to stimulate blood cells was 0.01 mg/
ml. At the end of the culture, 50 μl of the supernatant
from each culture was collected and TNFa, IL-6 and
IL-10 were measured using FlexSet kits (BDBiosci-
ence, Poland) according to the manufacturer’s instruc-
tions.
PBMC – the viability of those cells was higher after
incubation with the following conjugates: 19–21 and
22 (Fig. 2A). PBL – isolated leukocytes were the most
viable in the presence of the above mentioned com-
pounds (19–22) as well as conjugates 11 and 15
(Fig. 2B). Monocytes – only conjugates 14, 21 and 23
had promising impact on the viability of these phago-
cytes (Fig. 2C).
Statistical analysis
Median
A
25%-75%
Min-max
60
40
20
0
The data were evaluated by means of Mann-Whitney
U test using Statistica 8.1 (StatSoft, Poland). The
p value < 0.05 was considered statistically signifi-
cant. All the results obtained for the examined com-
pounds were compared to: control group (unstimu-
alted cells) and to native immunomodulators – MDP
and tuftsin, in order to estimate whether the new com-
pound surpass their activity.
control
tuftsin
MDP
16
23
2000
1600
1200
800
400
0
Median
B
25%-75%
Min-Max
Results and Discussion
The synthesized conjugates were tested for their bio-
logical activity. Viability test is a basic assay for the
assessment of immunomodulatory activity of any
compound. It reveals biologic activity of the com-
pounds and identifies those potentially toxic. Using
such a biological assay, we verified that some of the
analyzed chemical bonds between MDP and tuftsin
changed the activity of the conjugates towards immu-
nosuppression, while others resulted in immunostimu-
latory activity of the compounds. Apart from the na-
ture of the chemical bond, also the concentration of
particular conjugates and the subset of the examined
Fig. 3. The cytokine profile – selected results are presented. Results
of Flexset assay are presented for reference cultures (control, tuftsin
and muramyl dipeptide – MDP) and cultures with detected levels of
cytokines. A – Secretion of TNF . B – Secretion of IL-6. The results
throughout the figure are presented as medians (rectangles inside
the boxes), 25–75% percentiles (the boxes) and minimum-maximum
(error bars outside the boxes)
221

4. Dzierzbicka K, Ko³odziejczyk AM: Muramyl peptides –
synthesis and biological activity. Pol J Chem, 2003, 77,
373–395.
5. Dzierzbicka K, Ko³odziejczyk AM: Synthesis and antitu-
mor activity of conjugates of muramyldipeptide or nor-
muramyldipeptide with hydroxyacridine/acridone deriva-
tives. J Med Chem, 2003, 46, 183–189.
6. Dzierzbicka K, Trzonkowski P, Sewerynek P,
Ko³odziejczyk AM, Myœliwski A: Synthesis and biologi-
cal activity of tuftsin, its analogue and conjugates con-
taining muramyl dipeptides or nor-muramyl dipeptides.
J Pept Sci, 2005, 11, 123–135.
Cytokine profile
The examined conjugates had little impact on TNFa
production by PBMC, only compounds 16 and 23
slightly induced the secretion of this cytokine (Fig. 3A).
The other analyzed proinflammatory cytokine – IL-6
– was secreted in the presence of several conjugates:
11, 14 and 22, and the most efficient were 16 and 23
(Fig. 3B). The examined conjugates merely influ-
enced production of IL-10.
In conclusion, we described the synthesis and pre-
liminary biological activity of the new conjugates
MDP-containing tuftsin derivatives 10–23 (Scheme 1).
We have revealed that the majority of the conjugates
decreased the viability of the cells in PBMC cultures.
This effect seemed to be time- and dose-dependent.
Since PBMC are a heterogeneous population, it was
decided to perform the viability tests using separated
PBL and monocytes. These tests confirmed the inhibi-
tory effect of the examined compounds on the viabil-
ity of monocytes. The most efficient compounds were
11, 14 and 19–23.
The above mentioned analysis of new immuno-
modulating molecules is extremely important in
a continuing pursuit for potent antibacterial com-
pounds useful in the treatment of severe bacterial in-
fections. At this point, it is difficult to give definitive
statement on their usefulness or superiority over pre-
viously described conjugates. While preliminary in
vitro tests with presented conjugates are promising, it
is necessary to further verify their activity in vivo.
7. Dzierzbicka K, Wardowska A, Trzonkowski P:
Recent developments in the synthesis and biological
activity of muramylpeptides. Curr Med Chem, 2011, 18,
2438–2451.
8. Flowers HM, Jeanloz RW: The synthesis of 2-acetamido-
3-O-(D-1-carboxy-ethyl)-2-deoxy-a-D-glucose
(N-acetyl-muramic acid) and of benzyl glycoside deriva-
tives of 2-amino-3-O-(D-1-carboxy-ethyl)-2-deoxy-D-
-glucose (muramic acid). J Org Chem, 1963, 28,
2983–2986.
9. Granoth R, Vadai E, Burstein Y, Fridkin M, Tzehoval E:
Tuftsin-THF-g2 chimeric peptides: potential novel
immunomodulators. Immunopharmacology, 1997, 37,
43–52.
10. Khan MA, Nasti TH, Saima K, Mallick AI, Firoz A,
Wajahul H, Ahmad N, Mohammad O: Co-administration
of immunomodulator tuftsin and liposomised nystatin
can combat less susceptible Candida albicans infection
in temporarily neutropenic mice. FEMS Immunol Med
Microbiol, 2004, 41, 249–258.
11. Lang O, Mezo G, Hudecz F, Kohidai L: Efeect of tuftsin
and oligotufrsins on chemotaxis and chemotactic selec-
tion in Tetrahymena pyriformis. Cell Biol Int, 2006, 30,
603–609.
12. Lefrancier P, Choay J, Derrien M, Lederman I:
Synthesis of N-acetylmuramyl-L-alanyl-D-isoglutamine,
an adjuvant of the immune response and some N-acetyl-
muramyl peptide analogs. Int J Peptide Protein Res,
1977, 9, 249–257.
13. Mezo G, Szekerke M, Sarmay G, Gergely J: Synthesis
and functional-studies of tuftsin analogs containing
isopeptide bond. Peptides, 1990, 11, 405–415.
14. Najjar VA, Nishioka K: Tuftsin: a natural phagocytosis
stimulating peptide. Nature, 1970, 228, 672–673.
15. Nardin A, Lefebvre ML, Labroquere K, Faure O,
Abastado JP: Liposomal muramyl tripeptide phosphati-
dylethanolamine: Targeting and activating macrophages
for adjuvant treatment of osteosarcoma. Curr Cancer
Drug Targets, 2006, 6 123–133.
Acknowledgment:
This work was supported by the Polish State Committee
for Scientific Research (Grant No. N N405 181135).
References:
1. Azuma I, Seya T: Development of immunoadjuvants for
immunotherapy of cancer. Int Immunopharmacol, 2001,
1, 1249–1259.
2. Dzierzbicka K: Synthesis of conjugates of muramyl
dipeptide and nor-muramyl dipeptide with retro-tuftsin
(Arg-Pro-Lys-ThrOMe) as potential immunostimulants.
Pol J Chem, 2004, 78, 409–416.
3. Dzierzbicka K: Synthesis of new conjugates of
MDP and nor-MDP with retro-tuftsin derivatives as
potential immunomodulators. Pol J Chem, 2008, 82,
1431–1439.
16. Takada H, Yokoyama S, Yang SJ: Enhancement of endo-
toxin activity by muramyldipeptide. Endotoxin Res,
2002, 8, 337–342.
17. Traub S, von Aulock S, Hartung T, Hermann C: MDP
and other muropeptides – direct and synergistic effects on
the immune system. J Endotoxin Res, 2006, 12, 69–85.
18. Wardowska A, Dzierzbicka K, Myœliwski A: Tuftsin –
new analogues and properties (Polish). Post Biochem,
2007, 53, 60–65.
222

Conjugates of MDP and nor-MDP
19. Wardowska A, Dzierzbicka K, Szaryñska M,
D¹browska-Szponar M, Wiœniewska K, Myœliwski A,
Trzonkowski P: Analogues of muramyl dipeptide
(MDP) and tuftsin limit infection and inflammation in
murine model of sepsis. Vaccine, 2009, 27, 369–374.
20. Wardowska A, Dzierzbicka K, Trzonkowski P,
Myœliwski A: Immunomodulatory properties of new
conjugates of muramyl dipeptide and nor-muramyl
dipeptide with retro-tuftsin (Arg-Pro-Lys-Thr-OMe).
Int Immunopharmacol, 2006, 6, 1560–1568.
Received: June 2, 2011; in the revised form: September 19,
2011; accepted: October 20, 2011.
223