Novel fully protected muramic acid: A facile synthesis and structural study

Article abstract of DOI:10.1016/j.molstruc.2013.06.011

Synthesis and structural characterisation of novel fully protected muramic acid 2 (N-Boc-Mur-OMe, Mur = muramic acid) has been reported. N-Ac-Mur-OMe (1) prepared starting from commercially available N-acetylglucosamine, was treated with di-tert-butyl dicarbonate (Boc₂O) and N, N-dimethyl-4- aminopyridine (DMAP) in tetrahydrofuran. The intermediate mixed imide N-Ac-N-Boc-Mur-OMe was converted to N-Boc-Mur-OMe (2) upon in situ treatment with hydrazine hydrate in methanol. The structural analysis of 2, performed by IR and NMR spectroscopic methods and X-ray crystallography, was augmented by computational calculations including molecular and density functional theory studies (DFT) using M06/6-31G(d) computational model. The spectroscopic and DFT data obtained for novel Boc-protected 2 were compared with corresponding experimental values of its previously described Ac-protected analogue 1 in order to examine if the replacement of the protecting groups influences the conformational properties.

Full text of DOI:10.1016/j.molstruc.2013.06.011

Journal of Molecular Structure 1048 (2013) 349–356
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Novel fully protected muramic acid: A facile synthesis and structural
study
a
a
b
c
a,
ˇ
´
´
ˇ a
ˇ
´
⇑
Monika Kovacevic , Vladimir Rapic , Iva Lukac , Krešimir Molcanov , Ivan Kodrin , Lidija Barišic
a Department of Chemistry and Biochemistry, Faculty of Food Technology and Biotechnology, Pierottijeva 6, HR-10000 Zagreb, Croatia
b
-
ˇ
Division of Physical Chemistry, Ruder Boškovi´c Institute, Bijenicka cesta 54, HR-10000 Zagreb, Croatia
c Department of Chemistry, Faculty of Science, Horvatovac 102a, HR-10000 Zagreb, Croatia
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
ꢀ
ꢀ
N-Ac-Mur-OMe is transformed into
N-Boc-Mur-OMe.
Eight membered NHꢁ ꢁ ꢁOCester
intramolecular hydrogen bond is
preserved in solution upon alteration
of protecting group.
ꢀ
Intermolecular hydrogen bonding
occurs in the solid-state of N-Boc-
Mur-OMe.
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and structural characterisation of novel fully protected muramic acid 2 (N-Boc-Mur-OMe,
Mur = muramic acid) has been reported. N-Ac-Mur-OMe (1) prepared starting from commercially
available N-acetylglucosamine, was treated with di-tert-butyl dicarbonate (Boc2O) and N,N-dimethyl-4-
aminopyridine (DMAP) in tetrahydrofuran. The intermediate mixed imide N-Ac-N-Boc-Mur-OMe was
converted to N-Boc-Mur-OMe (2) upon in situ treatment with hydrazine hydrate in methanol. The
structural analysis of 2, performed by IR and NMR spectroscopic methods and X-ray crystallography,
was augmented by computational calculations including molecular and density functional theory studies
(DFT) using M06/6-31G(d) computational model. The spectroscopic and DFT data obtained for novel
Boc-protected 2 were compared with corresponding experimental values of its previously described
Ac-protected analogue 1 in order to examine if the replacement of the protecting groups influences
the conformational properties.
Received 21 March 2013
Received in revised form 5 June 2013
Accepted 5 June 2013
Available online 12 June 2013
Keywords:
Protected muramic acid
Structural analysis
X-ray crystallography
DFT calculations
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
The structural properties of bioorganometallic muropeptides I
[N-Ac-Mur-Ala-Fca; Fca = 10-aminoferrocene-1-carboxylic acid)]
Muropeptides are degradation products of peptidoglycans
that contain muramic acid (Mur) coupled to amino acids. They
are known as biologically active compounds, e.g. they exhibit
increased immunoadjuvant activity [1].
and II [N-Ac-Mur-NH-Fn-R; Fn = 1,10-ferrocenylene, R = H (IIa),
COOMe (IIb), NHAc (IIc)] have already been investigated in our
group [2,3] (Fig. 1). The detailed structural studies in solution con-
firmed strong influence of ferrocene moiety on conformational
properties of I and II. Moreover, the cyclic voltammograms of II
featured a one-electron oxidation for the ferrocene/ferrocenium
redox couple. Furthermore, Fca-containing peptides with Ala were
⇑
Corresponding author. Tel.: +385 1 46 05 069; fax: +385 1 48 36 082.
E-mail address: lbaris@pbf.hr (L. Barišic´).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.06.011

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M. Kovacevic et al. / Journal of Molecular Structure 1048 (2013) 349–356
OH
OH
OH
O
O
O
OH
OH
OH
NHAc
O
HO
NHAc
NH
HO
HO
O
O
O
H
N
O
H
N
N
H
COOMe
Fe
Fe
Fe
O
O
MeO
R
NHBoc
O
I
II (a, R = H; b, R = COOMe; c, R = NHAc)
III
Fig. 1. Ferrocene conjugates with muramic acid I–III.
O
O
O
O
Ph
Ph
H
H
N
O
N
OCH2Ph
O
OCH2Ph
(i)
O
O
Ac
Boc
(ii)
O
O
MeO
MeO
1
2
(i) (Boc)2O, DMAP/ THF, (ii) N2H4/ MeOH
Scheme 1. Transformation of N-Ac-Mur-OMe (1) to Boc-protected analogue 2. Intramolecular hydrogen bonds are shown by dashed lines.
found to exhibit quite different conformational properties depend-
ing on their primary structures [4]. Taking these findings into
account, our future research will be aimed to investigate confor-
mational and electrochemical properties of muropeptides III
(Fca-Mur) which contain an exchanged sequence of amino acids
relative to IIb (Fig. 1). Since the design of bioorganometallics III
is based on coupling of N-protected Fca with C-protected Mur,
there is a demand to provide a suitable N-protected muramic acid.
Taken into consideration that the cleavage of N-acetyl protecting
group of N-Ac-Mur-OMe (1) requires harsh conditions (refluxing
in HCl [5]) incompatible with other sensitive functionalities, its
conversion [6,7] into N-Boc-Mur-OMe (2) that requires milder
cleavage conditions (TFA/CH2Cl2) [5] is mandatory.
The aim of the study presented in this paper is to acquire addi-
tional insights about the novel compound N-Boc-Mur-OMe (2). The
synthesis of the N-Boc-Mur-OMe (2) is reported, the spectroscopic
and structural properties in solution and solid phase are deter-
mined and further supplemented with the computational studies
(DFT). Since conformational analysis of correlated N-Ac-Mur-OMe
(1) in solution indicated the presence of a strong 8-membered
NHAcꢁ ꢁ ꢁOCester intramolecular hydrogen bond (IHB) in equilibrium
with open forms [3] (Scheme 1), the focus of herein presented
research is to explore whether the replacement of Ac-protecting
group with more sterically demanding Boc-group will influence
the conformational properties of the resulted N-Boc-Mur-OMe (2).
mixture. Melting point was determined by using a Büchi appara-
tus. Infrared spectra were recorded as CH2Cl2 solutions between
NaCl windows or as KBr disks by using a Bomem MB 100 mid FTIR
spectrometer. The 1H and 13C NMR spectra were recorded on Bru-
kerAvance 600 MHz in CDCl3 with Me4Si as internal standard.
Spectral assignment was carried out by using standard 2D-
spectroscopy.
2.2. Synthesis of benzyl 2-tert-butoxycarbonyl-4,6-O-benzylidene-2-
deoxy-3-O-[(R)-1-(metoxycarbonyl)ethyl]-a-D-glucopyranoside (2, N-
Boc-Mur-OMe)
The conversion of N-Ac-Mur-OMe (1) [8] to N-Boc-Mur-OMe (2)
was performed following the procedure for amide to carbamate
transformation [7]. N-Ac-Mur-OMe (1) (1 g, 2.05 mmol), prepared
starting from N-acetyl-D-glucosamine [8], was dissolved in freshly
distilled THF (10 ml) and di-tert-butyl dicarbonate (Boc)2O (0.3 g,
1.37 mmol) and DMAP (58.2 mg, 0.48 mmol) were added. After
½ h stirring at room temperature, the second portion of (Boc)2O
(0.6 g, 2.74 mmol) was added and the stirring was continued at
50 °C for 1 h. Upon addition of third portion of (Boc)2O (0.6 g,
2.74 mmol), the reaction mixture was refluxed for 3 h and stirred
at room temperature for 36 h until no increasing of imide
intermediate1 had been detected by TLC analysis (hexane:ethylace-
tate = 3:1). The obtained crude imide was diluted with 20 ml of
MeOH and in situ treated with hydrazine hydrate (0.25 ml,
7.96 mmol). After 24 h of stirring at room temperature, the solvents
were removed under reduced pressure and the residue was dis-
solved in dichloromethane. The organic layer was washed with
10% aqueous solution of citric acid and brine, dried over Na2SO4
and evaporated in vacuo. After TLC-purification (hexane:ethylace-
tate = 3:1) the white crystals of pure N-Boc-Mur-OMe 2 (0.8 g,
2. Experimental and theoretical methods
2.1. General procedure
Most of the syntheses were carried out under argon. The CH2Cl2
used for synthesis and FT-IR was dried (P2O5), distilled over CaH2,
and stored over molecular sieves (4 Å). N-Acetyl-D-glucosamine
(Aldrich), the starting compound for preparation of benzyl 2-acet-
1
Small amount of intermediate was worked-up as it was described above and
subjected to NMR analysis in order to improve the proposed imide structure: 1H NMR
(CDCl3, 300 MHz): d (ppm) 7.49–7.29 (m, 10H, CHPh), 5.55 (s, 1H, CHbenzylidene), 4.91(s,
1H, H-1), 4.81(brs, 1H, H-2), 4.65 (d, 1H, J = 12.5 Hz, OCH2a-Ph), 4.50 (d, 1H,
J = 12.0 Hz, OCH2b-Ph), 4.31 (q, 1H,J = 6.7 Hz, CHLac), 4.15 (dd, 1H, J = 4.9 Hz, J = 9.8 Hz,
H-3), 3.87–3.83 (m, 1H, H-4), 3.74–3.67 (m, 2H,H-6b,H-6a), 3.64 (s, 3H, OCH3),3.58
(pt, 1H, H-5), 2.36 (s, 3H, COCH3), 1.40 (s, 9H, (C(CH3)3), 1.37 (d, 3H, J = 6.8 Hz,
CH3-Lac).
amido-4,6-O-benzylidene-2-deoxy-3-O-[(R)-1-(metoxycarbonyl)-
ethyl]-a-D-glucopyranoside (1, N-Ac-Mur-OMe), was used as
received [8]. All solvents were dried according to general proce-
dures for purification of solvents, unless indicated otherwise.
Product was purified by preparative thin layer chromatography
on silica gel (Merck, Kieselgel 60 HF254) by using EtOAc/hexane

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M. Kovacevic et al. / Journal of Molecular Structure 1048 (2013) 349–356
72%) were obtained. Mp 158.7–163.7 °C; Rf = 0.6 (hexane:ethylace-
tate = 3:1); IR (CH2Cl2, cmꢂ1):
3431(w, NHfree), 3356 (m, NHassoc.),
2.4. Theoretical methods
m
1732 (s, COester), 1709 (s, COBoc). 1H NMR (CDCl3, 300 MHz): d (ppm)
7.45–7.25 (m, 10H, CHPh), 6.02 (d, 1H, J = 5.1 Hz, NH), 5.56 (s, 1H,
CHbenzylidene), 5.27 (d, 1H, J = 2.6 Hz, H-1), 4.68 (d, 1H, J = 12.0 Hz,
OCH2a-Ph), 4.55 (d, 1H, J = 12.0 Hz, OCH2b-Ph), 4.50 (q, 1H,
J = 6.8 Hz, CHLac), 4.19 (dd, 1H, J = 4.2 Hz, J = 9.5 Hz, H-3), 3.84–3.81
(m, 1H, H-4), 3.79 (d, 1H, J = 9.6 Hz, H-6b), 3.73 (d, 1H, J = 9.6 Hz,
H-6a), 3.72 (brs, 4H, OCH3 + H-2), 3.65 (pt, 1H, H-5), 1.45 (s, 9H,
(C(CH3)3), 1.40 (d, 3H, J = 6.9 Hz, CH3-Lac); 13C NMR (CDCl3,
75 MHz): d (ppm) 174.48 (COester), 156.14 (COBoc), 137.38, 137.31
(CPh), 128.99–125.87 (CHPh), 101.27 (CHbenzylidene), 97.59 (C1),
83.33 (C5), 79.14 (C(CH3)3), 75.34 (CHLac, C3), 70.05 (CH2Ph), 69.02
(C6), 62.83 (C4), 54.92 (C2), 52.09 (OCH3), 28.43 (C(CH3)3), 18.78
(CH3-Lac). Anal. Calcd. [C29H37N1O9]: C 64.07, H 6.86, N 2.58%; Found:
C 64.04, H 6.89, N 2.56%.
The initial monomer geometry of N-Boc-Mur-OMe (2) was cut
out from the X-ray determined structure. The conformational anal-
ysis was performed with MacroModelv9.8 [14] molecular model-
ling program applying different search methods and force fields.
The class of the most stable conformers thus obtained, was opti-
mized with the three different functionals (B3LYP, M06 and
M06-2X) and 6-31G(d) basis set in vacuum and implicitly mod-
elled chloroform (using polarizable continuum model, PCM).
Vibrational frequencies were calculated to verify true minima on
the potential energy surface. All quantum mechanical calculations
were performed with Gaussian09 [15] program. The molecules
were visualized using GaussView [15] and Chem3D (Cambridge-
Soft, Cambridge, MA) programs. Root mean square deviations were
calculated by superposition of modelled over experimental struc-
ture using Maestro program [16]. The selected topological param-
eters were calculated with the help of AIM2000 program [17].
2.3. Determination of crystal structure
Crystals suitable for data collection were grown from ethanol by
vapour diffusion at low temperature (4 °C). Single crystal measure-
ment was performed on an Oxford Diffraction Xcalibur Nova R
(CCD detector, microfocus Cu tube) at room temperature [293(2)
K]. Since the absolute configuration was known from the synthetic
procedure, only the symmetry-independent part of the Ewald
sphere was measured. Program package CrysAlis PRO [9] was used
for data reduction. The structures were solved using SHELXS97
[10] and refined with SHELXL97 [10]. The models were refined
using the full-matrix least squares refinement; all non-hydrogen
atoms were refined anisotropically. Hydrogen atoms were treated
as constrained entities, using the command AFIX in SHELXL97
[10]. Molecular geometry calculations were performed by PLATON
[11], and molecular graphics were prepared using ORTEP-3 [12],
and CCDC-Mercury [13]. Crystallographic and refinement data for
the structures reported in this paper are shown in Table 1.
3. Results and discussion
3.1. Synthesis
N-Ac-Mur-OMe (1) was prepared starting from commercially
available N-acetyl-D-glucosamine [8]. The transformation of amide
1 to carbamate 2 was performed following the literature procedure
[7]. The reaction initiated by action of Boc2O/DMAP (ꢃ3.3 eq.)
under reflux was completed by stirring at room temperature leav-
ing crude imide N-Ac-N-Boc-Mur-OMe which was in situ treated
with hydrazine hydrate (4 eq.) to give N-Boc-Mur-OMe (2)
(Scheme 1).
3.2. IR and NMR spectroscopy
Conformational analysis of novel compound 2 in solution was
performed by IR and NMR spectroscopy. Since IR spectrum contains
two distinct sets of NH frequencies above and below
Table 1
Crystallographic, data collection and structure refinement details.
U ¼ 3400 cmꢂ1, the presence of both free and hydrogen-bonded
e
NH groups is indicated. Hydrogen bonding is additionally sup-
Compound
N-Boc-Mur-OMe (2)
C29H37N1O9
543.60
ꢂ1
e
Empirical formula
ported with low-energy COester vibration around U ¼ 1730 cm
as a result of its hydrogen-bond-accepting ability. In order to
determine if associated NH groups are engaged in intra- or intermo-
lecular manner, IR spectra were measured within the concentration
range c = 5 ꢄ 10ꢂ2 M to 1 ꢄ 10ꢂ3 M. The unchanged intensity ratio
of the NH vibrations upon dilution strongly supports intramolecular
hydrogen-bonding pattern (Fig. 2 and Scheme 1). These findings are
compatible with those obtained for Ac-protected analogue 1 [3]
suggesting that bulky Boc-group do not considerably interfere with
the hydrogen-bonding (Table 2). In addition, solid-state IR spectra
of both 1 and 2 contain exclusively signals of associated NH and
CO groups (Fig. 2 and Table 2).
Formula (wt./g molꢂ1
)
Crystal dimensions (mm)
Space group
a (Å)
b (Å)
c (Å)
0.15 ꢄ 0.04 ꢄ 0.03
P 212121
5.10930(10)
21.2302(5)
26.6982(5)
90
a
(°)
b (°)
90
c
(°)
90
Z
4
V (Å3)
2895.99(10)
1.247
0.765
Dcalc (g cmꢂ3
l
)
(mmꢂ1
)
H
T (K)
range (°)
3.31–76.15
293(2)
The suggested NHBocꢁ ꢁ ꢁOCester IHB was elucidated by NMR spec-
troscopy as a useful method to estimate a hydrogen-bond-donating
capacity of NH groups. Commonly, amide protons involved in
hydrogen bonds are shifted above d = 7 ppm in non-hydrogen-
bonding solvents. (Owing to the fast equilibration between hydro-
gen-bonded and free states, proton chemical shifts represent
weighted averages of the chemical shifts of all of the contributing
states). In comparison with Ac-protected 1, amide proton of
Boc-protected 2 is shifted upfield in non-competitive aprotic CDCl3
(d = 6.02 ppm) suggesting somewhat difficult involvement in
hydrogen-bonding. Since changing solvent to the strong hydrogen
bond acceptor DMSO (expected to induce a downfield shift of
solvent-exposed NH groups but not to affect the chemical shifts
of NH groups associated in intramolecular manner) did not cause
Diffractometer type
Range of h, k, l
Xcalibur Nova
ꢂ6 < h < 4
ꢂ26 < k < 24
ꢂ32 < l < 33
9515
5409
4621
Multi-scan
0.0220
Reflections collected
Independent reflections
Observed reflections (I P 2
Absorption correction
Rint
r
)
R (F)
0.0816
0.2566
Rw (F2)
Goodness of fit
H atom treatment
1.045
Constrained
351, 20
0.724; ꢂ0.409
No. of parameters, restraints
D
qmax
,
D
qmin (eÅꢂ3
)

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M. Kovacevic et al. / Journal of Molecular Structure 1048 (2013) 349–356
(a)
(b)
0
0
-2
-4
-6
-8
-5
-10
3600
3500
3400
3300
3200
3600
3500
3400
3300
3200 3150
Wavenumber (cm-1)
Wavenumber (cm-1)
Fig. 2. The NH-stretching vibrations in concentration-dependent IR spectra of 2 in CH2Cl2 (a) and KBr (b).
Table 2
IR spectroscopic data [c = 5 ꢄ 10ꢂ2 M in CH2Cl2 and as KBr disc, (cmꢂ1)], 1H NMR data and chemical shift variation (
D
d) of the NH protons 1 [3] and 2 [c = 5 ꢄ 10ꢂ2 M in CDCl3 and
DMSO, (ppm)].
mNH (free)
mNH (associated)
mCO (ester)
dNH
DdNH
1
2
CH2Cl2
CsI
3431 (w)
3356 (m)
3300 (m)
1732 (s)
1737 (s)
CDCl3
DMSO
7.49
7.83
0.34
CH2Cl2
KBr
3438 (w)
3365 (m)
3355 (m)
1733 (s)
1728 (s)
CDCl3
DMSO
6.02
6.59
0.57
significant chemical shift (
was indicated.
D
d = 0.57), the IHB of medium strength
in CDCl3 [20] revealed that amide proton temperature coefficients
are significantly influenced by the solvent polarity. Hence, contrary
to the case of the aqueous solution, a measured large temperature
Whereas hydrogen-bond-assisted self-assembly of neighbour-
ing molecules is subjected to disruption upon dilution or heating,
we measured concentration- and temperature-dependent NMR
spectra in order to clarify the pattern of hydrogen-bonding (intra-
or intermolecular) in 2. The chemical shifts of protons involved in
intermolecular hydrogen bonds are expected to be considerably
dependence (Dd/DT) in non-competitive CDCl3 is referred to ini-
tially shielded amide group that became exposed through dissoci-
ation of intermolecular aggregates or the disruption of
intramolecular hydrogen bonds [20]. Taken into account that IR
and concentration-dependent NMR analysis already excluded
intermolecular aggregations, relatively large temperature depen-
shifted upfield (
D
d = 1 ꢂ 4 ppm) upon heating or dilution. Since
successive dilution (50–6.25 mM) did not affect the chemical shift
of the amide proton (Fig. 3a), aggregation effects in CDCl3 are
excluded and intramolecular hydrogen bond is strongly supported.
In addition, temperature-dependent NMR spectra, showing
negligible higher-field shifting of amide protons (Fig. 3b), sustain
proposed intramolecular hydrogen-bonding motif. Namely, inter-
molecular hydrogen bonds and hydrogen bonds to solvent
undergo cleavage at increased temperatures which results in
discernible upfield shifting of the related amide protons [4,18].
Conformational studies of peptides in aqueous solutions [19] and
dence of examined amide proton (
participation in intramolecular hydrogen bond.
D
d/
D
T = ꢂ6 ppb) indicates its
3.3. Crystal structure
The molecular structure of N-Boc-Mur-OMe (2) determined by
single crystal X-ray diffraction is shown in Fig. 4. Its
stereochemistry is known from the synthesis, so determination
of absolute configuration was not necessary. There is no intramo-
lecular hydrogen bond, which was observed spectroscopically,
δ (ppm)
(a) δ (ppm)
(b)
6,5
7
6
6
5
5,5
0
10
20
30
40
50
300
305
310
315
320
325
c (mM)
T (K)
Fig. 3. (a) Concentration- and (b) temperature-dependent NH chemical shifts of 2.

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M. Kovacevic et al. / Journal of Molecular Structure 1048 (2013) 349–356
Fig. 4. ORTEP-3 [12] drawing of a molecule of N-Boc-Mur-OMe (2). Displacement ellipsoids are drawn for the probability of 50% and hydrogen atoms are shown as spheres of
arbitrary radii.
therefore the conformation in the solid state is different than in
solution. It also markedly differs from the calculated minimum-en-
ergy conformer (vide infra). In the crystal structure the intramolec-
ular hydrogen bond was not observed; instead an intermolecular
hydrogen bond NHBocꢁꢁꢁOCBoc is present. It links the molecules into
chains parallel with the direction [100] (Fig. 5). Therefore, it can be
argued that this hydrogen bond is responsible for the stabilisation
of the conformation found in the solid state, since there are no
other intermolecular interactions other than dispersion. While
the solid-state conformation of the molecule itself is not an energy
minimum, it can pack much more efficiently than the most stable
conformer; the energy of the crystal packing (especially of inter-
molecular hydrogen bonding) can apparently offset the difference.
The crystals were rather poor, and the quality of the measured
data was also poor, resulting with a relatively high R (Table 1).
Displacement ellipsoids illustrate conformational flexibility of the
molecule (Fig. 4), also indicating that the solid-state conformation
may not be very stable. All flexible parts of the molecule (i.e. those
where free rotation about single bonds is possible) have much lar-
ger ellipsoids than the relatively rigid central part. Especially large
and elongated ones belong to the Boc and methylpropanoate
groups (Fig. 4), indicating a disorder. The present measurement
cannot distinguish between static and dynamic disorder (i.e. many
similar, but stable conformations vs. large-amplitude motion),
therefore, a conformational analysis was performed by computa-
tional methods.
3.4. Computational study
Computational studies have already proven very useful for
acquiring additional information about the geometry of the most
stable conformers and for better understanding of interactions be-
tween the molecules. Some studies included research of very
similar N-Ac-Mur-OMe (1) itself or coupled to the ferrocene unit
[3]. Thereby, the synthesized N-Boc-Mur-OMe (2) was investigated
by the means of DFT calculations with the aim to find the most sta-
Fig. 5. Hydrogen bonded chain extending in the direction [100]. Parameters of the hydrogen bond: d(N1-H1A) = 0.86 Å; d(H1Aꢁ ꢁ ꢁO8i) = 2.13 Å; d(N1ꢁ ꢁ ꢁO8i) = 2.951(4) Å; (N1-
H1Aꢁ ꢁ ꢁO8i) = 159°. Symmetry operator (i) ꢂ1 + x, y, z.

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M. Kovacevic et al. / Journal of Molecular Structure 1048 (2013) 349–356
354
Table 3
Relative energies and the RMSD (calculated relative to the monomer unit from X-ray structure) of the most stable conformers of 2 in chloroform and in vacuum optimized with
M06 and M06-2X functionals with 6-31G(d) basis set. Types of conformers based on the geometries shown in Fig. 6.
M06/6-31G(d)
Chloroform
M06-2X/6-31G(d)
Chloroform
Vacuum
Type
Vacuum
Type
Type
D
E (kJ molꢂ1
)
RMSD
D
E (kJ molꢂ1
)
RMSD
Type
D
E (kJ molꢂ1
)
RMSD
D
E (kJ molꢂ1
)
RMSD
M1
M2
M3
M4
M5
0.00
0.33
1.18
1.48
3.14
1.125
2.199
M2
M1
M4
M3
M5
0.00
0.03
2.44
2.46
2.81
2.203
1.139
M2
M1
M4
M3
M5
0.00
0.05
2.16
2.27
3.15
2.247
1.132
M2
M1
M4
M5
0.00
1.04
3.51
3.60
3.78
2.248
1.142
*
–
*
Geometry different from the M1–M5.
Fig. 6. The most stable conformers of 2 optimized with M06/6-31G(d) in chloroform (PCM). The IHB with corresponding distances between hydrogen and hydrogen bond
acceptor atom are displayed.
ble conformer (or conformers) that existed (or coexisted) in solu-
tion and to compare them with the geometry from the crystal
structure. This might provide better explanation of the favourable
intra- and intermolecular interactions, mainly hydrogen bonds be-
tween the suitable hydrogen bond donors and acceptors of the
same or different molecules. Trying to find the optimal ratio
between the quality and the time of each calculation, for the fast
generating of the ensembles of the conformers, the molecular
mechanics with the conformational space search algorithms and
force-field included in MacroModel was applied. Only the most

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M. Kovacevic et al. / Journal of Molecular Structure 1048 (2013) 349–356
approximately 0.30 kJ molꢂ1in CHCl3 and 0.03 kJ molꢂ1 in vacuum.
More pronounced destabilization is the consequence of the phenyl
and benzyloxy groups rotations as it can be seen in Fig. 6 and in Ta-
ble 3. For example, the relative energy difference between M1 and
M3 conformers with different orientations of phenyl group is
approximately 1.20 kJ molꢂ1 in CHCl3 and 2.04 kJ molꢂ1 in vacuum.
All the intramolecular hydrogen bonds were verified according to
the suggested Koch and Popelier criteria [21] based on the topolog-
ical properties (like electron density and Laplacian of the electron
density) at the bond critical point between the hydrogen and
hydrogen bond acceptor atom. The calculated values are in the
range of 0.022–0.034 a.u. for electron density,
q(r), and 0.0714–
0.0745 for Laplacian of the electron density,
»
2q(r), which is also
confirmation of hydrogen bond presence. The slightly large values
correspond to the hydrogen bond of the conformers M1 and M3
with the HꢁꢁꢁO distance of 2.00 Å and the NꢂH–O angle of 162°.
For comparison, conformers M2 and M4, with different torsional
angle describing rotation around (H)N–C(O) bond, have HꢁꢁꢁO dis-
tance of 2.02 Å and the NꢂH–O angle of 158°.
Previously described approach with one monomer unit is prob-
ably adequate for the description of the intramolecular interactions
in solution, mainly hydrogen bonds. This is also supported by spec-
troscopic data, as it was already shown in preceding chapters. At
the same time, the non-existence of the intramolecular hydrogen
bond in the X-ray structure, prompted the idea to investigate the
energetics associated with the crystal packing of monomer units.
The simplest model for the crystal structure was used. The dimer
unit consisted of two monomer units connected by the intermolec-
ular hydrogen bond were cut out from the crystal structure and
optimized in vacuum at the same level of theory previously used
for the monomer unit optimization, i.e. M06/6-31G(d). These two
geometries are shown in Fig. 7. The accompanied RMSD value of
0.657 was determined for non-hydrogen atoms. The interaction
energy was calculated as the energy difference between the energy
of dimer and twice the energy of the monomer. In this way calcu-
lated interaction energy might have the inherent error known as
Basis Set Superposition Error (BSSE), so the correction by the coun-
terpoise method (CP) is usually needed [22–24]. The calculated
interaction energy is around ꢂ123 kJ molꢂ1 (ꢂ61 kJ molꢂ1 with
included BSSE correction) what implies considerable stabilisation
as a consequence of favourable interactions (mainly intermolecular
hydrogen bond) between two monomer units. The accompanied
deformation of the monomer units, which resulted from crystal
packing, was calculated as the energy difference between the
Fig. 7. The dimer of 2 (non-polar hydrogen atoms excluded) and its geometry
optimized at M06/6-31G(d) in vacuum. The intermolecular HB is displayed.
stable conformers (approximately 50 of them) were further opti-
mized with the Gaussian09 program. At first, the most widely ac-
cepted B3LYP functional with the 6-31G(d) basis set in vacuum
was used. The reliability of the computational model was tested
by comparison of two geometries. The referent geometry was
monomer unit of 2 cut out from X-ray determined structure and
it was compared with its optimized geometry. Upon the optimiza-
tion, the major difference between these two geometries was dif-
ferent orientation of the phenyl groups due to their rotation.
Consequently, newer functionals like M06 and M06-2X that are
capable to treat dispersion interactions more adequately were
decided to use. The slightly better agreement is achieved with
M06 functional as it can be seen from the RMSD calculated for
the non-hydrogen atoms by superposition of the optimized and
the corresponding crystal geometry (monomer or dimer). The cal-
culations were performed in vacuum and in chloroform. The data
calculated for the most stable conformers are presented in Table
3 and in Fig. 6. MOL files of selected optimized geometries are
available as Supplementary files.
The ensembles of the most stable conformers calculated with
two selected functionals (M06 and M06-2X) in vacuum and in
CHCl3 have very similar distributions. All the conformers include
intramolecular NHꢁ ꢁ ꢁOC hydrogen bond between the carbamate
nitrogen (from the amino protected group) and the carboxylic oxy-
gen (from the carboxyl protected group) forming 8-membered
ring. Several subsets of conformers were formed, depending on
the torsional angles that define rotation of groups attached to the
central bicyclic moiety. In all of the displayed conformers the ori-
entation of the lactate ester group is fixed by NHꢁꢁꢁOC hydrogen
bond with the protected amino group. The smallest destabilization
in energy arises from the different position of the Boc-protecting
group, which is determined by the value of H–N–C–O dihedral
angle – M1, M3 and M5 (around 170°), M2 and M4 (around
ꢂ10°). For example, this difference between two sets of the con-
formers (M1 and M2, M3 and M4) calculated at M06/6-31G(d) is
Fig. 8. The superposition of optimized X-ray monomer unit (blue colour, without
IHB) over the similar most stable conformer of 2 (red colour, with IHB) optimized at
M06/6-31G(d). The non-polar hydrogen atoms are not displayed. (For interpreta-
tion of the references to colour in this figure legend, the reader is referred to the
web version of this article.).

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M. Kovacevic et al. / Journal of Molecular Structure 1048 (2013) 349–356
356
energy of each monomer it acquired in dimer and the energy of the
most stable conformer calculated with the same computational
model. The corresponding changes in energy are approximately
19 kJ molꢂ1 for the monomer unit containing hydrogen bond
acceptor atom and 33 kJ molꢂ1 for the other monomer unit with
the hydrogen bond donor atom included. These energetically unfa-
vourable deformations were obviously largely overcome by the
energy gained through the favourable intermolecular interactions,
mostly hydrogen bonds. Alternatively, the energy gained from the
intramolecular hydrogen bond formation in one monomer was
roughly estimated as the energy difference of two optimized
geometries shown in Fig. 8. The energy of the conformer M1 (with
IHB) is about 17 kJ molꢂ1 lower than the energy of the conformer
(without IHB) optimized starting from the geometry of monomer
unit initially cut out from crystal structure. Using these simplified
models, it was demonstrated that intermolecular or intramolecular
hydrogen bonds notably contribute in stabilization of the investi-
gated system. Using these simplified models, it was demonstrated
that intramolecular hydrogen bond could also stabilize compound
2 (e.g. in solution) in the absence of the intermolecular hydrogen
bond (crystal lattice).
The computational resources provided by Isabella cluster
(isabella.srce.hr) at the Zagreb University Computing Centre (SRCE)
were used for this research.
Appendix A. Supplementary material
Supplementary crystallographic data for this paper can be
obtained free of charge via www.ccdc.cam.ac.uk/conts/retriev-
ing.html (or from the Cambridge Crystallographic Data Centre,
12, Union Road, Cambridge CB2 1EZ, UK; fax:+44 1223 336033;
or deposit@ccdc.cam.ac.uk). CCDC923167 contains the supplemen-
tary crystallographic data for this paper. Supplementary data asso-
ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.molstruc.2013.06.011.
These
data
include MOL files and InChiKeys of the most important compounds
described in this article.
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This research was supported by the Ministry of Science,
Education and Sports of the Republic of Croatia (Grant Numbers
058-1191344-3122, 119-1191342-1339 and 098-1191344-2943).