Amphiphilic desmuramyl peptides for the rational design of new vaccine adjuvants: Synthesis, in vitro modulation of inflammatory response and molecular docking studies

Article abstract of DOI:10.1016/j.ejmech.2020.112863

Nucleotide-binding oligomerization domain 2 (NOD2) is cytosolic surveillance receptor of the innate immune system capable of recognizing the bacterial and viral infections. Muramyl dipeptide (MDP) is the minimal immunoreactive unit of murein. NOD2 perceives MDP as pathogen-associated molecular pattern, thereby triggering an immune response with undesirable side-effects. Beneficial properties of MDP, such as pro-inflammatory characteristics for the rational design of new vaccine adjuvants, can be harnessed by strategically re-designing the molecule. In this work, a new class of amphiphilic desmuramylpeptides (DMPs) were synthesized by replacing the carbohydrate moiety (muramic acid) of the parent molecule with hydrophilic arenes. A lipophilic chain was also introduced at the C-terminus of dipeptide moiety (alanine-isoglutamine), while conserving its L-D configuration. These novel DMPs were found to set off the release of higher levels of tumour necrosis factor alpha (TNF-α) than Murabutide, which is a well-known NOD2 agonist. Molecular docking studies indicate that all these DMPs bind well to NOD2 receptor with similar dock scores (binding energy) through a number of hydrogen bonding and hydrophobic/π interactions with several crucial residues of the receptor. More studies are needed to further assess their immunomodulatory therapeutic potential, as well as the possible involvement of NOD2 activation.

Full text of DOI:10.1016/j.ejmech.2020.112863

Journal Pre-proof
Amphiphilic Desmuramyl Peptides for the Rational Design of New Vaccine Adjuvants:
Synthesis, In Vitro Modulation of Inflammatory Response and Molecular Docking
Studies
Farooq-Ahmad Khan, Nourina Nasim, Yan Wang, Alaa Alhazmi, Mehar Sanam,
Zaheer Ul-Haq, Damayanthi Yalamati, Marina Ulanova, Zi-Hua Jiang
PII:
S0223-5234(20)30835-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112863
EJMECH 112863
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 June 2020
Revised Date: 14 September 2020
Accepted Date: 16 September 2020
Please cite this article as: F.-A. Khan, N. Nasim, Y. Wang, A. Alhazmi, M. Sanam, Z. Ul-Haq, D.
Yalamati, M. Ulanova, Z.-H. Jiang, Amphiphilic Desmuramyl Peptides for the Rational Design of New
Vaccine Adjuvants: Synthesis, In Vitro Modulation of Inflammatory Response and Molecular Docking
Studies, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112863.
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2020 Elsevier Masson SAS. All rights reserved.

Amphiphilic Desmuramyl Peptides for the Rational Design of New
Vaccine Adjuvants: Synthesis, In Vitro Modulation of Inflammatory
Response and Molecular Docking Studies
H N
O
2
H
N
HO
n N
R
O
HN
H
N
O
O
H
O
OH
OH
H
NH
O
H2N
HN
O
12 steps
JH-H = 15Hz
O
O
Hydrophilicity Glycolic
Modulation
HO
OH
Pharmacophore
Desmuramyl peptides (DMPs)
Lipophilic Chain
Linker
H
N
O
O
O
R
H
(CH2)nOH
CH3CO
CH3(CH2)2CO
n = 2
n = 3
1
5
2
6
3
7
4
8
Muramyl dipeptide (MDP)

1
2
Amphiphilic Desmuramyl Peptides for the Rational Design of New
Vaccine Adjuvants: Synthesis, In Vitro Modulation of Inflammatory
Response and Molecular Docking Studies
3
4
5
a, f
*
a
a
b,c
a
d
Farooq-Ahmad Khan , Nourina Nasim , Yan Wang , Alaa Alhazmi , Mehar Sanam , Zaheer Ul-Haq ,
e
b
f
*
6
Damayanthi Yalamati , Marina Ulanova , Zi-Hua Jiang
7
8
9
a
H.E.J. Research Institute of Chemistry, International Center for Chemical & Biological Sciences, University of
Karachi-75270, Pakistan.
b
1
1
0
1
Medical Sciences Division, Northern Ontario School of Medicine, Lakehead University, 955 Oliver Road, Thunder
Bay, Ontario, P7B 5E1, Canada.
c
12
Medical Laboratory Technology Department, Jazan University, Jazan, Saudi Arabia.
d
1
1
3
4
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan.
e
f
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16
17
Alberta Research Chemicals Inc., 11421 Saskatchewan Drive, Edmonton, Alberta, T6G 2M9, Canada.
Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, P7B 5E1, Canada.
18
H2N
HN
O
H
N
HO
n N
R
O
H
N
O
O
H
O
OH
OH
H
NH
O
H2N
HN
O
12 steps
JH-H = 15Hz
O
O
Hydrophilicity Glycolic
Modulation
HO
OH
Pharmacophore
Desmuramyl peptides (DMPs)
Lipophilic Chain
Linker
H
N
O
O
O
R
H
(CH2)nOH
CH3CO
CH3(CH2)2CO
n = 2
n = 3
1
5
2
6
3
7
4
8
Muramyl dipeptide (MDP)
1
9
0
2
2
1
2
---------------------------------
* Corresponding authors
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2
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4
Tel: +92 111 222 292; fax: +92 21 348 19018-9; email: farooq.khan@iccs.edu
Tel: +1 807 766 7171; fax: +1 807 346 7775; email: zjiang@lakeheadu.ca
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Highlights
1. New type of desmuramyl peptides having hydrophilic aryl scaffolds were synthesized.
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30
2. Their preparation involves an efficient 12 step synthesis strategy.
3. They can effectively modulate the inflammatory response of THP-1 cells.
4. High levels of TNF-α – a major proinflammatory cytokine – were released.
5. Molecular docking studies indicate strong binding to NOD2 receptor.
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1
2
3
Key Words
Desmuramyl peptides, ICAM-1, Immunomodulatory agents, TNF-α, NOD2 ligands.
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Abstract
Nucleotide-binding oligomerization domain 2 (NOD2) is cytosolic surveillance receptor
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53
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of the innate immune system capable of recognizing the bacterial and viral infections. Muramyl
dipeptide (MDP) is the minimal immunoreactive unit of murein. NOD2 perceives MDP as
pathogen-associated molecular pattern, thereby triggering an immune response with undesirable
side-effects. Beneficial properties of MDP, such as pro-inflammatory characteristics for the
rational design of new vaccine adjuvants, can be harnessed by strategically re-designing the
molecule. In this work, a new class of amphiphilic desmuramylpeptides (DMPs) were
synthesized by replacing the carbohydrate moiety (muramic acid) of the parent molecule with
hydrophilic arenes. A lipophilic chain was also introduced at the C-terminus of dipeptide moiety
(alanine-isoglutamine), while conserving its L-D configuration. These novel DMPs were found
to set off the release of higher levels of tumour necrosis factor alpha (TNF-α) than Murabutide,
which is a well-known NOD2 agonist. Molecular docking studies indicate that all these DMPs
bind well to NOD2 receptor with similar dock scores (binding energy) through a number of
hydrogen bonding and hydrophobic/π interactions with several crucial residues of the receptor.
More studies are needed to further assess their immunomodulatory therapeutic potential, as well
as the possible involvement of NOD2 activation.
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1. Introduction
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The innate immune system is the first-line of defense against pathogenic infections [1],
which involves the evolutionary defense strategy via sentinel cells [2]. These cells are equipped
with numerous pattern recognition receptors (PRRs), which provide surveillance by sensing the
pathogen-associated molecular patterns (PAMPs) [3]. An anti-pathogen signaling cascade is
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triggered upon PAMP detection, thereby alerting the immune system for an inflammatory
response [4]. Bacterial peptidoglycan (PGN) and viral nucleic acids are common PAMP
examples. Muramyl dipeptide (MDP) is the smallest PGN fragment recognized by our immune
system via an intracellular NOD2 type PRR [5]. When MDP actuates NOD2, a downstream
signaling pathway activates the NF-κB transcriptional factor that results in the release of pro-
inflammatory cytokines, upregulation of adhesion molecules and nitric oxide (NO) secretion
[6,7].
Muramyl dipeptide (MDP, Figure 1) was first discovered in the laboratory of E. Lederer at
Université Paris-Sud in France [8]. It is composed of a dipeptide (L-alanine-D-isoglutamine)
attached to a carbohydrate moiety (N-acetyl glucosamine) via lactic acid linker [9]. MDP is a
potent immunostimulant due to its ability to induce the production of various pro-inflammatory
cytokines [10]. Previous studies have shown that pre-exposure to MDP enhances the immune
response to a later challenge [11]. Nevertheless, this molecule induces many undesirable
pharmacological effects too, such as: endotoxic sensitization, induction of arthritis, bone
resorption, transitory leukopenia and pyrogenicity [12]. Furthermore, poor cell membrane
penetration and rapid elimination limit MDP application in clinical settings [13]. Interestingly,
minor modification in the basic structure of MDP often leads to the separation of desirable
biological activities from unwanted side effects [14]. Therefore, numerous structural variations
of MDP have been performed to provide new chemical entities with improved therapeutic
potential, which can stimulate the immune defense against infectious pathogens and express
immunomodulatory activity without harmful side effects [11].
Many hydrophilic MDP derivatives have therefore been synthesized, leading to the
discovery of several useful molecules, such as Murabutide and Muramethide (Figure 1).
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Murabutide contains an aliphatic chain at the peptide end of hydrophilic MDP. This molecule
was found to be non-pyrogenic and safe immunomodulator. It was also able to enhance the non-
specific resistance against microbial infections without toxicity [15]. Besides, Nor-MDP and
Temurtide also reached the clinical development stages. They lack pyrogenicity and interact
safely with the immune system [16].
8
8
9
8
Figure 1. Hydrosoluble MDP derivatives
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Many lipophilic MDP analogs have also been developed by the pharmaceutical industry
(Figure 2), such as Romurtide, Mifamurtide, and MTP-Cholesterol [17,18]. Interestingly,
biological effects of some lipophilic derivatives extend far beyond their immunomodulatory
properties. For example, MTP-PE (Mifamurtide) originally developed by Novartis, is now being
used for osteosarcoma treatment [19].
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9
5
6
9
Figure 2. Lipophilic MDP derivatives developed by the pharmaceutical industry
In clinical settings, MTP-PE was shown to have immunomodulatory adjuvant properties in
humans [20]. Its prophylactic antiviral activity was explored against Influenza [21], HIV [22,23]
and Herpes Simplex [24] antigens. Other MDP derivatives, including the hydrosoluble ones,
were also investigated as vaccine adjuvant for the induction of humoral and cellular immune
response. Table 1 lists the selection of the derivatives used in experimental vaccines against
human viruses. Overall results from the clinical trials suggest that the lipophilicity could
potentially help these derivatives maintain their adjuvant activity while minimizing the
pyrogenicity of parent MDP molecule.
9
9
9
7
8
9
1
1
1
1
1
1
00
01
02
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04
05
Table 1. MDP derivatives used in Experimental Vaccines against Human Viruses
MDP Derivative
MTP-PE (Mifamurtide)
Threonyl-MDP
GMDP
Vaccine / Virus Type
Influenza [21], HIV [22,23] Herpes Simplex [24]
,
Influenza [25], HIV [26], Hepatitis B [27]
Influenza [28], HIV [29]
6
-O-acyl MDP
Influenza [30,31], Hepatitis B [30]
Tetanus [32], Hepatitis B [33]
Murabutide
1
06
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MDP analogs containing different carbohydrate moiety, such as galactose-, allose-,
mannose-, furanose- and xylose-containing molecules were synthesized and evaluated too [34–
37]. Only D-glucofuranose-containing MDP was reported to have better immunomodulatory
activity than the parent MDP molecule [36]. The carbohydrate moiety in MDP is apparently not
crucial for the immunomodulatory activity by this type of compounds [11]. Many MDP analogs
lacking the carbohydrate fragment have shown some interesting immunomodulatory properties.
These MDP analogs are commonly known as desmuramyl peptides (DMPs) in the literature [37–
39]. One such example is O-(L-alanyl-D-isoglutamine-L-alanyl)-glycerol-3-mycolate which can
stimulate the resistance to infection in mice, the results being comparable to the parent MDP
[38]. Many DMPs have also shown significant antitumor potency with remarkable
immunomodulatory properties [40]. Gang Liu and co-workers have done significant work in this
regard [18,41–43]. In one instance, they replaced the carbohydrate moiety of MDP with
®
hydrophobic arenes to afford paclitaxel (Taxol ) conjugated DMPs [42,44]. These conjugates
1
1
1
1
1
1
1
1
1
1
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21
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29
combine the effects of immunotherapy and chemotherapy for the cancer treatment [44].
Numerous other DMPs incorporating various molecular scaffolds to replace the carbohydrate
moiety have also been shown to display remarkable immunomodulatory activity [45–52] and
promising antitumor effect [45,46].
Previously reported DMPs are mostly devoid of hydrophilic character due to the
elimination of the carbohydrate fragment, and are lipophilic in nature. Lipophilicity is an
important variable which helps eliminate several problems associated with this type of
molecules, such as poor cell penetration and rapid elimination. Conversely, hydrophilicity is
another important parameter for the activation of NOD2, which is located in the cytosolic
aqueous environment. Additionally, the literature survey revealed that current protocols don’t
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mention the synthesis of desmuramyl peptides having lipophilic and hydrophilic characteristics
simultaneously.
Owing to limited efficacy of traditional immunomodulatory agents, the development of
non-toxic and multifunctional drugs having the ability to safely modify the immune response is
of vital importance [50]. In the design of new amphiphilic DMPs (Figure 3) as potential vaccine
adjuvants, we broke the molecule down to four fragments: 1) the dipeptide pharmacophore, 2)
the hydrophilic aryl amine moiety replacing the carbohydrate, 3) the lipophilic chain at the C-
terminus of the dipeptide, and 4) the linker between the aryl amine and the dipeptide. Firstly,
MDP recognition by the immune system is highly stereospecific with respect to the configuration
of the amino acid residues in the dipeptide moiety [10,53–55]. Conserving L-D configuration of
dipeptide is therefore essential for the design of new MDP mimics [56]. Secondly, interesting
biological activities can be obtained by adopting balanced hydrophilicity/lipophilicity of the
molecule, with Murabutide (Figure 1) being a good example. The hydrophilicity of these DMPs
could be modulated via hydroxylated N-alkyl group-containing aryl amine scaffolds (Figure 3) to
replace the carbohydrate moiety of MDP. Thirdly, a modest lipophilic chain was introduced at
the C-terminus of the dipeptide moiety to adjust the lipophilicity of these DMPs and assist their
penetration through the cell membrane and reach the cytosolic NOD2 receptors. Fourthly, a
glycolic acid linker was used in place of lactic acid to couple these hydrophilic aryl amines with
the N-terminus of the dipeptide moiety, thereby eliminating the chirality of the linker and
simplifying the structure of these DMPs. Although lactic acid chirality is known to influence the
stability as well as activity of MDP, nor-MDP analogs lacking this chirality is reported to have
comparable activity and lower toxicity [11].
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1
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Figure 3. Structural components of amphiphilic desmuramyl peptides (1–8).
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Amphiphilic DMPs have not been explored yet. Herein, we report the synthesis of a group
amphiphilic DMPs (1 – 8, Figure 3). The novel DMPs were examined for their effects on ICAM-
1 (CD54) expression in THP-1 cells and the release of proinflammatory cytokine TNF-α. These
amphiphilic DMPs demonstrated higher activity in the induction of TNF-α in THP-1
macrophages than Murabutide which is a well-known NOD2 agonist. Furthermore, molecular
docking studies indicate that all these DMPs bind NOD2 receptor well with similar docking
scores (binding energy).
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2. Results and Discussion
2.1. Syntheses
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Amphiphilic DMPs were prepared by employing an efficient convergent synthetic
approach. The synthesis began with 3-nitrophenol alkylation (Scheme 1). Briefly, tert-butyl 2-(3-
aminophenoxy)acetate 9 was obtained by refluxing a mixture of tert-butyl 2-bromoacetate and 3-
nitrophenol in acetone followed by catalytic hydrogenation of the nitro group via molecular
hydrogen (H
2
) and 10% palladium on charcoal.[57] Then, mono-N-alkylation of 9 was achieved
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by refluxing it with 2-bromoethanol or 3-bromo-1-propanol in the presence of N,N-
diisopropylethylamine (DIPEA) to afford mono-alkylated products 10 or 11 in good yield. Some
di-N-alkylated products were also formed in this process, with their yields being less than 10%.
Compound 10 was treated with sodium hydroxide (0.5 N) to give the free acid 20. Alternatively,
di-N-alkylated product 21 was obtained in high yield by refluxing 10 with an excess amount of
2-bromoethanol in the presence of DIPEA in acetonitrile, which was then hydrolysed to give
free-acid 22. Hydrophilicity modulation was achieved by reacting 10 and 11 with acetic
anhydride, which afforded the less hydrophilic compounds 12 and 13, respectively. The tert-
butyl ester hydrolysis gave free acids 16 and 17. NMR spectra of these compounds reveal
duplication of signals, indicating the presence of a mixture of stereoisomers or conformers in the
solution. Similarly, butyric acid reaction with 10 and 11 afforded 14 and 15, which upon
hydrolysis gave the free acids 18 and 19.
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Scheme 1. a) tert-butyl 2-bromoacetate, K CO , (CH ) CO, reflux, 81%; b) Palladium on charcoal, H ,
2
3
3 2
2
94%; c) Br(CH ) OH or Br(CH ) OH, iPr NEt, C H OH, reflux, 58% for n = 2, 41% for n = 3; d)
2
2
2 3
2
2
5
(CH CO) O or (C H CO) O, CH OH, >99%; e) Br(CH ) OH, iPr NEt, CH CN, reflux, 71%; f-g) 0.5N
3
2
3
7
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3
2 2
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3
NaOH, dioxane, 66% for 16, 65% for 17, >99% for 18-22.
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In the second phase of the convergent synthesis strategy, preparation of lipophilic
dipeptide moiety was achieved. Previously, we demonstrated the preparation of (R)-4-((S)-2-
1
aminopropanamido)-N -hexylpentanediamide 23 in five steps [57]. The free amine 23 was then
coupled with 20 using TBTU in conjugation with N-methylmorpholine in DMF to give mono-
alkylated DMP 1 in 38 % yield (Scheme 2). Similarly, di-alkylated DMP 2 was prepared by
reacting 23 with acid 22 in 28 % yield. The cause of the poor yields was due to reactive hydroxyl
groups present in the building blocks 20 and 22. Owing to the poor yield encountered, an
alternative strategy was adopted (Scheme 2). An equimolar mixture of free acid 20 or 22, N-
hydroxysuccinimide (NHS) and the free amine 23 were stirred in DMF-THF mixture at 0 °C.
1
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1
1
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Then, dicyclohexylcarbodiimide (DCC) was added. NHS ester containing intermediate of the
acid (20/22) was presumably generated by this new coupling method, which reacted more
favorably with the amine group than the hydroxyl groups, resulting in higher yields for 1–2 (58
% and 51 %).
O
NH2
O
H
N
H
N
HO
N
H
O
N
H
O
NH2
O
O
O
H
H2N
N
1
2
N
H
O
23
O
NH2
O
H
N
H
N
HO
HO
N
O
N
H
O
O
1
99
2
2
00
01
Scheme 2. a) TBTU, N-methylmorpholine, DMF, 38%; b) NHS, THF/DMF (3:2), DCC, 0 °C to rt, 58%;
c) TBTU, N-methylmorpholine, DMF, 28%; d) NHS, THF/DMF (3:2), DCC, 0 °C to rt, 51%.
2
02
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Similarly, an equimolar mixture of the free acid 16–19, N-hydroxysuccinimide (NHS), the free
amine 23, and dicyclohexylcarbodiimide (DCC) were stirred in DMF-THF mixture at 0 °C to
2
05
afford 3 and 4 and 7 and 8 in decent yield (Scheme 3).
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06
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Scheme 3. a) NHS, THF/DMF (3:2), DCC, 0 °C to rt, 45% for 3, 49% for 4, 51% for 7, and 48% for 8.
2
2
2
2
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10
11
Previously, we reported the synthesis of 5 and 6 using the same method (NHS/DCC coupling) to
give 53% and 48% yield, respectively [57]. The N-acylated target molecules (3, 4, 7, and 8) can
also be readily prepared from compounds 1 and 5. Thus, treatment of 1/5 with acetic anhydride
or butyric anhydride afforded DMP 3–4 and 7–8 in quantitative yields (Scheme 4).
2
2
12
13
Scheme 4. a) (CH CO) O or (C H CO) O, CH OH, rt, >99%.
3
2
3
7
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3
1
2
2
2
2
2
2
2
14
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H NMR studies of all compounds 1–8 display geminal proton-proton couplings ( JH-H) due to
methylenoxy (OCH ) protons of glycolic linker. This type of coupling usually appears as doublet
2
pair if there are chiral centers in the molecule. Geminal coupling constant strongly depends on
H–C–H bonds angle [58], and its value ranges from 0 Hz (for 125° angle) to 32 Hz (for 100°
2
angle). For DMPs 1–8, protons of the linker (OCH ) are diastereotopic in nature which emerge
2
as doublet pair at around 4.45 ppm, with J value being 15 Hz in all cases (Figure 4s–10s).
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Moreover, primary amide protons (NH of the D-iso-Gln residue) in compounds 1–8 are not
identical, since both protons appear as two singlets at δ 7.07 ppm and at δ 7.31 ppm due to
prohibited amide bond rotation, thereby confirming the existence of intramolecular hydrogen
bonding between glycolic carbonyl and α-carboxamide proton of D-iso-Gln [59].
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2.2. Biological Studies
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Innate immune cells contain specialized sensors, such as PRRs, to detect microbial
pathogens [3]. These sensors are expressed either on cell surface in the form of Toll-like
receptors (TLRs) or in the endosome e.g. NOD-like receptors (NLRs), which identify the
harmful bacteria and viruses via PAMPs [60]. Upon recognition, PRRs prompt a signaling
cascade setting off innate immune responses, including an increase in surface expression of the
adhesion molecule ICAM-1 and pro-inflammatory cytokine release [4]. Although synergism of
many NOD2 agonists, such as MDP or MDP-C with LPS, has been extensively reported in
recent years, a crosstalk between DMPs and LPS in the regulation of ICAM-1 has hardly been
explored [57,61,62]. Therefore, amphiphilic desmuramyl peptides were examined in combination
with LPS for the induction of ICAM-1 in monocytic THP-1 cells. In the wake of preliminary
results from DMP 5 and 6 [57], immunostimulatory prospects of the remaining compounds were
further evaluated in THP-1 macrophages. The results were then compared with the effect of a
well-known NOD2 agonist – murabutide.
In the first round of experiments, 20 µM concentration of murabutide or each compound
was employed to stimulate monocytic THP-1 cells for 21 hours. However, ICAM-1 expression
levels were significantly low (Supporting information, Figure 1s). Previously, MDP synergism
with LPS has been reported [62]. In our experiments, amplification of lipopolysaccharide-
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induced ICAM-1 in THP-1 monocytes was also observed by amphiphilic desmuramyl peptides
1–8 (Figure 2s–3s and Table 2s). Previously, 64% upregulation of ICAM-1 was observed at 16
µM concentration of compound 6 with LPS [57]. From the preliminary data, it was evident that
compound 6 could enhance the lipopolysaccharide-induced ICAM-1 expression more effectively
than other DMPs (Figure 3s). Further experiments were done with the macrophages, since
monocytic THP-1 cells are weakly responsive to the immunostimulatory signals. 20 ng/ml of
PMA (Phorbol 12-myristate-13-acetate) was used to ensure the sufficient differentiation of THP-
1 monocytes into macrophages. When 20 µM of each amphiphilic DMPs 1–8 was incubated with
differentiated THP-1 macrophages, the analysis of ICAM-1 expression indicated the highest
mean fluorescence intensity of 905.38 under the effect of 8, compared to a value of 602.95 for
untreated cells (Figure 5). Cells treated with murabutide demonstrated the average value of
1436.05, which means that compounds 1–8 induced lower level of ICAM-1 expression than
murabutide.
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60
Figure 4. Cell surface expression of ICAM-1 in THP-1 macrophages treated with 20 µM of murabutide,
or amphiphilic desmuramyl peptides 1–8. FACS analysis (Flow cytometry) was employed for ICAM-1
measurements. The results of 3 separate experiments are shown as mean fluorescence intensity ± standard
error mean (SEM). The one-way ANOVA was used for statistical analysis, whereas (∗) p < 0.05, and (∗∗)
p < 0.01 in comparison with murabutide treated cells.
2
2
61
62
Previously, we demonstrated the strong effect of 6 on LPS-stimulated macrophages with higher
ICAM-1 expression level than one induced by the well-known NOD2 agonist, murabutide [57].
In those experiments, 20 ng/mL of PMA was used to differentiate the monocytes, and the
resulting macrophages were treated with different concentrations of 6 prior to LPS stimulation.
Then, the stimulation with 8 µM concentration of 6 raised the overall expression of intracellular
adhesion molecule 1 by 33 % as compared to LPS-only treated cells. This value was higher than
that observed for 6 alone. Interestingly, similar ICAM-1 upregulation was also found in the case
of murabutide, but it was less evident than that observed for 6 at 8 μΜ [57]. When lower
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
concentrations (
ICAM-1 expression level was observed. However, at higher concentrations (
down regulation of ICAM-1 occurred. The expression of ICAM-1 is mediated by the NF-
transcriptional factor, and various inflammatory mediators such as TNF- -IFN and bacterial
≤
4 µM) of 6 were used to pre-treat the macrophages, no significant change in
≥
16 µM) of 6, the
κB
α, γ
LPS [63]. Hence, the observed ICAM-1 upregulation can be attributed to the lipophilic chain in
6, which enables its intracellular presence to potentiate LPS action via nuclear transcription
factor mediated cytokines. It was therefore essential to quantify the pro-inflammatory cytokines
released by the compounds 1–8 potentially mediated by the cytosolic NOD2 receptor.
2
2
2
78
79
80
When an agonistic ligand binds to its receptor, pro-inflammatory cytokines, nitric oxide and
immunomodulating mediators are released [64]. Anti-infectious activity is generally mediated by
a pro-inflammatory cytokine viz. tumour necrosis factor (TNF-
Page 15 of 47
α). In vitro studies suggest that

2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
numerous muropeptides induce TNF-
monocytes. For example, Boons et al. reported TNF-
lines by employing muramyl tripeptides [65]. Similarly, desmuramyl peptides and tuftsin/retro-
tuftsin containing nor-MDP derivatives were able to stimulate the secretion of TNF- in PBMCs
and lymphocytes [66]. Therefore, we decided to measure TNF- production in THP-1
macrophages to assess the immunostimulatory potential of amphiphilic DMPs (1–8). These
compounds were able to induce significant levels of TNF- . Maximum response level was
observed at 15 M concentration (Figure 6). This response decreased by further increase in the
concentration of the agonist. For example, 2-hydroxyethyl containing amphiphilic DMPs 1–4
used at a concentration of 15 M induced TNF- release of 3226 ± 297 pg/mL, 3111 ± 24
α
production in myeloid-derived cells and human
α
gene expression in human monocytic cell
α
α
α
μ
μ
α
pg/mL, 3342 ± 91 pg/mL and 2806 ± 146 pg/mL, respectively. These values are considerably
higher than those induced by 15 µM murabutide (2694 ± 271 pg/mL). Similarly, at 15 µM
concentration, 3-hydroxypropyl containing amphiphilic DMPs 5–8 demonstrate average TNF-
α
release values of 3622 ± 371 pg/mL, 3306 ± 231 pg/mL, 2902 ± 335 pg/mL and 3105 ± 400
pg/mL respectively, which were higher than those observed for all 2-hydroxyethyl containing
DMPs except 3. Interestingly, higher concentrations of amphiphilic DMPs (e.g. 30
μM)
dampened the release of TNF- (Figure 6). In the previous experiments, low values of LPS-
α
induced ICAM-1 expression were observed at higher concentrations of amphiphilic DMP 6,
although no indication of cell deaths was found [57]. Higher doses of MDP have also been
reported to show a similar effect [67]. Further in vivo studies are required to comprehend the
mechanistic details of the immunomodulatory activity by amphiphilic DMPs and their
therapeutic potential.
Page 16 of 47

303
304
305
306
307
308
309
Figure 5. TNF-α release in macrophages stimulated by DMPs (1–8). PMA concentration of 20ng/mL
was used to differentiate monocytes into macrophages. Then for the next 24 hours, stimulation was done
with 1, 15, 30 µM of each DMP. Enzyme-linked immunosorbent assay (ELISA) was used to estimate
TNF-α in the supernatant. Two separate experiments were used to express the results ± SEM. One-way
ANOVA was used to find the statistically significant differences, whereas (∗) p < 0.05, (∗∗) p < 0.01 vs
15 μM murabutide treated cells.
310
3
3
11
12
2.3.
Computational Studies
313
314
315
316
317
318
319
To get deeper insights into the structural requirements of amphiphilic
desmuramylpeptides (DMPs), molecular docking study was performed using Nucleotide-Binding
Oligomerization Domain-Containing Protein 2 receptor (NOD2). The crystal structure of human
NOD2 has not been reported yet. Therefore, homology modelling was carried out by using a
web-server, SWISS-MODEL [68]. For the identification of template structure, an NCBI
protein-Blast search [69] was performed against uniport reference sequence: Q9HC29. The
BLAST search identified rabbit (Oryctolague cuniculus) NOD2 protein (PDB ID: 5IRN) [70] as
Page 17 of 47

3
3
3
3
3
3
3
3
20
21
22
23
24
25
26
27
the best match for human NOD2 which shares 86% identity with the target protein (Figure 9s).
The quality of the model was assessed to confirm if it was suitable for performing further
studies. For this purpose, online quality evaluation tools, such as ERRAT, PROCHECK and
Verify-3D [71] were used (Figure 4s). The plot showed the overall refinement of the modelled
structures as most of the residues were present in the core region while only one outlier
Gln968 is found which is far from the active site. Superimposition of homology model with the
template is illustrated in Figure 7A. The calculated root means square deviation (RMSD) of
NOD2 model against 5IRN is 0.676 Å.
3
3
28
29
Page 18 of 47

3
30
Table 2. Binding energy score of DMPs (1–8) and their interactions with the residues of NOD2 receptor
DMP Dock Score Hydrogen bonding Hydrophobic/ interactions
π
(
Kcal/mol)
1
-7.95
Gly879 with
of D-iso-Gln
α
α
-carboxamide Aryl group of Trp931 with N-hexyl chain
Aryl group of Trp907 with (CH ) of D-iso-Gln
2
2
Gly905 with
of D-iso-Gln
-carboxamide
Cation-
π interaction between Arg823 and N-aryl ring
2
3
-7.92
-7.52
Arg823 with aryl amine
Aryl group of Phe903 with N-hexyl chain
Arg877 with
of D-iso-Gln
α
-carboxamide Aryl group of Trp907 with methyl of L-Ala
Arg823 with
of D-iso-Gln
α
-carboxamide Aryl group of Arg877 with N-hexyl chain
Aryl group of Phe851 with (CH ) of D-iso-Gln
2
2
Ser991 with acetyl group
Aryl group of Trp907 with (CH ) of D-iso-Gln
2
2
4
5
-7.94
-7.42
Arg823 with amide of
glycolic linker
Aryl group of Tyr799 with N-hexyl chain
Aryl group of Phe851 with methyl of L-Ala
Alkyl chain of Lys271 with N-aryl ring
Asn880 with butyryl group
Gly879 with
of D-iso-Gln
α
α
-carboxamide Alkyl chain of Lys271 with N-hexyl chain
Aryl group of Trp931 with methyl of L-Ala
-carboxamide
Gly905 with
of D-iso-Gln
Glu959 with aryl amine
6
7
-7.83
-7.77
Arg877 with N-hexyl chain Aryl group of Trp931 with N-hexyl chain
Asn880 with hydroxy ethyl Aryl group of Trp907 with methyl of L-Ala
group
Glu959 with amide of
glycolic linker
Aryl group of Phe851 with N-hexyl chain
Aryl group of Trp907 with (CH ) of D-iso-Gln
2
2
Trp931 with the amide of
L-Ala
Alkyl chain of Lys986 with methyl of acetyl group
8
-7.96
Arg877 with the amide of
L-Ala
Aryl group of Trp931 with N-hexyl chain
π - π Interaction between Phe851 and N-aryl ring
Trp907 with butyryl group
Lys989 with hydroxy propyl
group
3
31
Page 19 of 47

332
333
334
335
336
Figure 6. (A) Superimposition of crystal structure (brown) and modeled structure (blue); (B) Surface
representation of the NOD2-LRR domain with compound 1 (in blue) showing the concave binding
pocket.
337
3
38
The docking results indicate that all DMPs (1–8) bind well to NOD2 receptor, with their dock scores
(binding energy) ranging from -7.96 to -7.42 kcal/mol. Although these compounds show similar dock
scores, they display different binding modes through a number of hydrophilic and hydrophobic
interactions with the crucial residues in the binding site of the receptor (Table 2, Figure 8). The majority
of hydrogen bond interactions were observed between L-Ala-D-iso-Gln residue of DMPs and the
surrounding amino acids of NOD2 binding pocket, which means that this dipeptide moiety is essential for
339
340
341
342
343
344
345
346
347
348
349
350
351
352
the activity of 1–8. The
H-bonding with the residues Gly823, Gly879 and Gly905. In contrast, L-alanine in compound 7 and 8
formed H-bonding with Arg877 and Trp931 residues. The -CH CH - moiety of D-iso-Gln in the dipeptide
α-carboxamide group of D-iso-Gln in compound 1, 2, 3 and 5 was implicated in
2
2
was also involved in hydrophobic/π interactions with Phe851 and Trp907 in compound 1, 3 and 7, while
the methyl group of L-Ala in 2 and 4–6 interacted with Phe851, Trp907 and Trp931. Hydrophilic arene in
compound 2 and 5 was found interacting with Arg823 and Glu959 via H-bonding. The aryl amine in 1
established the cation-π interaction with Arg823 in NOD2 binding pocket. Furthermore, hydrophobic
interactions involving the lipophilic N-hexyl chain were observed in all DMP molecules (1–8). The hexyl
chain in compound 5 interacted with the lipophilic alkyl chain of Lys271 while in all other compounds
Page 20 of 47

3
3
3
3
53
54
55
56
the hexyl chain was interacting with an aromatic ring of several Phe, Trp, and Arg residues. Interestingly,
besides the cation-π interaction mentioned above for compound 1, hydrophobic/π interactions involving
the aryl ring of the DMPs were only found in compounds 4 and 8, both of which bearing a butyryl group
on the nitrogen atom of the aryl amine.
3
3
3
3
3
3
3
3
3
57
58
59
60
61
62
63
64
65
Taken together, the results of docking studies suggest that all DMPs (1 – 8) bind into the concave
hydrophobic cavity of NOD2-LRR domain and interact with the crucial residues of the NOD2. In the
cavity, they adopt different conformations due to their hydroxyalkyl and the lipophilic chains, which
consequently leads to their different modes of interaction with the receptor. Overall, docking results
provide structural insight into the binding mode of newly synthesized DMPs, which highlights the
importance of the dipeptide moiety (L-Ala-D-iso-Gln) in conjugation with other structural elements
including the alkyl amide chain, the aryl amine group, the hydroxylalkyl group, and the N-acyl group.
These findings will be useful for designing new DMPs with higher binding affinity to the NOD2 receptor,
ultimately leading towards improved biological activities.
3
66
Page 21 of 47

367
368
369
370
371
Figure 7. Molecular docking of DMPs (1–8). The stick model of different colors shows the compounds,
and the key amino acid residues of NOD2 receptor around compounds have been displayed in grey stick
model.
Page 22 of 47

3
72
3.
Conclusion
3
3
3
3
3
3
3
3
3
3
3
3
3
3
73
74
75
76
77
78
79
80
81
82
83
84
85
86
In summary, new class of desmuramyl peptides containing hydrophilic arene moiety and
lipophilic chain were prepared. These compounds were primarily assessed in vitro by
investigating their synergistic effect on LPS-induced expression of surface glycoprotein viz.
ICAM-1 (CD54). In addition, pro-inflammatory cytokine (TNF-
macrophages was measured. The novel compounds were found to upregulate the expression of
LPS-induced ICAM-1 (CD54), and could trigger the release of higher TNF- levels than
α) release in THP-1
α
murabutide. The overall similitude in the immunostimulatory activity of the amphiphilic DMPs
and murabutide is remarkable, especially when there are huge structural differences among them.
In silico studies also revealed that all the ligands interacted with crucial residues of NOD2-LRR
binding domain. Additional work is needed to demonstrate whether these compounds reveal the
immunomodulatory effect via NOD2 receptor activation. Their immunomodulating potential
needs further assessment through the profiling of other cytokines in different immune cells. In
vivo studies may be ensued to investigate the therapeutic potential of the amphiphilic DMPs as
immunomodulatory/immunostimulatory agents.
3
3
87
88
4.
Experimental Section
4.1. Reagents and instruments
3
3
3
3
3
89
90
91
92
93
Most of the chemicals, obtained from Sigma-Aldrich (Merck KGaA), were used without further
purifications. Analytical grade solvents were employed, and the air-sensitive compounds were
handled using the standard Schlenk techniques. Murabutide was obtained from Sigma-Aldrich,
and LPS E. coli O111:B4 was purchased from InvivoGen, San Diego, CA. DMSO was used to
dissolve the synthetic DMPs followed by their water reconstitution and storage at -20 °C.NMR
Page 23 of 47

®
®
3
3
94
95
spectra were recorded with Bruker Avance (400 MHz) and on a Varian Unity Inova (500
MHz) spectrometer in deuterated solvents.
3
96
4.2. Syntheses procedures and structure characterization
3
3
3
4
4
4
4
4
4
4
4
4
4
4
97
98
99
00
01
02
03
04
05
06
07
08
09
10
For the cleavage of tert-Butoxycarbonyl group, ice-cooled dichloromethane solution of Boc
containing compound was treated with trifluoroacetic acid (TFA). Stirring was continued until
consumption of all the starting material which was confirmed via TLC monitoring. The solution
was then concentrated in vacuo followed by its neutralization with an aqueous 10% NaHCO3
solution. Subsequent solvent removal gave the crude product. Whenever the desired product was
an amine salt, the TFA containing solvent was evaporated in vacuo. Then methanol co-
evaporation was then used to eliminate the residual TFA and get the product quantitatively.
Similarly, ester hydrolysis of 10 – 15 and 21 was done by dropwise addition of aqueous NaOH
(0.5 N, 2 ml) to an ice-cooled solution of dioxane (3 mL) containing the above-mentioned
compounds. Hydrolysis was monitored with TLC until consumption of all the starting material.
The reaction mixture was subsequently neutralized with dilute HCl (0.5 N) and the solvent
removed under reduced pressure. Absolute ethanol was employed to filter off the salt. Procedure
for the synthesis of compounds 5, 6, 9, 11, 21, 23, 25 and 26 can be traced back from our
previously published work [57].
1
4
4
11
12
4.2.1. (R)-N -hexyl-4-((S)-2-(2-(3-((2-hydroxyethyl)amino)phenoxy)acetamido)
propanamido)pentanediamide (1)
4
4
4
13
14
15
A mixture of DMF (3 mL) and 23 (0.1 g, 0.33 mmol) was added to TBTU (0.12 g, 0.36 mmol),
N-methylmorpholine (0.11 ml, 1.0 mmol) and 20 (0.07 g, 0.33 mmol) containing DMF solution
(5 mL). After vigorous stirring (18 h, room temperature), the solvent was removed in vacuo. Air-
Page 24 of 47

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
sensitive 1 (0.062 g, 38 %) was obtained as white powder after the residue purification over flash
silica column (Chloroform/Methanol, 5:1). Alternatively, DMF (2 mL) solution of 20 (0.07 g,
0.33 mmol) was mixed to an ice-cooled THF (3 mL) containing 1-hydroxypyrrolidine-2,5-dione
(0.04 g, 0.33 mmol) and 23 (0.1 g, 0.33 mmol). This was followed by the addition of
dicyclohexylcarbodiimide (0.07 g, 0.33 mmol). Overnight stirring at room temperature resulted
in dicyclohexyl urea precipitates. After filtration, the solvents were removed in vacuo. Residue
thus obtained was purified over flash silica column (Chloroform/Methanol, 5:1), which afforded
1
air-sensitive 1 (0.095 g, 58 %) as white powder: R
0.84 (t, 3H, J = 7 Hz, CH CH ), 1.21 (m, 6H, CH (CH
(quint, 2H, J = 8 Hz, CH (CH ) CH ), 1.68 (m, 1H, CHCHH), 1.92 (m, 1H, CHCHH), 2.05 (t,
f
= 0.42; H NMR (500 MHz, (CD
3
)
2
SO):
δ
3
2
3
2 3 3
) ), 1.24 (d, 3H, J = 7 Hz, CHCH
), 1.34
3
2 3
2
2H, J = 7 Hz, CHCH CH ), 2.98 (m, 2H, CONHCH ), 3.04 (q, 2H, J = 6 Hz, CH OH), 3.52 (q,
2
2
2
2
2H, J = 6 Hz, NHCH CH OH), 4.10 (td, 1H, J = 5, 8 Hz, CHCH ), 4.35 (quint., 1H, J = 7 Hz,
2
2
2
CHCH ), 4.40 (dd, 2H, J = 15 Hz, OCH ), 4.68 (t, 1H, J = 5 Hz, OH), 5.54 (t, 1H, J = 5 Hz,
3
2
NHCH CH CH OH), 6.10 (d, 1H, J = 8 Hz, OCCHCHCH), 6.15 (s, 1H, CCHC), 6.20 (d, 1H, J =
2
2
2
2, 8 Hz, OCCHCHCH), 6.94 (t, 1H, J = 8 Hz, OCCHCHCH), 7.08 (s, 1H, NH
NH ), 7.77 (t, 1H, J = 5 Hz, CONHCH ), 8.07 (d, 1H, J = 7 Hz, CH CHNH), 8.22 (d, 1H, J = 8
Hz, CHNHCO). C NMR (125 MHz, (CD ) SO): 13.98 (CH CH ), 18.39 (CH CH), 22.11
2
), 7.31 (s, 1H,
2
2
3
1
3
δ
3
2
3
2
3
(CH CH ), 26.13 (CH ), 27.70 (CH ), 29.10 (CH ), 31.05 (CH ), 31.81 (CH ), 38.53 (NCH ),
2
3
2
2
2
2
2
2
45.55 (NHCH ), 48.16 (CHCH ), 52.23 (CHCH ), 59.61 (CH OH), 66.66 (OCH ), 98.36
2
3
2
2
2
(CHCHCH), 101.61 (CHCHCH), 106.05 (CCHC), 129.64 (CHCHCH), 150.35 (NCCH), 158.83
(NCCHC), 167.73 (CH CHC=O), 171.33 (CH C=O), 171.95 (OCH C=O), 173.30 (NH C=O);
3
2
2
2
+
MALDI-TOF (m/z) Calcd for C24
H
39
N
5
O
6
[M+Na] : 516.2798, found: 516.2229.
Page 25 of 47

1
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4.2.2. (R)-4-((S)-2-(2-(3-(bis(2-hydroxyethyl)amino)phenoxy)acetamido)propanamido)-N -
hexylpentanediamide (
2
)
A mixture of DMF (3 mL) and 23 (0.1 g, 0.33 mmol) was added to TBTU (0.12 g, 0.36 mmol),
N-methylmorpholine (0.11 ml, 1.0 mmol) and 22 (0.08 g, 0.33 mmol) containing DMF solution
(5 mL). After vigorous stirring (18 h, RT), the solvent was removed in vacuo. Air-sensitive 2
(0.052 g, 28 %) was obtained as white powder after the residue purification over flash silica
column (Chloroform/Methanol, 5:1). Alternatively, DMF (2 mL) solution of 22 (0.08 g, 0.33
mmol) was mixed to an ice-cooled THF (3 mL) containing 1-hydroxypyrrolidine-2,5-dione (0.04
g, 0.33 mmol) and 23 (0.1 g, 0.33 mmol). This was followed by the addition of
dicyclohexylcarbodiimide (0.07 g, 0.33 mmol). Overnight stirring at room temperature resulted
in dicyclohexyl urea precipitates. After filtration, the solvents were removed in vacuo. Residue
thus obtained was purified over flash silica column (Chloroform/Methanol, 5:1), which afforded
1
an air-sensitive compound 2 (0.091 g, 51 %) as white powder: R = 0.37; H NMR (500 MHz,
f
(CD ) SO):
δ
0.84 (t, 3H, J = 7 Hz, CH CH ), 1.22 (m, 6H, CH (CH ) ), 1.24 (d, 3H, J = 7 Hz,
3
2
3
2
3
2 3
CHCH
CHCHH), 2.06 (t, 2H, J = 7 Hz, CHCH
3.50 (q, 4H, J = 6 Hz, NCH ), 4.10 (td, 1H, J = 5, 9 Hz, CHCH ), 4.35 (quint., 1H, J = 7 Hz,
3
), 1.33 (quint, 2H, J = 8 Hz, CH
3
(CH
2
)
3
CH
2
), 1.69 (m, 1H, CHCHH), 1.93 (m, 1H,
2
CH
2
), 2.98 (m, 2H, NHCH
2
), 3.37 (m, 4H, CH OH),
2
2
2
CHCH ), 4.43 (d, 2H, J = 15 Hz, OCH ), 4.73 (t, 2H, J = 5 Hz, OH), 6.15 (d, 1H, J = 8 Hz,
3
2
OCCHCHCH), 6.23 (s, 1H, J = 2 Hz, CCHC), 6.29 (d, 1H, J = 8 Hz, OCCHCHCH), 7.01 (t, 1H,
J = 8 Hz, OCCHCHCH), 7.06 (s, 1H, NH ), 7.29 (s, 1H, NH ), 7.75 (t, 1H, J = 5 Hz,
2
2
1
3
CONHCH ), 8.09 (d, 1H, J = 7 Hz, CHNH), 8.21 (d, 1H, J = 8 Hz, CHNH). C NMR (125
2
MHz, (CD
3
)
2
SO):
δ
14.39 (CH
), 31.46 (CH ), 32.25 (CH
Page 26 of 47
3
CH
2
), 18.79 (CH
3
CH), 22.51 (CH
2
CH
3
), 26.55 (CH
2
), 28.13
),
(CH ), 29.52 (CH
2
2
2
2
), 38.97 (NCH
2
), 48.64 (CHCH
3
), 52.68 (CHCH
2

4
4
4
4
61
62
63
64
2 2 2
53.78 (NHCH ), 58.60 (CH OH), 67.23 (OCH ), 98.77 (CHCHCH), 101.52 (CHCHCH), 105.54
(CCHC), 130.22 (CHCHCH), 149.76 (NCCH), 159.42 (NCCHC), 168.17 (CH CHC=O), 171.76
3
(CH C=O), 172.38 (OCH C=O), 173.70 (NH C=O); MALDI-TOF (m/z) Calcd for C H N O
7
2
2
2
26 43
5
+
[M+Na] : 560.3060, found: 560.2590.
1
4
4
65
66
4.2.3. (R)-N -hexyl-4-((S)-2-(2-(3-(N-(2-hydroxyethyl)acetamido)phenoxy)acetamido)
propanamido)pentanediamide (3)
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
DMF (2 mL) solution of 16 (0.08 g, 0.33 mmol) was mixed to an ice-cooled THF (3 mL)
containing 1-hydroxypyrrolidine-2,5-dione (0.04 g, 0.33 mmol) and 23 (0.1 g, 0.33 mmol). This
was followed by the addition of dicyclohexylcarbodiimide (0.07 g, 0.33 mmol). Overnight
stirring at room temperature resulted in dicyclohexyl urea precipitates. After filtration, the
solvents were removed in vacuo. Residue thus obtained was purified over flash silica column
(Chloroform/Methanol, 5:1), which afforded an air-sensitive compound 3 (0.08 g, 45 %) as white
powder. Alternatively, methanol (5 mL) containing 1 (0.05 g, 0.1 mmol) was stirred with acetic
f
anhydride (0.01 ml, 0.1 mmol) at room temperature, which afforded 3 in quantitative yield: R =
1
0.40; H NMR (500 MHz, (CD ) SO):
δ 0.81 (t, 3H, J = 7 Hz, CH CH ), 1.19 (m, 6H,
3 2
3
2
CH (CH ) ), 1.23 (d, 3H, J = 7 Hz, CHCH ), 1.32 (m, 2H, CH (CH ) CH ), 1.66-1.70 (m, 4H,
3
2 3
3
3
2 3
2
CHCHH, CH CO), 1.90 (m, 1H, CHCHH), 2.05 (t, 2H, J = 7 Hz, CHCH CH ), 2.96 (m, 2H,
3
2
2
CONHCH ), 3.41 (m, 4H, CH OH, NHCH CH OH), 4.07 (td, 1H, J = 5, 8 Hz, CHCH ), 4.31
2
2
2
2
2
(quint., 1H, J = 7 Hz, CHCH ), 4.54 (dd, 2H, J = 15 Hz, OCH ), 4.78 (s, 1H, OH), 6.89-6.99 (m,
3
2
3H, CHAr), 7.07 (s, 1H, NH
NHCH
2
), 7.33 (s, 1H, NH
2
), 7.36 (m, 1H, CHAr), 7.82 (t, 1H, J = 5 Hz,
1
3
2
3 2 3 2
), 8.25 (t, 2H, J = 8 Hz, CHNH). C NMR (125 MHz, (CD ) SO): δ 14.31 (CH CH ),
18.54 (CH CH), 22.43 (CH CH ), 22.87 (CH ), 26.45 (CH ), 27.98 (CH ), 29.37 (CH CO),
3
2
3
2
2
2
3
Page 27 of 47

4
4
4
4
4
83
84
85
86
87
31.37 (CH
2 2 2 3 2 2
), 32.18 (CH ), 38.97 (NCH ), 48.74 (CHCH ), 51.15 (CHCH ), 52.67 (CH OH),
58.34 (NCH ), 67.18 (OCH ), 114.38 (CHCHCH), 115.03 (CHCHCH), 121.32 (CCHC), 130.70
2
2
(CHCHCH), 144.72 (NCCH), 158.68 (NCCHC), 167.89 (CH CHC=O), 170.04 (CH C=O),
3
2
172.02 (OCH C=O), 172.46 (CH C=O), 173.88 (NH C=O); MALDI-TOF (m/z) Calcd for
2
3
2
+
C H N O [M+Na] : 558.2904, found: 558.2421.
26
41
5
7
1
4
4
88
89
4.2.4. (R)-N -hexyl-4-((S)-2-(2-(3-(N-(2-hydroxyethyl)butyramido)phenoxy)acetamido)
propanamido) pentanediamide (
4)
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
DMF (2 mL) solution of 18 (0.09 g, 0.33 mmol) was mixed to an ice-cooled THF (3 mL)
containing 1-hydroxypyrrolidine-2,5-dione, (0.04 g, 0.33 mmol) and 23 (0.1 g, 0.33 mmol). This
was followed by the addition of dicyclohexylcarbodiimide (0.07 g, 0.33 mmol). Overnight
stirring at room temperature resulted in dicyclohexyl urea precipitates. After filtration, the
solvents were removed in vacuo. Residue thus obtained was purified over flash silica column
(Chloroform/Methanol, 5:1), which afforded an air-sensitive compound 4 (0.092 g, 49 %) as
white powder. Alternatively, methanol (5 mL) containing 1 (0.05 g, 0.1 mmol) was stirred with
butyric anhydride (0.017 ml, 0.1 mmol) at room temperature, which afforded 4 in quantitative
1
yield: R = 0.38; H NMR (500 MHz, (CD ) SO):
δ 0.76 (t, 3H, J = 6 Hz, CH (CH ) CO), 0.84
3 2 2
f
3 2
(t, 3H, J = 7 Hz, CH CH ), 1.24 (m, 8H, CH (CH ) ), 1.26 (d, 3H, J = 7 Hz, CHCH ), 1.35 (m,
3
2
3
2 4
3
2H, CH (CH ) CH ), 1.45 (m, 2H, CH CH CH CO), 1.72 (m, 1H, CHCHH), 1.96 (m, 1H,
3
2 3
2
3
2
2
CHCHH), 2.07 (t, 2H, J = 7 Hz, CHCH CH ), 3.00 (m, 2H, CONHCH ), 3.44 (q, 2H, J = 6 Hz,
2
2
2
CH
2
OH), 3.65 (t, 2H, J = 6 Hz, NHCH
2
CH
2
2
OH), 4.13 (td, 1H, J = 5, 8 Hz, CHCH ), 4.37
3 2
(quint., 1H, J = 7 Hz, CHCH ), 4.57 (dd, 2H, J = 15 Hz, OCH ), 4.74 (s, 1H, OH), 6.89-6.97 (m,
3H, CH ), 7.07 (s, 1H, NH ), 7.33 (s, 1H, NH ), 7.35 (t, 1H, J = 3 Hz, CH ), 7.82 (t, 1H, J = 5
Ar
2
2
Ar
Page 28 of 47