Synthesis of biologically active biotinylated muramyl dipeptides

Article abstract of DOI:10.1016/j.bmcl.2010.08.056

Muramyl dipeptide (MDP) is believed to interact with an innate immune receptor, Nod2. To identify the cellular receptor for MDP, we have synthesized biotinylated MDP isomers and tested the ability of these compounds to activate Nod2 in a cell-based assay. We found that the modification of MDP does not perturb its ability to activate Nod2. These tagged versions of MDP will be useful to identify the cellular receptor of the immunostimulatory molecules.

Full text of DOI:10.1016/j.bmcl.2010.08.056

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6061–6063
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of biologically active biotinylated muramyl dipeptides
Catherine Leimkuhler Grimes a, Daniel K. Podolsky b, Erin K. O’Shea a,c,
*
a Harvard University, Department of Molecular and Cellular Biology and Department of Chemistry and Chemical Biology,
Faculty of Arts and Sciences Center for Systems Biology, 52 Oxford Street, Cambridge, MA 02138, USA
b UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
c Howard Hughes Medical Institute, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2010
Revised 9 August 2010
Accepted 11 August 2010
Available online 17 August 2010
Muramyl dipeptide (MDP) is believed to interact with an innate immune receptor, Nod2. To identify the
cellular receptor for MDP, we have synthesized biotinylated MDP isomers and tested the ability of these
compounds to activate Nod2 in a cell-based assay. We found that the modification of MDP does not per-
turb its ability to activate Nod2. These tagged versions of MDP will be useful to identify the cellular recep-
tor of the immunostimulatory molecules.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Muramyl dipeptide
Nod2
Innate immunity
Affinity reagent
The innate immune system is important for detecting and
responding to microbial invasion.1–3 The primary innate immune
sensors, pattern recognition receptors (PRRs), are activated by
pathogen associated molecular patterns (PAMPs), triggering a com-
plex cascade of events known as the inflammatory response.4 The
Nod-like receptors (NLR) are a family of PRRs that reside in the
cytosol5 and are proposed to use their leucine rich region (LRR)
to sense the presence of a pathogen.6 However, no evidence for a
direct interaction between NLRs and PAMPs has been reported.7
Nod2 is a member of the NLR family that is expressed in mono-
cytes, granulocytes, dendritic, and intestinal epithelial cells.8,9
Nod2 activation by PAMPs results in the production of immunostim-
ulatory molecules and anti-microbial peptides through activation of
Figure 1. Muramyl dipeptide isomers: 1 is the proposed ligand for Nod2. The
isomer, 2, does not activate Nod2.
the NF-jB and the mitogen activated (MAP)-kinase (p38) path-
ways.10,11 The proposed ligand (or PAMP) for Nod2 is a small piece
of bacterial cell wall that has long been known to have immunostim-
ulatory properties, muramyl dipeptide (MDP) (1, Fig. 1).12–14 A direct
interaction between MDP and Nod2 has not been shown, so it re-
mains unclear if MDP is sensed directly by Nod2 or by a different
protein.
for interaction, we reasoned that a modification would best be made
at the six-position of the carbohydrate, distant from the potential
binding site (12, Scheme 1). In addition, we desired a version of
MDP that could be easily modified with crosslinkers, biotin tags
and/or fluorophores. We therefore reasoned that an amino group
on the six-position of MDP would be a good chemical handle for
our purposes.
Sansonetti and co-workers have shown that MDP activates the
Nod2 pathway in a stereo-specific manner.15 Replacement of
L-isog-
lutamate for
D-isoglutamate, the natural stereoisomer found in
MDP, eliminated the ability of MDP to stimulate Nod2 (2, Fig. 1). This
finding suggests that the peptide portion of MDP is important for
recognition. To keep the recognition region of the molecule available
C6-Amino versions of both MDP-L-D (12, Scheme 1) and MDP-L-L
(13, Scheme 1) were synthesized from protected N-acetyl-muramic
acid (3, Scheme 1) in six steps, using slight modifications from a
previously reported synthesis.16,17 Our synthesis differed from
the previously published protocol in two ways: (1) the peptide
coupling was accomplished using the mixed anhydride method;
* Corresponding author. Tel.: +1 617 495 4328; fax: +1 617 496 5425.
E-mail address: erin_oshea@harvard.edu (E.K. O’Shea).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.056

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C. L. Grimes et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6061–6063
Scheme 1. Synthetic route of 6-amino MDP isomers: 12 and 13. Reagents and conditions: (a) IRA H+ Resin, MeOH, quantitative; (b) p-toluene sulfonic acid, methyl sulfonyl
chloride, pyridine, 92%; (c) sodium azide, DMF, 87%; (d) potassium hydroxide; (e) (i) N-methylmorpholine, isobutyl chloroformate, DMF; (ii) 8 or 9, 63%; (f) H2, Pd–C, MeOH/
AcOH 82%.
and (2) the azido coupling was accomplished without prior protec-
tion of the 4-hydroxyl group. We found the protecting group on the
4-OH difficult to remove and the peptide coupling was achieved
with minimal internal ester formation. Briefly, treatment with
IRA H+ resin in methanol afforded 4 in which the benzylidene
group was deprotected and the methyl protecting group installed.
The azido group was then installed via mesylation on the six-posi-
tion of the carbohydrate in 90% yield over two steps. Following
deprotection of the carboxylate, the appropriately protected dipep-
tide (8 or 9) was coupled using standard peptide coupling condi-
tions18 to yield the protected 6-amino muramyl dipeptide, 10 or
11 in 60% yield. Global deprotection using H2, Pd–C yielded 12 or
13 in 41% overall yield.
ity to activate Nod2 in an established NF-
(Fig. 2). Briefly, HEK-293 cells were co-transfected with Nod2 and
an NF- B driven firefly luciferase reporter. In the presence of its li-
gand, Nod2, will activate NF- B which in turn stimulates expres-
sion of the luciferase reporter. As an internal control for
transfection efficiency, all cells were transfected with Renilla
luciferase.
j
B luciferase assay9,13
j
j
Figure 2a indicates that all
Nod2, suggesting that the modification at the six-position did not
interfere with the activity of MDP. In addition, none of the iso-
L-D isomers were able to activate
L-L
mers were active (Fig. 2a), indicating that the biotinylation of
muramyl dipeptide does not afford any additional, non-specific
biological activity. Furthermore, the ability of the modified MDPs
Compound 12 was reacted with a variety of succinimide-mod-
to trigger NF-jB in a robust manner was dependent on Nod2—
ified biotin groups to yield the biotin-6-amino-MDP-
L
-D
derivatives
when Nod2 was not transfected into HEK-293 cells, only modest
(14, 16, and 18, Table 1). In addition, the biotin-6-amino-MDP-L-L
activity was observed (Fig. 2b). However, it should be noted that
series (15, 17, and 19, Table 1) was synthesized. 6-Amino muramyl
dipeptide derivatives containing biotin linkers of various lengths
(20, 32, and 56 Å) and one cleavable linker (14 and 15, Table 1)
were synthesized. Table 1 represents a set of biotinylated MDP
the L-D isomers also effect low level activation of NF-jB in a Nod
2 independent fashion, given the activation in HEK-293 control
cells which lack Nod2.9 The biotinylated MDPs could be useful in
identifying such off target proteins.
compounds to test the activation of Nod2. The
an important control, as they should not activate the Nod2
pathway.
To determine if the modification at the six-position of MDP
would alter Nod2 dependent NF-jB activation, the biotin MDP
L
-
L
derivatives are
Of all the linkers tested, compounds 14 and 16 remained the
most active (Fig. 2a). The long 56 Å linker, 18, only had minimal
activity (Fig. 2a). We reasoned that the addition of the longer linker
may cause the molecule to aggregate in cell culture medium or
that the linker may fold in a conformation that prevents the MDP
from binding to its receptor. Thus, 14 and 16 represent the MDP
derivatives and the 6-amino derivatives were tested for their abil-
Table 1
MDP probes
Compound
Stereochemistry
Biotin linker
O
14
15
L
L
-
-
D
L
O
HN
O
S S
NH
N
H
S
O
16
17
L
L
-
-
D
L
O
HN
O
O
NH
NH
O
S
S
4
O
18
19
L
L
-
-
D
L
O
HN
NH
NH
12
O

C. L. Grimes et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6061–6063
6063
Figure 2. Nod2 dependent response to MDP: (a) Nod2 DNA was added to the transfection mixture; (b) empty vector in place of Nod2 DNA was added to the transfection
mixture.
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derivatives that retained the most biological activity; their respec-
tive isomers, 15 and 17, were not active. We anticipate that these
reagents will be useful tools in studies to identify the receptor for
MDP, be it Nod2 or another protein.
Nod2 plays an important role in detecting the presence of bac-
teria. Additionally, mutations in Nod2 have been associated with
two chronic inflammatory disorders: Blau syndrome and Crohn’s
disease.19–22 Our synthesis of a modifiable MDP provides a tool
for use in studies to better understand the molecular mechanisms
by which bacteria cause inflammation. We are currently using 14–
17 to determine if MDP interacts directly with Nod2 or other pro-
teins and to gain knowledge at the molecular level of the initial sig-
naling events involved in Nod2 activation.
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Acknowledgments
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C.L.G. was supported by a postdoctoral fellowship from the
Irvington Institute Fellowship Program of the Cancer Research
Institute. E.K.O. is an Investigator of the Howard Hughes Medical
Institute. D.K.P. was supported by NIH Grant (5R01DK069344).
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Supplementary data
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H.; Moran, T.; Karaliuskas, R.; Duerr, R. H.; Achkar, J. P.; Brant, S. R.; Bayless, T.
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.056.
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