Ten-membered rings as key interaction motifs in folding of desmuramyl di-, tri-, and tetrapeptides

Article abstract of DOI:10.1007/s11224-018-1234-x

Muropeptides are fragments of polymeric peptidoglycan, unique constituent of bacterial cell walls, acting as immunostimulators (adjuvants). The smallest biologically active unit of peptidoglycan is N-acetylmuramyl-L-alanyl-D-isoglutamine. In this paper, the synthesis and conformational analysis of desmuramyl peptides, derivatives lacking the N-acetylmuramyl moiety, is described. Systematic conformational analysis (bottom-up approach) of peptides in zwitterionic and non-zwitterionic forms of corresponding desmuramyl di-, tri-, and tetrapeptides was performed and results were compared with experimental data obtained by NMR study. Ten-membered ring (C10, N_1bH?O₃) formed between isoGln amide group and C=O of AdGly was found to be a key interaction that constitutes the central structural spine and initiates β-turn-like folding in our model peptide systems.

Full text of DOI:10.1007/s11224-018-1234-x

Structural Chemistry (2019) 30:743–754
https://doi.org/10.1007/s11224-018-1234-x
ORIGINAL RESEARCH
Ten-membered rings as key interaction motifs in folding of desmuramyl
di-, tri-, and tetrapeptides
Rosana Ribić1 & Ivan Kodrin2 & Ivana Biljan2 & Marija Paurević3 & Srđanka Tomić2
Received: 4 October 2018 /Accepted: 7 November 2018 /Published online: 17 November 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Muropeptides are fragments of polymeric peptidoglycan, unique constituent of bacterial cell walls, acting as immunostimulators
(adjuvants). The smallest biologically active unit of peptidoglycan is N-acetylmuramyl-L-alanyl-D-isoglutamine. In this paper,
the synthesis and conformational analysis of desmuramyl peptides, derivatives lacking the N-acetylmuramyl moiety, is described.
Systematic conformational analysis (bottom-up approach) of peptides in zwitterionic and non-zwitterionic forms of correspond-
ing desmuramyl di-, tri-, and tetrapeptides was performed and results were compared with experimental data obtained by NMR
study. Ten-membered ring (C10, N1bH⋯O3) formed between isoGln amide group and C=O of AdGly was found to be a key
interaction that constitutes the central structural spine and initiates β-turn-like folding in our model peptide systems.
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.
.
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Keywords Desmuramyl peptides Adamantane Adjuvants Conformational analysis β-Turn-like folding
Introduction
of pharmacological properties of muramyl and desmuramyl
peptides [2]. For example, MDP derivative mifamurtide
(muramyl tripeptide phosphatidylethanolamine) used in
clinics as a drug for the treatment of osteocarcinoma has fatty
acid chains attached to Glu residue [3]. Furthermore, previous
investigations indicated that sugar N-acetylmuramyl moiety is
not required for immunostimulating activity and it can be
substituted by different acyl groups in order to enhance lipo-
philicity [4–8]. The biological properties of MDP and its mi-
metics have engendered extensive studies of primary
structure-activity relations whereas less attention has been
paid to conformational characteristics of these compounds.
Conformational analyses of C-modified MDP derivatives,
such as murabutide (butyl ester of MDP), were performed
[9, 10] while literature data for N-modified peptides is scarce.
Herein, we describe a comprehensive structural study of pre-
ferred solution conformations of core desmuramyl dipeptide
(L-Ala-D-isoGln) and its tripeptide and tetrapeptide deriva-
tives with incorporated unnatural bulky adamantane amino
acid (AdGly). In this analysis, previously synthesized and
biologically evaluated MDP-mimetic, (S)-AdGly-L-Ala-D-
isoGln tripeptide was used [4]. Adamantyl tripeptide exhibited
immunomodulating activity in experiments in vivo, in the
mouse model [4]. Tetrapeptide derivative used in this study,
L-Ser-(S)-AdGly-L-Ala-D-isoGln (Fig. 1), is serine derivative
of the adamantyl MDP-mimetic, the synthesis of which is
reported here for the first time.
Muropeptides are fragments of polymeric peptidoglycan,
unique constituent of bacterial cell walls, acting as
immunostimulators (adjuvants). The smallest biologically ac-
tive unit of peptidoglycan is MDP (N-acetylmuramyl-L-ala-
nyl-D-isoglutamine) [1]. Considerations about the structure-
activity relationships of muropeptides suggest that the core
structure for the immunostimulating activity is desmuramyl
dipeptide part (L-Ala-D-isoGln), while substituents at either
the Ala or Gln sides are required for activity. Increase of lipo-
philicity is an especially important factor for the enhancement
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11224-018-1234-x) contains supplementary
material, which is available to authorized users.
* Rosana Ribić
rosana.ribic@unin.hr
1
University Center Varaždin, University North, Jurja Križanića 31b,
HR-42000 Varaždin, Croatia
2
Faculty of Science, University of Zagreb, Horvatovac 102a,
HR-10000 Zagreb, Croatia
3
Department of Chemistry, University of Osijek, Ulica cara Hadrijana
8/A, HR-31000 Osijek, Croatia

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Struct Chem (2019) 30:743–754
the Bruker AV-600 spectrometer at 300 MHz and 75.5 MHz,
respectively. 1H and 2D COSY, NOESY, 1H-13C HSQC, and
1H-13C HMBC NMR spectra of final peptides were recorded
on the Bruker Avance III HD 400 NMR spectrometer
equipped with a broadband observed (BBO) 5-mm Prodigy
cryoprobe and z-gradient accessories in D2O and DMSO-d6.
All NMR experiments were performed at 298 K. Chemical
shifts were referenced with respect to tetramethylsilane.
Fig. 1 Chemical structure of target tetrapeptide composed of four amino
acids
Computational details
To identify the ensemble of the most stable conformers, we
carried out an initial screening for possible conformation
minima using the MacroModel v10.3 [13, 14]. Several search
methods and force fields were employed, but mostly
OPLS_2005 was used [15]. The starting structures obtained
from MacroModel (approximately between 200 and 400 for
each compound) were used as input structures for further
optimization in Gaussian09 [16] with B3LYP-D3/6-31+
G(d,p) computational model [17–19]. Solvent effects were
described by IEFPCM [20, 21]. Harmonic vibrational fre-
quencies were calculated in order to confirm that these struc-
tures correspond to energy minima on the potential energy
surface. Thermochemical calculations allowed the calcula-
tion of the Gibbs free energy values, at 298.15°. Molecules
were visualized with Maestro [22], Chem3D [23], and
GaussView 5 [24] programs. The topological analysis was
performed with AIM2000 [25, 26].
Having in mind that H-bond is one of the key intramolec-
ular interaction in living systems, Ser was introduced to in-
crease structure complexity in terms of H-bonding (hydroxyl
group in the side-chain of serine as additional H-bond donor)
in order to test the proposed methodology (theoretical mod-
el). It is also important to note that Ser is often used for the
connection of peptide and carbohydrate parts in biologically
active glycoconjugates, including synthetic adjuvants and
immunologically active compounds [11, 12] and that in the
continuation of our research, application of this approach in
design of novel class of adjuvants is planned. In order to get
better insight into conformational properties of desmuramyl
peptide pharmacophore and therefore deeper understanding
of structure-activity relationship important for the design of
novel more active MDP-mimetics, a comprehensive confor-
mational investigation of corresponding desmuramyl dipep-
tide, tripeptide, and tetrapeptide in solution was undertaken.
Systematic conformational analysis of peptides in zwitterion-
ic and non-zwitterionic forms was performed based on the
theoretical model. In-depth conformational analysis was per-
formed and afterward calculated conformers were compared
with experimental results of the NMR study obtained in deu-
terated water and DMSO.
Synthetic procedures
L-Alanyl-D-isoglutamine benzyl ester trifluoroacetate
Boc-L-alanyl-D-isoglutamine benzyl ester (400 mg,
0.98 mmol) was suspended in anhydrous CH2Cl2 (2 mL)
and TFA (1 mL) was added. The reaction mixture was stirred
at room temperature for 1 h (until the disappearance of Boc-L-
Ala-D-isoGln-OBn was confirmed via TLC) and then concen-
trated in vacuo. The residue was purified by flash chromatog-
raphy on silica gel (CHCl3/MeOH 2:1) to yield L-Ala-D-
isoGlnOBn TFA salt (414 mg, 0.98 mmol) as viscous oil in
Experimental section
Materials and methods
Boc-L-Ala-D-isoGln-OBn was obtained from Bachem
(Switzerland), (S)-Boc-1-adamantyl-glycine from Chem-
Impex International Inc. (USA) and other reagents and sol-
vents for the synthesis of compounds were obtained from
Sigma-Aldrich Corp. Organic solvents were further purified
and/or dried using standard methods. Thin layer chromatog-
1
quantitative yield. Rf = 0.49 (CHCl3/MeOH 2:1). H NMR
(MeOH-d4, 300 MHz) δ 7.36–7.30 (m, 5H, Harom), 5.13 (s,
2H, CH2; Bn), 4.41 (dd, J = 5.1, 9.0 Hz, 1H, CH-α; isoGln),
3.87 (q, J = 7.0 Hz, 1H, CH-α; Ala), 2.48 (pt, J = 7.5, 7.7 Hz,
2H; CH2-γ; isoGln), 2.24–2.13 (m, 1H, CH-β; isoGln), 2.03–
1.91 (m, 1H CH-β; isoGln), 1.44 (d, J = 7.0 Hz, 3H, CH3;
Ala). 13C NMR (MeOH-d4, 75.5 MHz) δ 193.85, 192.40
(C=O), 129.61, 129.30 (CHarom), 67.56 (CH2; Bn), 53.72,
50.56 (CH-α; Ala, isoGln), 31.42 (CH2-γ), 28.43 (CH2-β),
raphy (TLC) was performed on Fluka silica gel (60 F254
)
plates (0.25 mm). Visualization was achieved using UV light
at 254 nm and ninhydrin. Column chromatography was per-
formed on Merck silica gel 60 (size 70–230 mesh ASTM).
Mass spectra were recorded on Agilent 6410 MS instrument.
1H and 13C NMR spectra of all precursors were recorded on
+
18.32 (CH3; Ala). MS (ESI) m/z: [M]+ calcd for C15H22N3O4
308.2, found 308.1.

Struct Chem (2019) 30:743–754
745
L-Alanyl-D-isoglutamine
1 h (until the disappearance of Boc-(S)-AdGly-L-Ala-D-
isoGlnOBn was confirmed via TLC) and then concentrated in
vacuo. The residue was purified by flash chromatography
(CHCl3/MeOH 2:1) to yield TFA×(S)-AdGly-L-Ala-D-
isoGlnOBn (266 mg, 0.43 mmol) as viscous oil in quantitative
L-Ala-D-isoGlnOBn TFA salt (81 mg, 0.19 mmol) was dis-
solved in 1,4-dioxane (1 mL) and neutralized with N-
ethylmorpholine. After flash chromatography (CHCl3/MeOH
2:1), obtained residue was dissolved in MeOH (8 mL), 10%
Pd/C (30 mg) was added and it was subjected to hydrogen at-
mosphere under 30 PSI at room temperature for 24 h. Then the
catalyst was filtrated, solvent removed in vacuo and residue pu-
rified by column chromatography on silica gel (CHCl3/MeOH =
1:1). L-Ala-D-isoGln (16 mg, 0.07 mmol) was obtained as a
colorless solid in 39% yield. Rf = 0.15 (CHCl3/MeOH 1:1).
MS (ESI) m/z: [M-H]− calcd for C8H15N3O4 217.2, found 216.0.
1
yield. Rf = 0.62 (CHCl3/MeOH 2:1). H NMR (MeOH-d4,
300 MHz) δ 7.36–7.30 (m, 5H, Harom), 5.12 (s, 2H, CH2; Bn),
4.38 (dd, J = 4.7, 9.4 Hz, 1H, CH-α; isoGln), 4.29 (q, J = 7.1 Hz,
1H, CH-α; Ala), 3.35 (s, 1H, CH-α; AdGly), 2.47 (pt, J = 7.4,
7.7 Hz, 2H; CH2-γ; isoGln), 2.32–2.21 (m, 1H, CH-β; isoGln),
1.99–1.94 (m, 4H, 3CH; Ad and CH-β; isoGln), 1.86–1.75 (m,
12H, Ad), 1.36 (d, J = 7.1 Hz, 3H, CH3; Ala). 13C NMR
(MeOH-d4, 75.5 MHz) δ 176.20, 175.00, 174.17, 172.70
(C=O), 137.59 (Carom), 129.58, 129.27, 129.25 (CHarom), 67.48
(CH2; Bn), 64.58 (CH-α; AdGly), 53.61, 51.19 (CH-α; Ala,
isoGln), 39.43, 37.79 (CH2; Ad), 36.77 (C; Ad), 31.52
(CH2-γ), 28.13 (CH2-β), 29.80 (CH; Ad), 17.54 (CH3; Ala).
MS (ESI) m/z: [M]+ calcd for C27H39N4O5+ 499.3, found 499.4.
Tert-butyloxycarbonyl-(S)-1-adamantyl-glycyl-L-
alanyl-D-isoglutamine benzyl ester
(S)-BocAdGly (113 mg, 0.37 mmol) was dissolved in anhydrous
CH2Cl2 (5 mL) and cooled at 0 °C. EDC hydrochloride (85 mg,
0.44 mmol) and HOBt hydrate (49 mg, 0.37 mmol) were added
to the solution and the mixture was stirred for 0.5 h at 0 °C. Then
this mixture was added to the solution of L-Ala-D-isoGlnOBn
TFA salt (154 mg, 0.37 mmol), in anhydrous 1,4-dioxane
(5 mL), followed by triethylamine (65 μL, 0.74 mmol). The
reaction mixture was stirred for 1 h at 0 °C and then for 48 h at
room temperature. The mixture was diluted with AcOEt (20 mL)
and washed with water (10 mL). The organic layer was dried
over anhydrous MgSO4 and after filtration, concentrated in
vacuo. The residue was purified via column chromatography
on silica gel (CHCl3/MeOH 9:1) to yield Boc-(S)-AdGly-L-
Ala-D-isoGlnOBn (180 mg, 0.30 mmol) as viscous oil in 82%
(S)-1-Adamantyl-glycyl-L-alanyl-D-isoglutamine
(S)-AdGly-L-Ala-D-isoGlnOBn TFA salt (50 mg, 0.08 mmol)
was dissolved in 1,4-dioxane (1 mL) and neutralized with N-
ethylmorpholine. After flash chromatography (CHCl3/MeOH
2:1), obtained residue was dissolved in MeOH (5 mL), 10%
Pd/C (25 mg) was added and it was subjected to hydrogen at-
mosphere under 30 PSI at room temperature for 24 h. Then the
catalyst was filtrated, solvent removed in vacuo and residue pu-
rified by column chromatography on silica gel (CHCl3/MeOH
2:1). (S)-AdGly-L-Ala-D-isoGln (14 mg, 0.03 mmol) was ob-
tained as a colorless solid in 38% yield. Rf = 0.21 (CHCl3/MeOH
2:1). MS (ESI) m/z: [M + H]+ calcd for C20H32N4O5 408.5,
found 409.3.
1
yield. Rf = 0.76 (CHCl3/MeOH 9:1). H NMR (MeOH-d4,
300 MHz) δ 7.35–7.29 (m, 5H, Harom), 6.36 (d, J = 8.6 Hz, 0.5
ili < 1H, NH), 5.11 (m, 2H, CH2; Bn), 4.35 (dd, J = 4.5, 9.8 Hz,
1H, CH-α; isoGln), 4.21 (q, J = 7.1 Hz, 1H, CH-α; Ala), 3.66 (s,
1H, CH-α; AdGly), 2.52–2.41 (m, 2H; CH2-γ; isoGln), 2.31–
2.25 (m, 1H, CH-β; isoGln), 2.01–1.89 (m, 1H CH-β; isoGln),
1.95 (br s, 3H, CH; Ad), 1.71–1.59 (m, 12H, CH2; Ad), 1.34 (d,
J = 7.1 Hz, 3H, CH3; Ala). 13C NMR (MeOH-d4, 75.5 MHz) δ
176.27, 175.27, 174.12, 173.10 (C=O), 137.62 (Carom), 129.58,
129.29, 129.23 (CHarom), 80.68 (C; Boc), 67.43 (CH2; Bn),
64.47 (CH-α; AdGly), 53.60, 51.31 (CH-α; Ala, isoGln),
39.76, 37.91 (CH2; Ad), 37.35 (C; Ad), 31.57 (CH2-γ), 27.98
(CH2-β), 29.87 (CH3; Boc), 28.80 (CH; Ad), 17.38 (CH3; Ala).
MS (ESI) m/z: [M + H]+ calcd for C32H46N4O7 598.7, found
599.4.
N-(9-Fluorenylmethyloxycarbonyl)-(O-benzyl)-L-seryl-(S)
-1-adamantyl-glycyl-L-alanyl-D-
isoglutamine benzyl ester
Fmoc-L-Ser(OBn)-OH (70 mg, 0.17 mmol) was dissolved in
anhydrous CH2Cl2 (5 mL) and cooled at 0 °C. EDC hydrochlo-
ride (39 mg, 0.34 mmol) and HOBt hydrate (23 mg, 0.17 mmol)
were added to the solution and the mixture was stirred for 0.5 h
at 0 °C. Then this mixture was added to the solution of com-
pound (S)-AdGly-L-Ala-D-isoGlnOBn (104 mg, 0.17 mmol),
in anhydrous 1,4-dioxane (5 mL), followed by N-
ethylmorpholine (43 μL, 0.34 mmol). The reaction mixture
was stirred for 1 h at 0 °C and then for 48 h at room temperature.
The mixture was diluted with AcOEt (20 mL) and washed with
water (10 mL). The organic layer was dried over anhydrous
MgSO4 and after filtration, concentrated in vacuo. The residue
was purified via column chromatography on silica gel (CHCl3/
MeOH 15:1) to yield Fmoc-L-Ser(OBn)-(S)-AdGly-L-Ala-D-
isoGlnOBn (52 mg, 0.06 mmol) as viscous oil in 33% yield.
(S)-1-Adamantyl-glycyl-L-alanyl-D-isoglutamine benzyl ester
trifluoroacetate
Boc-(S)-AdGly-L-Ala-D-isoGlnOBn (260 mg, 0.43 mmol) was
suspended in anhydrous CH2Cl2 (1 mL) and TFA (0.5 mL) was
added. The reaction mixture was stirred at room temperature for

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Struct Chem (2019) 30:743–754
Rf = 0.31 (CHCl3/MeOH 15:1). 1H NMR (DMSO-d6,
300 MHz) δ 8.33 (d, J = 6.0 Hz, 1H, NH), 7.93–7.19 (m,
18H, Harom; Fmoc, 2Bn), 5.08 (m, 2H, CH2; Bn), 4.51 (m,
2H, CH2; Bn), 4.46–4.08 (m, 9H, CH; Fmoc, CH-α; Ala,
isoGln, Ser, AdGly, CH2; Fmoc, Bn), 3.64 (m, 2H, CH2; Ser),
2.39–2.31 (m, 1H, CH-β; isoGln), 1.83–1.71 (m, 4H, 3CH; Ad
and CH-β; isoGln), 1.57–1.50 (m, 12H, Ad), 1.18 (d, J =
6.7 Hz, 3H, CH3; Ala). 13C NMR (DMSO-d6, 75.5 MHz) δ
172.73, 172.04, 171.93, 169.21, 169.16 (C=O), 155.92 (CO;
Fmoc), 143.71, 143.62, 140.59, 138.08, 136.05 (Carom; Fmoc,
2Bn), 128.31, 128.04, 127.87, 127.78, 127.55, 127.38, 127.31,
127.28, 127.18, 126.99, 125.14, 120.02 (CHarom; Fmoc, 2Bn),
71.98, 69.69, 65.78, 65.39 (CH2; Ser, Fmoc, 2Bn), 60.12,
54.69, 51.26, 48.80, 46.51 (CH; Fmoc, CH-α; AdGly, Ser,
Ala, isoGln), 37.87, 36.26 (CH2; Ad), 36.05 (C; Ad), 29.88
(CH2-γ; isoGln), 27.60 (CH; Ad), 27.01 (CH2-β; isoGln),
17.37 (CH3; Ala). MS (ESI) m/z: [M + H]+ calcd for
C52H59N5O9 898.1, found 899.4.
The starting compound in the preparation of desmuramyl pep-
tides was Boc-L-alanyl-D-isoglutamine benzyl ester. Simplest
model compound, dipeptide L-Ala-D-isoGln, was obtained by
the removal of N- and C-protection groups (Scheme 1). After
Boc-group was removed using TFA, terminal NH2 was
deprotonated by N-ethylmorpholine (NEM) treatment.
Synthesis of tripeptide, as well as investigation of its
immunomodulating activity, is already described in previous
paper [4], but here we report a more efficient method for its
preparation. Protected tripeptide was synthesized by the con-
densation of (S)-Boc-1-adamantyl-glycine with L-Ala-D-
isoGln-OBn, using the optimized coupling method (EDC/
HOBt) coupling reagents in the mixture of solvents dichloro-
methane/1,4-dioxane 1:1). After N-Boc-deprotection obtained
compound was neutralized with NEM and then debenyzlated,
giving the tripeptide (S)-AdGly-L-Ala-D-isoGln (Scheme 1).
Tripeptide was prepared in 34% overall yield while previously
described synthetic procedure resulted with 17% overall yield.
Tetrapeptide was synthesized also using the EDC/HOBt
coupling method. N-Fmoc L-serine with and benzyl group
on side chain (Fmoc-L-Ser(OBn)-OH) was coupled to (S)-
AdGly-L-Ala-D-isoGln-OBn (Scheme 2). All protecting
groups (N-Fmoc and O-benzyl on Ser and benzyl on peptide
C-terminus) from tetrapeptide were cleaved in one step by
catalytic hydrogenolysis, giving L-Ser-(S)-AdGly-L-Ala-D-
isoGln.
L-Seryl-(S)-1-adamantyl-glycyl-L-alanyl-
D-isoglutamine
Fmoc-L-Ser(OBn)-(S)-AdGly-L-Ala-D-isoGlnOBn (51 mg,
0.06 mmol) was dissolved in MeOH (10 mL), 10% Pd/C
(25 mg) was added and it was subjected to hydrogen atmo-
sphere under 35 PSI at room temperature for 24 h. Then the
catalyst was filtrated, solvent removed in vacuo and residue
purified by column chromatography on silica gel (CHCl3/
MeOH = 1:1). Tetrapeptide L-Ser-(S)-AdGly-L-Ala-D-isoGln
(8 mg, 0.02 mmol) was obtained as a colorless solid in 29%
yield. Rf = 0.15 (CHCl3/MeOH 1:1). MS (ESI) m/z: [M-H]−
calcd for C23H37N5O7 495.6, found 494.0.
Molecular modeling
To rationalize the effect of increase of amino acids number on
structural preferences of modeling compounds, we performed
an exhaustive conformational search of the corresponding
zwitterionic and non-zwitterionic forms of desmuramyl dipep-
tide, tripeptide, and tetrapeptide. These compounds were
modeled in a solution by using polarizable continuum model
mimicking two solvents water as probably the most natural
environment and DMSO as the frequently used solvent for
NMR analysis.
Results
Synthesis
Synthesis of desmuramyl peptides was performed in order to
enable the NMR structural study of solution conformations.
We decided to follow the bottom-up approach, starting with
the simplest compound, desmuramyl dipeptide (L-Ala-D-
Scheme 1 Synthesis of di- and
tripeptide

Struct Chem (2019) 30:743–754
747
Scheme 2 Synthesis of
tetrapeptide
isoGln), having reduced number of H-bond donors and accep-
tors in comparison with the structurally most complex deriva-
tive, tetrapeptide (L-Ser-(S)-AdGly-L-Ala-D-isoGln). The aim
of this computational study was to locate the lowest-energy
conformers and to analyze the structural preferences for partic-
ular H-bonding patterns. With addition of amino acids, the total
number of potential H-bond donors and acceptors is also in-
creased. Therefore, an H-bonding network in the tetrapeptide
may significantly differ from H-bonding networks set up in the
lower homologues like tripeptide and dipeptide. However,
some of these H-bonds may be sturdy enough to withstand
the engagement of additional amino acids, thus showing a ca-
pacity to form the central structural spine and to initiate peptide
folding. We expected four sets of H-bond donor (NH) and
acceptor (C=O) groups due to the maximum number of four
amino acids chained in tetrapeptide L-Ser-(S)-AdGly-L-Ala-D-
isoGln. However, as isoGln and Ser have more than two het-
eroatoms, the total number of atoms engaged in H-bonding
increases. To make the labeling scheme as simple as possible,
we have decided to number atoms from C-terminus toward N-
terminus, because additional amino acids are linked in this par-
ticular way to its predecessor of the peptide series in the syn-
thetic procedure described. All H-bond donors and acceptors of
the same amino acids are labeled with the same number, for
example in isoGln as N1 and O1, because it is the first amino
acid at C-terminus. If the amino acid has more than one H-bond
donating or accepting group, additional letter is used, for exam-
ple, N1a and N1b for each NH donor group in isoGln. Details of
the proposed labeling scheme are shown in Fig. 2.
Figures 3, 4, and 5 show representative geometries of the
most stable conformers of all investigated derivatives in their
non-zwitterionic and zwitterionic forms. As expected, differ-
ent conformational minima can be sorted into conformational
families based on the determined H-bonding patterns. These
H-bonding patterns are shown for each conformer separately
unless more than one represented geometry has the same num-
ber and type of H-bonds. For example, all the non-zwitterionic
conformers of dipeptide L-Ala-D-isoGln belong to the same
conformational family having one five-membered (C5) and
one nine-membered (C9) ring. All of the H-bonds shown by
Fig. 2 Chemical structures of desmuramyl dipeptide, tripeptide, and
tetrapeptide in their non-zwitterionic (left side) and zwitterionic forms
(right side) showing the atom-numbering scheme for potential H-bond
donors (blue color) and H-bond acceptors (red color). Some of the col-
ored atoms can be H-bond donors and acceptors simultaneously

748
Struct Chem (2019) 30:743–754
Fig. 3 Energy level diagrams of the most stable conformers of dipeptide
L-Ala-D-isoGln in their non-zwitterionic (left side) and zwitterionic
forms (right side). Blue lines correspond to the relative free energies of
the most stable conformers optimized in water, and red in DMSO. Dashed
lines represent H-bonds verified by the QTAIM analysis. The summary of
the H-bonding patterns (n-membered rings labeled as Cn) for L-Ala-D-
isoGln modeled in water and DMSO is shown in blue (for non-
zwitterionic form) and in red-colored rectangulars (for zwitterionic form).
Non-polar hydrogens are omitted for clarity
dotted curves in Figures 3, 4, and 5 are validated by QTAIM
analysis. Values of topological parameters at bond critical
points between H-bond acceptors and H-bond donors, like
electron density ρ(r), Laplacian of the electron density
∇2ρ(r) and the energy density H(r), were calculated and com-
pared with the Koch and Popelier criteria for characterization
of H-bonds [27, 28]. Details about geometric parameters for
each H-bond and corresponding values of topological param-
eters are given in Tables S1–S6 in Supporting information.
The H-bonding patterns observed in dipeptide L-Ala-D-
isoGln are unique and differ in comparison with its higher
structural analogues. The conformational families contain
two or only one H-bond. The non-zwitterionic conformers
mostly differ in torsional angle describing rotation of the N-
terminal amino and isoGln amide group. All of the most stable
conformers have two H-bonded rings, C5 and C9. A new
structural feature, the ten-membered ring (C10) connecting
N-terminus with C-terminus, is found in the conformers of

Struct Chem (2019) 30:743–754
749
Fig. 4 Energy level diagrams of the most stable conformers of tripeptide
(S)-AdGly-L-Ala-D-isoGln in their non-zwitterionic (left side) and zwit-
terionic forms (right side). Blue lines correspond to the relative free en-
ergies of the most stable conformers optimized in water, and red in
DMSO. Dashed lines represent H-bonds verified by the QTAIM analysis.
The summary of the H-bonding patterns (n-membered rings labeled as
Cn) for (S)-AdGly-L-Ala-D-isoGln modeled in water and DMSO is
shown in blue (for non-zwitterionic form) and in red colored rectangulars
(for zwitterionic form). Non-polar hydrogens are omitted for clarity
the zwitterionic dipeptide. The same structural characteristic is
also present in higher analogues, thus confirming the favor-
able interaction between two ends in all of the investigated
derivatives. However, it should be taken into consideration
that all calculations have been performed using implicit sol-
vent model. The explicit model would be probably a better
choice for description of solute-solvent interactions, but com-
putationally more expensive. While neither of these ap-
proaches is definite, they do show trends and, therefore, de-
serve discussion. In principle, the H-bonding network formed
between adjacent groups in tripeptide and tetrapeptide is more
complex in comparison with their lower analogue. In
tripeptide, a specific interaction is formed between the amide
group of isoGln and C=O of AdGly (N1bH⋯O3) as ten-
membered ring (C10). The same type of H-bond exists in
the most stable conformers of tetrapeptide. According to the
conformational analysis, it seems that this bond is a key inter-
action in higher analogues and strong enough to resist engage-
ment of other H-bond donors and H-bond acceptors into the
peptide sequence.
The conformational families of tripeptide mostly contain
three hydrogen bonds. Except for the previously mentioned
C10 ring, there are also one thirteen-membered ring (C13,
O1bH⋯N3 or N3H⋯O1b) between terminal groups of the
peptide chain and one seven-membered ring (C7,
N1aH⋯O1) between NH amide and carboxylic group of

750
Struct Chem (2019) 30:743–754
Fig. 5 Energy level diagrams of the most stable conformers of
tetrapeptide L-Ser-(S)-AdGly-L-Ala-D-isoGln in their non-zwitterionic
(left side) and zwitterionic forms (right side). Blue lines correspond to
the relative free energies of the most stable conformers optimized in
water, and red in DMSO. Dashed lines represent H-bonds verified by
the QTAIM analysis. The summary of the H-bonding patterns (n-mem-
bered rings labeled as Cn) for L-Ser-(S)-AdGly-L-Ala-D-isoGln modeled
in water and DMSO is shown in blue (for non-zwitterionic form) and in
red-colored rectangulars (for zwitterionic form). Non-polar hydrogens are
omitted for clarity
isoGln. The last ring is only partially preserved in the
tetrapeptide. However, the extension of the peptide chain with
the fourth amino acid, Ser, triggers multiple H-bonding. In
zwitterionic conformers, both peptide ends are connected by
a sixteen-membered (C16) ring. Simultaneously, side chain of
Ser forms a seventeen-membered (C17) H-bonded ring with
tetrapeptide C-terminus. The conformational families of
tetrapeptide also contain some turn-like structures commonly
observed in natural peptides. Thus, a seven-membered (C7,
N2H⋯O4a) and a ten-membered (C10, N1aH⋯O4a) ring are
formed between neighboring amino acids. These turns are
usually labeled as γ- and β-turns. β-Turns can be further
classified according to values of the four torsion angles (ϕi+1
,
ψi+1, ϕi+2, ψi+2) of two central residues in sequence of four
amino acids. If these values are around − 60°, − 30°, − 90°,
and 0°, a corresponding β-turn is usually classified as type I.

Struct Chem (2019) 30:743–754
751
Based on the comparison of these values with the calculated
values of torsion angles (Table S7) in (S)-AdGly-L-Ala se-
quence of the tetrapeptides, all of the observed β-turns can be
described as type I.
Variations in distribution of conformers caused by different
solvents (modeled as a polarizable continuum) are mostly ob-
served within ensembles of the most stable conformers of the
smallest derivative, dipeptide. In tripeptide and tetrapeptide,
different solvent causes only slight changes in energies of the
reported conformers.
Discussion
In continuation of our research of peptidoglycan based
immunostimulators, we now report the synthesis and confor-
mational analysis of desmuramyl peptides. In the present
study, we placed our focus on the study of geometric prefer-
ences of the most stable conformers of the selected di-, tri-,
and tetrapeptides according to the performed computational
study and regarding results of the NMR analysis.
Considering the polarity of both solvents (water and
DMSO), we investigated possible trends on H-bonding pat-
terns regarding increase of amino acids number. While it is
natural to assess the presence of the specific interactions be-
tween molecules of solvent and solute, such approach can be
computationally very expensive. Although PCM approach ac-
counts only for the electrostatic part of the solvent interac-
tions, it also shows trends regarding H-bonding patterns, es-
pecially when robust backbone H-bonds are sturdy enough to
persist, independent of increase in number of potential H-bond
donors and acceptors.
Based on the in-depth computational study, we have
observed that H-bond network in higher peptide homo-
logues becomes more complex in comparison with lower
homologues, due to the increase in number of H-bond do-
nors and acceptors. Despite the complexity of the H-bond
network in model system, preferred structural interactions
can be noticed in zwitterionic and non-zwiterionic forms.
In both forms of di-, tri-, and tetrapeptides interactions
between terminal amino acids leading to folded structures
are present. The most significant structural feature ob-
served in model of tri- and tetrapeptide is ten-membered
ring (C10, N1bH⋯O3) between isoGln amide group and
C=O of AdGly. Based on the literature survey of the sim-
ilar compounds, this ten-membered ring could be consid-
ered as mimetic of key conformational feature of
muropeptides (MDP and its derivatives) characterized by
ten-membered ring formed between Ala-NH and N-
acetylcarbonyl group of MurNAc.
To be able to discuss the obtained results, we compared
our results with literature data and found that very similar
observations regarding the formation of the ten-membered
ring. S-shaped three-dimensional structure of MDP with
two successive β-turns involving the MurNAc, L-Ala, D-
Lac, and D-isoGln was proposed by Fermandjian based on
1H NMR study [9, 29]. First β-turn was formed between
NAc carbonyl and the L-Ala NH and the second β-turn in-
volved H-bonding between the D-Lac carbonyl and the D-
isoGln NH2. Harb’s group found some Benergy criteria^ ev-
idence for S-shaped conformation using molecular dynamics
calculation but all results were not in full agreement with
those obtained in the previous reports, indicating that exis-
tence of second β-turn is doubtful [10]. Research of
Boulanger et al. also confirmed the existence of the first β-
NMR study
1H and 13C resonances of desmuramyl dipeptide, tripeptide,
and tetrapeptide have been assigned by analysis of 1H, COSY,
1H-13C HSQC, and 1H-13C HMBC spectra. Full listing of 1H
and 13C chemical shifts in D2O and DMSO-d6 is available in
Table 1. The labeling scheme is shown in Fig. 6.
2D NOESY spectra of desmuramyl dipeptide, tripeptide,
and tetrapeptide were also recorded to confirm the assign-
ments and in order to provide spatial relationship between
protons. In D2O, only the side-chain amide protons of
isoGln in tripeptide and tetrapeptide could be detected in the
1D and 2D 1H spectra. The absence of signals of other amide
protons can be attributed to fast exchange with deuterons from
D2O, which are Binvisible^ in the proton frequency range. On
the other hand, all amide protons are well resolved in spectra
recorded in DMSO. Dipeptide, tripeptide, and tetrapeptide
displayed a single set of amide protons suggesting that only
one conformer is present on the NMR time scale.
In the 2D NOESY spectrum of dipeptide recorded in D2O,
in addition to several intraresidue NOE contacts, NOE cross-
peaks from isoGln CH-α, CH2-β, and CH2-γ to Ala CH3-β
protons were observed. In DMSO, only intraresidue NOE
correlations were detected.
The NOESY spectrum of tripeptide in D2O displayed NOE
contacts between CH2-γ protons of isoGln and CH2 protons
of adamantane. Sequential NOE correlations from the AdGly
CH-α and isoGln CH2-γ to the Ala CH3-β are also present in
the spectrum. In DMSO, isoGln NH showed NOE contacts
with CH2 protons of adamantane and sequential NOE contacts
with Ala CH3-β protons (Fig. S1). In addition, NOE cross-
peaks between NH2-E and NH2-Z protons of isoGln and Ala
CH3-β are observed in the spectrum.
The NOESY spectrum of tetrapeptide recorded in D2O
showed NOE correlations from the Ser CH-α and CH2-β
protons to isoGln CH2-β and adamantane CH2 protons. In
addition, NOE contacts between adamantane CH and isoGln
CH2-β protons are also detected in the spectrum. In the
NOESY spectrum of tetrapeptide in DMSO, mostly
intraresidue NOE are observed with exception of correlations
between the Ala CH-α to adamantane CH2 and isoGln NH
protons.

752
Struct Chem (2019) 30:743–754
Table 1 1H and 13C chemical shift data (in ppm)
Dipeptide
Tripeptide
D2O
Tetrapeptide
D2O
DMSO
DMSO
D2O
DMSO
δ1H
δ13C
δ1H
δ13C
δ1H
δ13C
δ1H
δ13C
δ1H
δ13C
δ1H
δ13C
D-isoGln
NH
8.47
4.09
8.63
4.09
9.09
3.96
CH-α
CH2-β
4.20
53.5
52.8
4.19
53.5
52.9
4.19
53.4
53.9
2.00
1.88
2.21
27.7
1.80
1.74
2.04
29.1
2.05
1.86
2.18
27.6
1.92
1.75
2.15
27.2
2.05
1.86
2.19
27.6
1.81
1.75
1.98
28.6
CH2-γ
COOH
C-δ
33.5
181.4
176.6
33.5
176.1
174.0
33.5
181.2
175.9
31.9
175.8
173.7
33.6
181.2
176.7
34.9
177.0
174.5
NH2-E
NH2-Z
L-Ala
NH
7.53
6.98
7.56
7.06
7.21
7.12
7.93
6.47
7.35
7.00
8.43
4.12
1.26
8.35
4.09
1.20
CH-α
CH3-β
CO
3.78
1.35
49.4
18.1
3.08
1.11
57.7
19.7
4.32
1.35
50.0
16.1
49.3
17.7
4.26
1.32
49.9
16.3
49.5
17.7
176.5
174.7
174.4
171.9
174.9
172.1
(S)-AdGly
NH
8.19
4.08
CH-α
CO
3.46
62.0
3.01
63.1
3.97
62.2
59.9
167.8
171.2
171.2
170.0
(Ad)C-α
(Ad)CH2-β
1.66
1.55
1.96
35.7
1.62
1.55
1.93
36.9
1.63
1.53
1.91
35.9
1.60
1.55
1.89
36.9
(Ad)CH-γ
(Ad)CH2-δ
27.6
37.5
28.1
38.5
27.9
38.2
28.2
38.5
1.56
1.63
1.50
1.54
1.55
L-Ser
CH-α
CH2-β
OH
3.57
3.66
55.7
63.4
3.27
3.51
57.2
64.5
CO
174.6
173.2
turn, but did not support the formation of the second β-turn
because of the lack of consistency with the NMR data and
with optimal structure of the lowest energy [30].
lysozyme model binding site [31], confirmed the presence
of the 10-membered ring as a preferred conformation of
MDP which was in good agreement with previous results
[30, 32] and showed that efficient binding of an aliphatic
desmuramyl analogue to binding site can be accomplished
Presence of ten-membered ring as dominant pattern was
confirmed in MDP analogues lacking the MurNAc moiety as
well. For example, ten-membered rings were observed in
desmuramyl derivatives which have MurNAc ring replaced
by 2-(acetamidocyclohexyloxy)acetyl groups [8]. In these
peptides, the first ring was formed between L-Ala (H-bond
donor) and acetamidocarbonyl group (attached at N-
terminus) and the second one between L-Ala and isoGln
side-chain carboxyl group, showing the strong stabilization
of Ala. It is important to note that Pristovšek’s group during
the investigation on 3D peptide pharmacophore for MDP-
type immunostimulants, by computer docking to T4
Fig. 6 The labeling scheme for NMR assignments

Struct Chem (2019) 30:743–754
753
even though it does not possess MurNAc moiety. These re-
sults indicate that (i) specific binding of this class of com-
pounds closely corresponded to efficient binding of Ala and
isoGln moieties, while loss of the contact with MurNAc moi-
ety can be compensated by additional binding modes with
substituents at N- and C-terminus in nonspecific manner and
(ii) probably one of the important structural feature of
desmuramyl and muramyl derivatives is stabilization of the
N-terminal amino acid by formation of ten-membered ring.
The presence of ten-membered ring (first β-turn) was like-
wise confirmed in immunologically active derivatives ester-
ified at C-terminus such as murabutide, while that conforma-
tion of Glu or isoGln side chain was less defined [9]. These
results suggest that stabilizations of biologically active con-
formations at N-terminus have important structural roles.
The same trend was also noticed in larger peptidoglycan
fragments such as peptidoglycan monomer (PGM).
Calculations of PGM yielded a set of conformers showing a
well-defined N-terminal peptide part connected to a rigid
carbohydrate unit, while the C-terminal part exhibits more
conformational freedom in accordance with experimental re-
sults [33, 34]. The recently published simulation of
gluсosaminyl-muramyl-dipeptide (GMDP) confirmed the
relatively compact structure in the N-terminal region [35].
Model compounds used in this research have incorporated
unnatural amino acid with bulky adamantane residue which
similarly causes rigidity of peptide backbone in the N-
terminal region which consequently leads to formation of
local structural features in this class of immunostimulating
compounds. The obtained results indicate that ten-membered
ring (C10, N1bH⋯O3) between isoGln amide group and
C=O of AdGly is a key interaction that forms the central
structural spine and initiates peptide folding in our model
systems. This observation about favorable peptide folding
is in good accordance with the reported NOE contacts of
CH2 protons of adamantane with CH2 and NH protons of
neighboring isoGln. Furthermore, NOE correlations between
Ser and isoGln side-chain protons also suggest the formation
of H-bonds bringing together two ends of tetrapeptide. We
can emphasize that starting from core dipeptide, engagement
of additional peptide bonds (amido groups acting as potential
H-bond donors and acceptors) initiates a formation of differ-
ent H-bonding networks while the presence of ten-membered
ring between isoGln and AdGly in tri- and tetrapeptides stays
conserved. H-bond acceptor groups in model system (for
example, C=O of the Ala or AdGly) are expected to compete
with other H-bond donor groups that can be attached to the
reported tetrapeptide. Therefore, a future study will include
syntheses and detailed conformational analyses of target
tetrapeptide functionalized with sugar moiety to investigate
its effect on the central backbone regarding the most impor-
tant H-bonding interactions, ten-membered ring initiating β-
turn-like motif.
Conclusion
Systematic conformational analysis (bottom-up approach) of
corresponding desmuramyl di-, tri-, and tetrapeptides in zwit-
terionic and non-zwitterionic forms was performed, based on
the theoretical model and compared to the experimental NMR
spectroscopic data. Interactions between terminal residues
were present in all compounds, in zwitterionic and non-
zwiterionic forms. Based on obtained results, we conclude that
ten-membered ring (C10, N1bH⋯O3) between isoGln amide
group and C=O of AdGly is the key structural pattern that
initiates peptide folding in described tri- and tetrapeptides
containing bulky adamantane residue. In continuation, synthe-
ses and conformational analyses of a larger model system of
muramyl peptide mimetics, like tetrapeptides functionalized
with sugar moieties, will be performed in order to investigate
effects of saccharide on the formation of ten-membered rings
initiating β-turn-like motifs.
Funding This work was funded by the Croatian Science Foundation
under project numbers 7444 and 7899. Computational resources were
provided by the Croatian National Grid Infrastructure (www.cro-ngi.hr)
at Zagreb University Computing Centre (SRCE).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical statement All ethical guidelines have been adhered to.
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