Conjugates of 1′-aminoferrocene-1-carboxylic acid and proline: Synthesis, conformational analysis and biological evaluation

Article abstract of DOI:10.3390/molecules190812852

Our previous studies showed that alteration of dipeptides Y-Fca-Ala-OMe (III) into Y-Ala-Fca-OMe (IV) (Y = Ac, Boc; Fca = 1′-aminoferrocene-1- carboxylic acid) significantly influenced their conformational space. The novel bioconjugates Y-Fca-Pro-OMe (1, Y = Ac; 2, Y = Boc) and Y-Pro-Fca-OMe (3, Y = Boc; 4, Y = Ac) have been prepared in order to investigate the influence of proline, a well-known turn-inducer, on the conformational properties of small organometallic peptides with an exchanged constituent amino acid sequences. For this purpose, peptides 1-4 were subjected to detailed spectroscopic analysis (IR, NMR, CD spectroscopy) in solution. The conformation of peptide 3 in the solid state was determined. Furthermore, the ability of the prepared conjugates to inhibit the growth of estrogen receptor-responsive MCF-7 mammary carcinoma cells and HeLa cervical carcinoma cells was tested.

Full text of DOI:10.3390/molecules190812852

Molecules 2014, 19, 12852-12880; doi:10.3390/molecules190812852
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Conjugates of 1'-Aminoferrocene-1-carboxylic Acid
and Proline: Synthesis, Conformational Analysis and
Biological Evaluation
Monika Kovačević 1, Krešimir Molčanov 2, Kristina Radošević 3,*, Višnja Gaurina Srček 3,
Sunčica Roca 4, Alan Čače 1 and Lidija Barišić 1,*
1
Department of Chemistry and Biochemistry, Faculty of Food Technology and Biotechnology,
Pierottijeva 6, Zagreb 10000, Croatia; E-Mails: mkovacevic@pbf.hr (M.K.);
alan.cace@gmail.com (A.C.)
2
Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54,
Zagreb 10000, Croatia; E-Mail: Kresimir.Molcanov@irb.hr
3
Department of Biochemical Engineering, Laboratory for Cell Technology,
Aplication and Biotransformation, Faculty of Food Technology and Biotechnology, Pierottijeva 6,
Zagreb 10000, Croatia; E-Mail: vgaurina@pbf.hr
4
NMR Centre, Ruđer Bošković Institute, Bijenička cesta 54, Zagreb 10000, Croatia;
E-Mail: sroca@irb.hr
* Authors to whom correspondence should be addressed; E-Mails: lbaris@pbf.hr (L.B.);
kristina.radosevic@pbf.hr (K.R.); Tel.: +385-1-4605-069 (L.B.); Fax: +385-1-4836-082 (L.B.).
Received: 1 July 2014; in revised form: 12 August 2014 / Accepted: 13 August 2014 /
Published: 21 August 2014
Abstract: Our previous studies showed that alteration of dipeptides Y-Fca-Ala-OMe (III)
into Y-Ala-Fca-OMe (IV) (Y = Ac, Boc; Fca = 1'-aminoferrocene-1-carboxylic acid)
significantly influenced their conformational space. The novel bioconjugates Y-Fca-Pro-OMe
(1, Y = Ac; 2, Y = Boc) and Y-Pro-Fca-OMe (3, Y = Boc; 4, Y = Ac) have been prepared
in order to investigate the influence of proline, a well-known turn-inducer, on the
conformational properties of small organometallic peptides with an exchanged constituent
amino acid sequences. For this purpose, peptides 1–4 were subjected to detailed
spectroscopic analysis (IR, NMR, CD spectroscopy) in solution. The conformation of
peptide 3 in the solid state was determined. Furthermore, the ability of the prepared
conjugates to inhibit the growth of estrogen receptor-responsive MCF-7 mammary
carcinoma cells and HeLa cervical carcinoma cells was tested.

Molecules 2014, 19
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Keywords: ferrocene; proline; peptidomimetic; intramolecular hydrogen bond;
conformational analysis; X-ray crystallography; anticancer activity
1. Introduction
Peptides and proteins play crucial role in cellular signalling processes and account for numerous
targets in medicine [1]. Despite their enormous diversity in biological function and structure, the use of
peptides and proteins as drugs is limited due to: (i) their low metabolic stability towards proteolysis in
the gastrointestinal tract and in serum; (ii) poor absorption after oral administration; (iii) rapid
excretion through liver and kidneys and (iv) undesired effects caused by interaction of the
conformationally flexible peptides with various receptors. The improvement or alteration of
unfavourable structural and biological properties of peptide and proteins can be realized by using their
mimetics [2]. A peptidomimetic is a peptide or non-peptide compound capable of mimicking the
properties or biological activity of a peptide owing to the presence of secondary structure and other
features that are analogous to those of the original peptide [2–5]. There are different approaches in the
design and synthesis of peptidomimetics, i.e., the incorporation of the conformational constraints
(non-peptide moieties) in order to reduce conformational flexibility, the amide bond replacement, the
conjugation of peptides with small molecules and the backbone cyclization. 1,1'-Disubstituted
ferrocene templates [ferrocene-1,1'-dicarboxylic acid (Fcd), ferrocene-1,1'-diamine (Fcda) and
1'-aminoferrocene-1-carboxylic acid (Fca)] were recognized as useful and efficient
bioorganometallic constraints designed to induce chirally organized structures upon conjugation with
natural amino acids {Fn-[CO-(AA)m-OMe]2 (I) [6–13]}, {Fn-[NH-(AA)m-OMe]2 (II) [14,15]},
{Y-Fca-(AA)m-OMe (III) [16–21]} and {Y-(AA)n-Fca-OMe (IV) [22]} [Fn = ferrocenylene;
AA = amino acid; Y = di-tert-butyldicarbonate (Boc), acetamide (Ac); m = 1, 2; n = 1]. Namely, the
introduction of the chiral peptide chains into the ferrocene scaffold enables their communication
through intramolecular hydrogen bonds required for 3D structure formation and function of biological
systems. Recently, Donoli et al. reported intramolecularly hydrogen-bonded series of 310-helical
peptides of different length containing terminal ferrocenyl units and based on the strongly foldameric
achiral α-aminoisobutyric (Aib) acid [23,24].
Our group demonstrated that the attachment of alanine-containing sequences at N- or C- terminus of
Fca [25] lead to peptidomimetics III [16–21] and IV [22] able to adopt key structural features of
protein secondary structure. Certainly, the number and position of hydrogen bond donating sites as
well as protecting groups on the N- and C-termini strongly influenced their conformational
preferences. The peptide III was stabilized in an interchain intramolecular manner, i.e., 9-membered
NHFca···OCAla (IIIA) and 8-membered NHAla···OCY hydrogen-bonded rings (IIIB). Upon the
alteration of peptides III to IV, hydrogen bonding pattern was quite perturbed. Thereat, NHFca was
engaged in intrachain manner through contacts with hydrogen-bond-accepting acetamide carbonyl
groups (γ-turn, IVA) or alanine nitrogen (5-membered hydrogen-bonded ring, IVB). The interchain
IHB, a motif common for peptides III, was realized in conformer IVC through 9-membered
NHAla···OCFca hydrogen-bonded ring (Table 1).

Molecules 2014, 19
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Table 1. Hydrogen bonding patterns of peptides derived from Fca and Ala (the dotted lines
represent the hydrogen bonds).
Type
Hydrogen Bonding Patterns *
H
N
O
X
8
N
H
9
7
X
O
Fe
A
Fe
B
1
O
III
IV
O
6
H
N
4
H
N
3
5
2
OMe
OMe
O
O
O
H
N
O
H
N
O
H
N
H
X
N
N
H
X
N
H
O
X
O
Fe
C
O
O
Fe
Fe
A
O
O
OMe
OMe
OMe
(X = OtBu, Me)
B
(X = Me)
(X = OtBu, Me)
* The hydrogen bonding patterns were systematized in accordance with the nomenclature of
1,n'-disubstituted ferrocene peptides [26].
The present study covers the synthesis, conformational features and biological evaluation of
Y-Fca-Pro-OMe (V) [1, Y = Ac; 2, Y = Boc] and Y-Pro-Fca-OMe (VI) [3, Y = Boc; 4, Y = Ac],
analogues of peptides III and IV. The role of proline in the process of peptide folding, which is
necessary to achieve the physiologically active 3D structure, is attributed to its unique structural
properties. While the most peptide bonds exist in trans configuration to keep the side chains apart, the
peptide imide bond that involves the NH group of the rigid five-membered pyrrolidine ring appears in
both trans and cis forms. Cis-trans isomerization of proline-containing sequences affects numerous
biological processes performed by peptides and proteins (cellular uptake, oligomerization, folding,
catalysis). Furthermore, proline has been recognized as a turn inducer, e.g., proline can induce β-turns,
especially if preceded or followed by aromatic amino acids [27–29].
Considering that peptide sequence and protecting groups at N- and C-terminal positions
strongly influence the conformational features, molecular recognition, assembly and folding of small
peptides [30], our goal is to determine the impact of proline unit on the conformational and biological
properties of the corresponding bioorganometallics V and VI depending on amino acid sequence and
employed protecting groups (Boc or Ac).
2. Results and Discussion
2.1. Chemistry
Ac-Fca-Pro-OMe (1) and Boc-Fca-Pro-OMe (2) were prepared by coupling of N-protected Fca [25]
with C-protected proline following the well-established procedure [16–21]. Therefore, HCl·Pro-OMe
was treated with an excess of NEt3 to give the free base which was coupled in situ with Y-Fca-OH
(Y = Ac, Boc), previously activated by using the standard HOBt/EDC protocol.

Molecules 2014, 19
12855
Boc-Pro-Fca-OMe (3) and Ac-Pro-Fca-OMe (4), with an exchanged sequences of the constituent
amino acids regarding to 1 and 2, were obtained starting from Boc-Fca-OMe [25], applying the
procedure used for the preparation of the recently described alanine analogues IV [22]. Upon
Boc-deprotection in the presence of gaseous HCl, the resulting HCl·Fca-OMe was processed with an
excess of NEt3 to liberate the N-terminus, followed with coupling with activated Boc-Pro-OH. The
obtained Boc-Pro-Fca-OMe (3) was: (i) Boc-deprotected and (ii) Ac-protected upon treatment with
acetyl chloride to give Ac-Pro-Fca-OMe (4) (Scheme 1).
Scheme 1. Synthesis of bioconjugates 1–4.
O
O
N
OtBu
N
H
Y
N
H
(c)
Fe
Fe
O
(Y = OtBu)
OMe
OMe
O
O
Y-Fca-OMe (Y = Me, OtBu)
3
(a)
(d)
O
O
N
Me
N
H
Y
N
H
Fe
Fe
O
OH
OMe
O
O
4
Y-Fca-OH (Y = Me, OtBu)
(b)
(b)
(Y = Me)
O
(Y = OtBu)
O
N
H
Me
N
H
OtBu
Fe
Fe
O
O
N
N
OMe
OMe
O
O
1
2
Reagents and conditions: (a) NaOH/H2O, MeOH; (b) 1. EDC; HOBt, 2. HCl·Pro-OMe/Et3N;
(c) 1. HClgas, 2. Et3N, 3. Boc-Pro-OH/EDC, HOBt; (d) 1. HClgas, 2. Et3N, 3. AcCl.
The structural properties of the synthesized compounds were examined in solution (for 1–4) and in
the solid state (3). The solution-phase analysis was based on the well-established strategy [16–22], i.e.,
the comparison of the spectroscopic values of a reference, a model and the new compounds. The
monosubstituted analogues X–XII [17], unable to participate in IHBs, were selected as reference
compounds. Since the required feature of the model substances is their structural resemblance to the
tested compounds including the ability to form IHBs, proline-containing peptides VII–IX [31–34] and
alanine-containing peptides III and IV [17,22] were employed as model compounds.

Molecules 2014, 19
12856
2.2. Conformational Analysis of 1–4 in Solution
Proline-containing bioorganometallics 1–4 were examined in solution by standard spectroscopic
techniques (IR, NMR and CD spectroscopy) to assess their hydrogen-bonding potential which is of
crucial importance for formation of ordered structures. Therefore, the impact of the ferrocene unit on
the conformational properties of Ac-Fca-Pro-OMe (1), Boc-Fca-Pro-OMe (2), Boc-Pro-Fca-OMe (3)
and Ac-Pro-Fca-OMe (4) was explored by comparison of their spectral data with those corresponding
to the model peptides Boc-Phe-Pro-OMe (VII) Boc-Pro-Gly-OMe (VIII) and Ac-Pro-Gly-OMe (IX)
reported by Ikawa and co-workers [31–34] (to the best of our knowledge, there is no literature data on
Ac-AA-Pro-OMe, (AA = amino acid), an analogue of Ac-Fca-Pro-OMe (1)). The required feature of
the model compounds is their structural semblance to the analyzed compounds, including their
hydrogen-bonding potential. Model peptides VIII and IX were found to adopt 7-membered intramolecular
NHGly···OCNHY hydrogen-bonded ring (Y = Boc, Ac), while the NH group of phenylalanine-containing
peptide VII was free of hydrogen-bonding interactions.
Furthermore, we explored the influence of proline incorporated at N- or C-terminus of Fca,
respectively, on hydrogen-bonding capability of the resulted peptides 1–4 by comparison with
previously described alanine-containing model peptides Ac-Fca-Ala-OMe (IIIa), Boc-Fca-Ala-OMe
(IIIb) [17], Boc-Ala-Fca-OMe (IVa) and Ac-Ala-Fca-OMe (IVb) [22]. The most stable conformations
of IIIa and IIIb were established through interchain IHBs: NHFca···OCCOOMe (type A, 9-membered) or
NHAla···OCNHY (type B, 8-membered ring) (Y = Boc, Ac). Their constitutional isomers IVa and IVb
were stabilized through intrachain IHBs: NHFca···OCNHY (type A, 7-membered ring) or NHFca···NNHY
(type B, 5-membered ring). An additional conformation C was embedded through interchain
NHNHY···OCCOOMe IHB (9-membered ring) (Table 1). The IR spectroscopic data of model compounds
III, IV, VII–IX is listed in Table 2.
Table 2. IR spectroscopic data (cm−1) of model compounds III [a], IV [a], VII-IX [b].
νNH
νNH
νCO
νCO
νCO
Compound
IIIa
Formula
(Free)
(Assoc.)
(Ester)
(Amide I) (Amide II)
1682 m
1514 m
1657 m
Ac-Fca-Ala-OMe
Boc-Fca-Ala-OMe
Boc-Ala-Fca-OMe
3434 m
3433 m
3423 m
3351 w
3327 m
3324 w
1739 s
1731 s
1714 s
1518 m
1655 m
IIIb
1557 m
1504 m
1536 m
IVa
1709 brs
1664 m
IVb
Ac-Ala-Fca-OMe
3425 m
3290 w
1709 s
1562 m
1507 m
1538 m
[c]
[c]
[c]
[c]
[c]
[c]
[c]
VII
Boc-Phe-Pro-OMe
Boc-Pro-Gly-OMe
Ac-Pro-Gly-OMe
~3450 m
~3430 m
~3430 m
[c]
[c]
VIII
~3310 w
~3300 m
IX
IR spectra were recorded in CH2Cl2 (c = 5 × 10−2 M);
IR spectra were recorded in CDCl3
[a]
[b]
(c = 1.25 × 10−3 M); [c] No data available.

Molecules 2014, 19
12857
2.2.1. IR Spectroscopy
The amide NH and carbonyl stretching regions of the IR spectra of the peptides 1–4 indicate the
presence of both associated and free states. Generally, the absorption bands below 3400 cm−1 are
assigned to the hydrogen-bonded NH groups, while the higher absorptions are assigned to non-bonded
NH groups. The intensification of the bands below 3400 cm−1 in IR spectra of peptides 1, 2 and 4
suggests that the amount of their hydrogen-bonded states increases in population in comparison with
peptide 3. Moreover, two hydrogen-bonded internal rotamers of 1, 3 and 4 are seen as two separate
signals below 3300 cm−1 (Figure 1, Table 3).
Table 3. IR spectroscopic data [a] (cm−1) of reference (X–XII) and goal compounds 1–4.
νNH
νNH
νCO
(Ester)
1711
νCO
νCO
Compound
Formula
(Free)
(Assoc.)
(Amide I) (Amide II)
X
Fc-COOMe
Fc-NHAc
XI
3436 m
3436 m
1684
1723
XII
Fc-NHBoc
3351 m
3231 m
3356 sh
3315 m
3290 m
3234 w
3272 m
3229 m
1676
1535
1604
1
2
3
Ac-Fca-Pro-OMe
Boc-Fca-Pro-OMe
Boc-Pro-Fca-OMe
Ac-Pro-Fca-OMe
3434 w
3430 m
1741
1741 sh
1706
1718
1532
1604
3419 sh
3405 m
1694
1532
1655
1690
1563
1623
4
3416 w
1709
[a]
IR spectra were recorded in CH2Cl2 (c
=
5
×
10−2 M). Fc
=
ferrocenyl,
Fca = 1'-amino-ferrocene-1-carboxylic acid.
In order to determine whether the associated NH groups are engaged in an intra- or intermolecular
manner, the concentration dependence of the solution NH absorption bands was tested. Since the
ratio of the associated and free NH bands of 1–4 revealed unaffected upon successive dilution from
c = 5 × 10−2 M to 5 × 10−4 M, their intramolecular employment is strongly indicated (Figure 1).
Furthermore, the ratio of hydrogen-bonded and non-bonded NH peak intensities depends on the
employed protecting group. The bulky Boc groups of 2 and 3 interfere with hydrogen bonding and
reduce the content of their associated NH bands.
The carbonyl stretching frequencies indicate if the corresponding ester or amide groups are involved
in hydrogen bonding. In order to assign the peaks observed in the carbonyl region, we compared the
spectroscopic data of a reference and the new compounds. The corresponding monosubstituted analogues
Fc-COOMe (X), Fc-NHAc (XI) and Fc-NHBoc (XII) (Fc = ferrocenyl) [17] served as a reference
compounds due to their inability to form intramolecular hydrogen bonds (IHBs) (Table 3).

Molecules 2014, 19
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Figure 1. The NH stretching vibrations in concentration-dependent IR spectra of 1–4 in
CH2Cl2 (solid line: c = 5 × 10−2 M, dashed line: c = 5 × 10−4 M).
0
0
-1
-2
-3
-1
-2
-3
-4
-5
-6
-4
3500
3400
3300
3200
3500
3400
3300
3200
Wavenumber (cm-1)
Wavenumber (cm-1)
Ac-Fca-Pro-OMe (1)
Boc-Pro-Fca-OMe (3)
0
-2
-4
-6
-8
0
-1
-2
-3
-4
-10
-12
-5
3500
3500
3400
3300
3200
3400
3300
3200
Wavenumber (cm-1)
Wavenumber (cm-1)
Boc-Fca-Pro-OMe (2)
Ac-Pro-Fca-OMe (4)
Since the carbonyl stretches of esters 1 and 2 at 1741 cm−1 correspond to the bands of associated
model esters III, their engagement in hydrogen bonding is indicated. Contrarily, the carbonyl
stretching absorptions of esters 3 and 4 that are correlated to those of non-bonded esters IV and X,
indicate their non-involvement in hydrogen-bonding. Considering that hydrogen-bond-accepting
urethane [30,35,36] and acetamide group [19] absorb at lower wavenumbers, the urethane carbonyl
group of Boc-Pro-Fca-OMe (3) and acetamide carbonyl group of Ac-Pro-Fca-OMe (4), registered
around 1690 cm−1, are expected to participate in hydrogen bonding. Boc and Ac carbonyl groups,
attached at upper cyclopentadienyl ring (Cp) of Ac-Fca-Pro-OMe (1) and Boc-Fca-Pro-OMe (2), are
precluded from participating in IHBs because of the unavailability of hydrogen-bonding-donating
groups at the lower Cp ring. Therefore, their absorption frequencies are correlated with those belonging
to the hydrogen-bonding free XI and XII (Table 3).
2.2.2. NMR Spectroscopy
The hydrogen bonding engagement of the bioconjugates 1–4, strongly indicated by the
corresponding NH stretching absorptions below 3400 cm−1, was corroborated by the downfield shifts
(δ > 7 ppm) of their NH groups in non-polar CDCl3. Certainly, the hydrogen-bonded NH groups of the

Molecules 2014, 19
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previously described model compounds III, IV and VII–IX were found to resonate downfield in
similar positions, while NH groups of the HB-free reference compounds XI and XII were upfield
shifted (Table 4).
1
[a]
Table 4. H-NMR chemical shifts
of the NH protons (ppm) of a model (III, IV,
VII-IX), a reference (XI, XII) and the goal compounds 1–4. The proposed IHB patterns of
a model and a reference compounds VII-XVI. The possible IHB patterns of the goal
compounds 1–4.
δ/ppm
δ/ppm
Compound
IIIa
Formula
IHB Pattern
IHB Ring Size
(NHAA) [b]
(NHFca/Fc)
NHFca···OCAla
NHAla···OCAc
NHFca···OCAla
NHAla···OCBoc
NHFca···NAla
NHAla···OCFca
NHFca···OCAc
NHFca···NAla
9-membered
8-membered
9-membered
8-membered
5-membered
9-membered
7-membered
5-membered
9-membered
Ac-Fca-Ala-OMe
Boc-Fca-Ala-OMe
Boc-Ala-Fca-OMe
6.71
6.77
5.14
7.93
6.40
7.64
IIIb
IVa
IVb
Ac-Ala-Fca-OMe
6.41
8.04
NHAla···OCFca
[c]
[d]
VII
Boc-Phe-Pro-OMe
Ac-Pro-Gly-OMe
7.25(t)
6.23(c)
7.28(t)
6.49(c)
VIII
NHGly···OCAc
NHGly···OCBoc
7-membered
7-membered
IX
Boc-Pro-Gly-OMe
[d]
[d]
XI
Fc-NHAc
6.49
XII
Fc-NHBoc
5.55
8.23(t)
8.38(c)
7.12(t)
6.80(c)
8.78(t)
7.53(c)
8.81(t)
7.29(c)
1
2
3
4
Ac-Fca-Pro-OMe [e]
Boc-Fca-Pro-OMe [e]
Boc-Pro-Fca-OMe [e]
Ac-Pro-Fca-OMe [e]
NHFca···OCPro
NHFca···OCPro
NHFca···OCPro
NHFca···OCPro
9-membered
9-membered
7-membered
7-membered
[a] NMR spectra were measured in CDCl3 (c = 5 × 10−2 M); [b] AA = natural amino acid; [c] No data available;
[d]
[e]
IHB was not indicated; The occurrence of both isomers [(c) = cis, (t) = trans] in solution at 298 K is
confirmed by 13C-NMR on the basis of the well-documented shift differences observed for Cβ- and
Cγ-atoms [37,38].
The goal of this paper was to determine the conformational consequences of: (i) the replacement of
natural amino acid (phenylalanine or glycine) belonging to the model proline-containing peptides
VII–IX with bioorganometallic amino acid (Fca) and (ii) the replacement of alanine units of
Fca-containing model peptides III and IV with proline. Due to the lack of an amide hydrogen, proline
residues of 1–4 are not able to donate hydrogen bonding. Their possible involvement in hydrogen
bonding might be realized through inter- (9-membered) or intrachain (7-membered) IHB rings

Molecules 2014, 19
12860
upon accepting NHFca (Table 4). Considering that the model peptides III, IV and VII–IX were
stabilized through IHBs, the expected IHB patterns of the novel peptides 1–4 were explored by
concentration- and temperature-dependent NMR analyses. The considerable changes in chemical shifts
(Δδ = 1–4 ppm) upon dilution or heating are expected for intermolecular hydrogen bonding. Since the
successive decreasing of concentration (50–6.25 mM) did not cause any discernible changes in
chemical shifts of amide protons, the intramolecular hydrogen bonds, proposed by IR experiments,
were corroborated (Figure 2). Furthermore, the results of temperature-dependent NMR spectroscopy
were in accordance with these findings. The examined conjugates 1–4 demonstrated negligible
dependence of the chemical shifts (Δδ < 0.7 ppm) on temperature (Figure 3).
Figure 2. Concentration dependent NHtrans chemical shifts of peptides 1–4. [1H-NMR
measurements were performed for a series of 6.25, 12.5, 25 and 50 mM solutions].
δ (ppm)
4
3
1
8.8
8.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
7.0
2
0
5
10 15 20 25 30 35 40 45 50
c (mM)
Figure 3. Temperature dependent NHtrans chemical shifts of peptides 1–4 (c = 2.5 × 10−2 M)
in the temperature range of 258–328 K.
δ (ppm)
9.0
δΔ
δT
8.7
8.4
8.1
7.8
7.5
7.2
6.9
4
= -3.71 ppb
= -6.86 ppb
= -6.71 ppb
δΔ
δT
3
1
δΔ
δT
δΔ
δT
= -10.3 ppb
2
260 270 280 290 300 310 320 330
Temperature (K)

Molecules 2014, 19
12861
The measurements of the temperature dependences of chemical shifts (Δδ/ΔT) in hydrogen bonding
solvents enable differentiation of the solvent-exposed and solvent-shielded peptide NH groups [39–45].
Since DMSO causes rapid decomposition of ferrocene peptides, their temperature dependences were
determined in CDCl3 solution [22,46,47]. Generally, the law Δδ/ΔT values (−2.4 ± 0.5 ppb/K) are
measured for both exposed and shielded amide protons of short peptides. The larger temperature
dependencies are characteristic for NH groups which were shielded from solvent at starting state, but
became exposed through dissociation of the self-associated aggregates or unfolding of ordered
conformations at increased temperatures [48]. Accordingly, larger temperature dependencies of
concentration-independent amide protons of 1–4 emanate from the initially shielded species switched to
the unshielded environment by unfolding of conformations organized through IHBs (Figure 3).
Polarity and hydrogen bonding potency of a solvent are of outstanding significance in the
evaluation of hydrogen bond strength. Owing to its accepting capability, DMSO disrupts peptide
hydrogen bonds causing the unfolding of ordered structures [49]. Taking that in consideration, the
strength of IHBs indicated by the results of IR and NMR analyses was explored by DMSO titration
experiments (Figure 4) [16–22,50]. The highest chemical shift value of NHFca belonging to
Ac-Pro-Fca-OMe (4) and the lowest chemical shift alteration upon addition of DMSO (Δδ = 0.42 ppm)
indicate its engagement in a strong IHB. A similar behaviour in DMSO experienced amide protons of
Ac-Fca-Pro-OMe [1, (Δδ = 0.85 ppm)] and Boc-Pro-Fca-OMe [3, (Δδ = 0.73 ppm)] suggesting their
participation in IHBs of medium strength. Contrarily, upfield shifting (δ = 7.12 ppm) and the highest
chemical shift variation (Δδ = 1.12 ppm) of NHFca belonging to Boc-Fca-Pro-OMe (2) indicate its
involvement in weak IHB.
Figure 4. Solvent dependence of NHtrans chemical shifts at varying concentrations of
DMSO in CDCl3 (c = 2.5 × 10−2 M, 298 K) to probe exposed vs. hydrogen-bonded amides.
δ (ppm)
9.4
9.2
9.0
8.8
8.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
7.0
4
3
1
2
-5
0
5 10 15 20 25 30 35 40 45 50 55 60
% DMSO
1
The IHB patterns of peptides 1–4, proposed by IR and H-NMR analyses (Table 4), are supported
by their NOESY spectra. The interchain NOE contacts between amide protons of Fca and proline
belonging CHα and COOMe protons strongly support the proposed 9-membered NHFca···OCPro IHB
rings in peptides 1 and 2 (Table 4, Figure 5).

Molecules 2014, 19
12862
Figure 5. The proposed IHBs in peptides 1 (a) and 2 (c) are depicted with dashed lines and
the interchain NOE connectivities are presented with arrows; the interchain NOE
connectivities between the C- and N-termini of 1 (b) and 2 (d) are depicted with dashed lines.
COOMe
CH3Ac
CHDProCHFc
CHFc
O
N
7.96
8.00
8.04
8.08
8.12
8.16
8.20
8.24
8.28
8.32
8.36
8.40
8.44
8.48
N
H
Me
Fe
O
NHFca
OMe
O
1 (Ac-Fca-Pro-OMe)
PPM (F1
PPM (F2) 8.0
7.6 7.2
6.8 6.4 6.0 5.6
5.2 4.8 4.4 4.0
3.6
3.2
2.8 2.4 2.0
(a)
(b)
NHFca
COOMe
CHFc
3.2
3.6
4.0
4.4
4.8
5.2
5.6
6.0
6.4
6.8
7.2
O
N
H
OtBu
Fe
O
CHD
Pro
N
OMe
O
2 (Boc-Fca-Pro-OMe)
PPM (F1)
PPM (F2)
7.60
7.50
7.40
7.30
7.20
7.10
7.00
6.90
6.80
6.70
(c)
(d)
Furthermore, NOE contacts of amide protons of Fca within the peptide chain attached at upper Cp
of 3 and 4, promote the suggested intrachain NHFca···OCPro IHBs (γ-turn) (Table 4, Figure 6). NMR
investigations of the solvent influence on the cis-trans isomerization of small peptides [51–59] revealed
the increased fraction of the trans form in less polar or non-polar solvents. It was also found that
intramolecular hydrogen bonds are consistent with the trans form [60]. Multiple resonances detected
for the same proton during the course of NMR analysis are attributed to the presence of rotamers
if the rate of the interconversion (e.g., amide cis-trans isomerization) is slow relative to the NMR
scale [37,40,61]. On heating, isomerization rate increases causing the coalescence of signals. The fully
coalesced peaks at higher temperatures correspond to the weighted average chemical shift value for the
two conformers on the NMR timescale. Contrary, slow proline isomerization at higher temperatures
reveals the peaks resolved due to the persistence of cis and trans conformers at NMR timescale [62–65].

Molecules 2014, 19
12863
Figure 6. The proposed IHBs in peptides 3 (a) and 4 (c) are depicted with dashed lines and
the intrachain NOE connectivities are presented with arrows; the intrachain NOE
connectivities of 3 (b) and 4 (d) are depicted with dashed lines.
NHFca
CH3Boc
1.6
2.0
2.4
2.8
3.2
3.6
4.0
4.4
4.8
5.2
5.6
6.0
6.4
6.8
7.2
7.6
8.0
8.4
O
N
OtBu
N
H
Fe
O
OMe
CHD
Pro
O
3 (Boc-Pro-Fca-OMe)
PPM (F1
PPM (F2) 8.76
8.72
8.68
8.64
8.60
8.56
8.52
8.48
8.44
8.40
8.36
8.32
(a)
(b)
Works 3: L. Barisic 2602 50 mM
CH3Ac + CHJ
Pro
CHD
Pro
CHFc
O
8.52
8.56
8.60
8.64
8.68
8.72
8.76
8.80
8.84
8.88
8.92
8.96
9.00
N
Me
N
H
Fe
O
NHFca
OMe
O
4 (Ac-Pro-Fca-OMe)
PPM (F1)
PPM (F2)
8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0
3.6 3.2 2.8 2.4
(c)
(d)
The NMR spectra recorded at: (i) decreased temperatures and (ii) an increased amount of DMSO,
respectively, identified the cis and trans isomers of the new peptides 1–4. The trans:cis ratio was
calculated from integrated intensities of the appropriate resonances. Upon heating to 328 K, the two
peaks of the trans and cis isomers of 1–4 coalesced into a single peak due to the medium
(compound 3) and rapid (compounds 1, 2 and 4) isomerization. In DMSO, peptides 3 and 4 underwent
to slow or medium isomerization, respectively, while peptide 1 displayed rapid isomerization.
Completely coalesced amide resonances of peptide 2 upon titration with DMSO reflect the average
chemical shift of rapidly exchanged cis and trans rotamers as a consequence of weak IHB ineligible to
induce isomer locking. In contrast, the abundance of the trans amide in peptide 4 is attributed to its
stabilization through stronger IHB in comparison to those proposed for the bioconjugates 1–3
(Table 5). These finding are consistent with the above-mentioned conclusions [51–65] concerning the
cis-trans isomerization of a proline imide bond of small peptides.

Molecules 2014, 19
12864
Table 5. The trans-cis ratios of imide bonds of peptides 1–4 measured by 1H-NMR.
T (K) [a]
DMSO (%) [b]
T (K) [a]
DMSO (%) [b]
Ac-Fca-Pro-OMe (1)
Boc-Fca-Pro-OMe (2)
228
328
0
36
228
328
0
36
cis
8.67
8.51
8.39
8.23
91:9
9.00
8.92
90:10
cis
7.14
7.57
6.80
7.13
93:7
8.04
6.83
7.99
δ (ppm)
trans
δ (ppm)
trans
trans:cis
90:10
trans:cis
90:10
T (K)
DMSO (%) [a]
T (K)
DMSO (%) [a]
Boc-Pro-Fca-OMe (3)
Ac-Pro-Fca-OMe (4)
228
7.38
8.67
66:33
328
0
36
228
7.63
8.98
94:6
328
0
36
cis
9.00
9.10
56:44
cis
7.29
8.81
93:7
9.41
9.21
74:26
8.21
8.53
8.72
δ (ppm)
trans
δ (ppm)
trans
trans:cis
trans:cis
[a] The temperature-dependent NMR spectra were measured in CDCl3 (c = 2.5 × 10−2 M); [b] 1H NMR titration
of CDCl3 solutions of 1–4 (c = 2.5 × 10−2 M) with DMSO was performed at 298 K.
2.2.3. CD Spectroscopy
Circular dichroism is a valuable tool for evaluating the secondary structure, folding and binding
properties of proteins [66]. The insertion of ferrocene into the chiral peptide environment induces
Cotton effect in the region of ferrocene-based transitions around 480 nm owing to an ordered
structures driven by hydrogen bonding between podand peptide chains [6–13,16–22] or within the
same peptide chain [12,67]. Furthermore, it was shown that the sign of the CD signal of ferrocene
peptides (which reflects an average of the entire molecular population) is influenced by the sequence
of the natural peptide fragment [17,19,68], the type of N-protected groups attached at ferrocene
core [19] and the type of the employed solvent [17,19].
Although the proposed hydrogen bonding patterns for peptides 1 and 2 are very similar, their CD
spectra are quite different due to chromophoric reorientation induced by hydrogen bonding in the
presence of different protecting groups [69]. The different intensities of the Cotton effects in the CD
spectra of the bioconjugates 1–4 indicate the different levels of chiral organization. The absolute
values of Cotton effects of 2–4, ranged between 500 and 1,000 deg·cm2·dmol−1, are in a good
agreement with previously described alanine analogues III [17] and IV [22]. The increased
CD-activity of peptide 1 indicates higher level of chiral organization established through interchain
NHFca···OCCOOMe IHB (Figure 7). Moreover, the CD-silent ferrocene region in the spectrum of peptide
2, obtained upon titration with DMSO, is attributed to the loss of the chiral organization owing to the
cleavage of weak IHB. The decreasing of the CD intensity in a strong hydrogen-bond forming solvent,
demonstrated herein for peptides 1 and 4, has also been shown for previously described ferrocene
peptides [16,19,20]. However, the addition of DMSO to CH2Cl2 solution of 3 caused the increment of
the CD signal. This exception, reported for the several ferrocene ureidopeptides by Heinze et al. arose
from substantial alteration of the chromophore setting of the examined peptide in CH2Cl2 and
CH2Cl2/DMSO. Heinze reached the conclusion that the solvent dependent Cotton effects observed are
greatly influenced by other ordering phenomena, while the stable conformations established through
IHBs in non-polar solvents have only a minor influence [47].

Molecules 2014, 19
12865
Figure 7. The Cotton effects in chirality-organized ferrocene peptides 1 and 2 (a) and 3 and
4 (b) in CH2Cl2 (c = 5 × 10−3 M) and CH2Cl2 (c = 5 × 10−3 M) containing 20% of DMSO.
2
500
2 + 20% DMSO
0
0
-500
1 + 20% DMSO
-1000
-500
1
4 + 20% DMSO
-1500
-2000
4
3
3 + 20% DMSO
-1000
400
450
500
550
600
400
450
500
550
600
λ (nm)
λ (nm)
(a)
(b)
2.2.4. Crystal Structure of Boc-Pro-Fca-OMe (3)
Single crystal X-ray diffraction confirmed existence of an intrachain hydrogen bond in
Boc-Pro-Fca-OMe (3) which was established by NMR spectroscopy. Conformation of the compound
is stabilised by one N-H···O and four weak C-H···O intramolecular hydrogen bonds (Figure 8, Table 6).
An additional stabilization is achieved through interchain dipolar interaction between a carbonyl group
bound to one Cp ring and NH group bound to another Cp ring (Figure 9): the covalent bonds are
approximately parallel (torsion angle between them being 5.9°), and distance between their midpoints
is 3.33 Å. However, there are no intermolecular hydrogen bonds present, so the packing is dominated
by dispersion interactions, only.
Figure 8. Conformation of Boc-Pro-Fca-OMe (3) in the crystal stabilised by intrachain
intramolecular hydrogen bonding.

Molecules 2014, 19
12866
Table 6. Geometric parameters of hydrogen bonding.
D–H···A
D–H/Å
0.86
H···A/Å
2.10
D···A/Å
2.810(10)
2.906(12)
2.827(14)
2.969(14)
3.031(16)
D–H···A/°
139
Symm. op. on A
x, y, z
N1–H1···O2
C2–H2···O1
0.93
2.46
110
x, y, z
C8–H8B···O1
C13–H13C···O2
C15–H15C···O2
0.97
2.47
101
x, y, z
0.96
2.37
120
x, y, z
0.96
2.45
119
x, y, z
Figure 9. An additional stabilization of Boc-Pro-Fca-OMe (3) through interchain
intramolecular dipolar interaction between CO and NH groups.
2.3. Biological Evaluation of Bioconjugates 1–4
The use of peptidomimetics is one of the most recent methods of drug design and development in
medicinal chemistry due to various types of pharmacological activities displayed by them. Due to their
versatile activities (antimicrobial, anticancer, antiviral, antimalarial, antioxidant, immunosuppressive
activity, immunodetection, selectivity for DNA receptors etc. [70]), peptidomimetics are utilized to
design and develop novel drug molecules which can be effective in the treatment of various human
diseases. Since cancer is considered as the most serious health problem all over the world, the
discovery of new potent, safe and selective antitumor agents is strongly needed. Peptidomimetics have
potential as antitumor agents due to their anti-cancer activity reported in the literature. For example,
anticancer peptidomimetics can bind to target proteins and induce cancer cells to enter apoptotic cell
death by mimicking key interactions that activate apoptotic pathway in the specified cells [71]. Also,
Liao et al. synthesized novel unnatural amino acid-substituted (hydroxyethyl)urea peptidomimetics
which inhibited secretase and interfered with the malignancy of neuroblastomas and therefore showed
that these peptidomimetics can be used as lead compounds for further development of novel anticancer
drugs [72].
U.S. National Cancer Institute (NCI) phased out its murine leukemia P388 anticancer drug
screening program in 1990 and developed in vitro primary screen based upon a panel of 60 different
human tumor cell lines [73]. Only in first five years of that program 30,000 compounds involved in
cancer researches worldwide have been tested according to NCI screen. Compounds interesting as

Molecules 2014, 19
12867
potential anticancer drug are being tested over a defined range of concentrations to determine the
relative degree of growth inhibition or cytotoxicity against each cell line.
In the ongoing research, evaluation of biological activity of novel Fca and proline conjugates 1–4
was done against human tumour cell lines. Since conventional anticancer drug discovery and
development is focused on the search for cytotoxic agents by this preliminary in vitro screen we aimed
to determine potential of compounds 1–4 as cytotoxic i.e., antitumor agents.
The biological activity of peptides 1–4, with emphasis to possible anti-cancer activity, was tested
in vitro and evaluated on the basis of their ability to inhibit growth of MCF-7 mammary carcinoma
cells and HeLa cervical carcinoma cells. Cytotoxicity of the synthesized compounds was assessed on
the basis of in vitro growth in 96-well plates measured by cell-mediated reduction of tetrazolium salt
WST-1 and the results are expressed as cell viability (%) and presented in Figure 10.
Figure 10. Effects of four different compounds on MCF-7 (a) and HeLa (b) cells viability
assessed by the WST-1 assay. Values were represented as the mean ± SD from three
independent experiments.
(a)
(b)

Molecules 2014, 19
12868
Compound 3 does not possess antiproliferative potential toward MCF-7 cell line, while other tested
compounds showed some potential to inhibit growth of MCF-7 cells (Figure 10a). The 50% inhibition
of cell growth was not detected by the compounds 1, 3 and 4 in the range of tested concentrations
(1–500 µM) and therefore their IC50 values are considered to be higher than 500 µM. The highest
cytotoxicity in MCF-7 cells is obtained by compound 2 with IC50 value of 357.72 µM. Similar results
were obtained with HeLa cells (Figure 10b). The trend of cytotoxicity is the same as in MCF-7 cells;
the weakest effect was provoked by compound 3 and the most pronounced inhibition of HeLa cells
growth was observed in the treatment with a compound 2. The compounds 1, 2 and 4 have resulted
with 50% inhibition of cell growth after the 72-hour treatment and therefore IC50 values for HeLa cells
were calculated to be: 474.99 µM (compound 1), 463.64 µM (compound 4) and 203.27 µM
(compound 2). Considering that the peptide 2 is stabilized by the weakest IHB in comparison with
conjugates 1, 3 and 4, these results are quite unexpected. But, having in mind that polarity or
lipophilicity of the compound is a prerequisite for potential pharmaceutical applications, the obtained
results might be attributed to the increased lipophilicity of peptide 2 (Rf = 0.50) with regard to more
polar analogues 1 (Rf = 0.30), 3 (Rf = 0.33) and 4 (Rf = 0.22). Obtained IC50 values are relatively high
when compared with compounds with well known anti-cancer activity, like doxorubicine or cisplatin,
as the reference drugs. IC50 values for doxorubicin and cisplatin determined in different human cancer
cell lines and obtained from literature survey varies mostly from 0.1 µM to 15 µM [74,75] depending
on cell type. Since previously described conjugates of Fca with amino acids had not been subjected to
biological evaluation, we were not able to explore how the introduction of proline into conjugates 1–4
influences the cytotoxicity of this class of peptidomimetics. The obtained results on cytotoxic activity
of herein described bioorganometallics 1–4 represents the initial step in the future investigation of
ferrocene-containing peptidomimetics.
Therefore, peptide 2, which exibited the highest inhibitory effect among tested bioconjugates
actually has poor cytotoxic activity toward MCF-7 and HeLa cells and can not be considered as
potential anticancer drug candidate. Nevertheless, the obtained results are valuable as a guide how to
further direct the synthesis of similar analogues with improved biological properties.
The cells were daily examined by inverted light microscopy to observe if there are any visible
morphological changes in MCF-7 cells. After treatment with synthesized compounds, cells were
stained with crystal-violet and photographed (Figure 11).
Figure 11. Light microscopy of MCF-7 cells stained with cystal-violet after 72 h of
treatment with tested compounds 1–4. Control cells (a) and cells treated with 500 µM
1 (b); 2 (c); 3 (d) and 4 (e). Magnification 400×.
Exposure to the highest concentration of the tested compounds 1–4 (500 µM) for 72 h resulted in
reduction of cell number and decreased monolayer density (Figure 11b–e) when compared to control

Molecules 2014, 19
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MCF-7 cells (Figure 11a) which were well attached and has typical epithelial morphology. Evidently,
the proliferation of the MCF-7 cancer cell was inhibited by the testing compounds in vitro and
observed morphological changes correspond with obtained cytotoxicity results, but further
investigation is needed to explore possible mechanism of action and type of cell death.
3. Experimental Section
3.1. General Information
The syntheses were carried out under argon. The CH2Cl2 used for synthesis and FTIR was dried
(P2O5), distilled over CaH2, and stored over molecular sieves (4 Å). EDC (Acros Organics, Geel,
Belgium), HOBt (Aldrich, Schnelldorf, Germany), acetyl chloride (Aldrich, Schnelldorf, Germany)
and proline (Acros Organics, Geel, Belgium), were used as received. The syntheses of Y-Fca-OMe and
Y-Fca-OH have been previously described [16–21,25]. The protection of N- or C-termini of proline
was carried out by using sodium hydroxide, aqueous dioxane and di-tert-butyldicarbonate to give
Boc-Pro-OH or gaseous HCl in MeOH to obtain HCl·Pro-OMe. Products were purified by preparative
thin layer chromatography on silica gel (Kieselgel 60 HF254, Merck, Darmstadt, Germany) by using
CH2Cl2 or mixture EtOAc/CH2Cl2 as eluents. Infrared spectra were recorded as CH2Cl2 solutions
between NaCl windows by using a Bomem MB 100 mid FTIR spectrometer. (m) = medium,
1
(w) = weak, (sh) = shoulder. The H and 13C NMR spectra were recorded at 600 MHz on Bruker
1
Avance spectrometer and were referenced to the residual solvent peak (CDCl3, H: 7.26 ppm, 13C:
77.16 ppm). In the case of the CDCl3/d6-DMSO mixture, the calibration was done using Me4Si as
internal standard due to the overlapping of the residual solvent peak of d6-DMSO with proline peaks.
Double resonance experiments (COSY, NOESY and HMBC) were performed in order to assist in
signal assignment. (s) = singlet, (d) = doublet, (m) = multiplet or (br) = broad. Unless otherwise noted,
all spectra were recorded at 298 K. CD spectra were recorded using a Jasco-810 spectropolarimeter in
CH2Cl2. Molar ellipticity coefficients, [θ], are in degrees, concentration, c, is given in mol·L−1, and
path length, l, is given in cm, to give units for [θ] of deg·cm2·dmol−1. NMR titrations were performed
by adding of 10 µL portions of the d6-DMSO in a NMR-tubes containing solutions of the examined
peptides 1–4 in CDCl3 (c = 2.5 × 10−2 M). The spectra were recorded after each addition and
d6-DMSO was added until no further change in the chemical shifts of the NHs were observed. CD
titrations were performed in a similar manner by stepwise addition of DMSO into cuvettes containing
solutions of the examined peptides 1–4 in CDCl3 (c = 5 × 10−3 M). The mass spectra were acquired
using 4800 MALDI TOF/TOF-MS Analyzer. Preparation of intact samples. Peptide samples 1–4, each
were dissolved in deionized water to concentration of 0.05 mg/mL. Volume of 10 µL of each sample
solution was purified using ZipTip C4 pipette tips (Millipore, Bedford, MA, USA) and dried, then
dissolved in CHCA matrix (α-cyano-4-hydroxycinnamic acid, 5 mg/mL, Sigma–Aldrich) and dried
onto the MALDI plate (volume of 1 µL). At the end, 0.05 µg of peptide was deposited onto the
MALDI plate. MALDI TOF/TOF-MS. For direct profiling and MS/MS fragmentation study of
peptides a 4800 Plus MALDI TOF/TOF analyzer (Applied Biosystems Inc., Foster City, CA, USA)
equipped with a 200 Hz, 355 nm Nd:YAG laser was used. Acquisition was performed in positive ion
reflector mode. Instrument parameters were set using the 4000 Series Explorer software version

Molecules 2014, 19
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(V 3.5.3, Applied Biosystems Inc.). Mass spectra were obtained by averaging 1000 laser shots
covering the mass range between m/z 9 and 4500. MS/MS was achieved by 1 or 2 kV collision induced
dissociation (CID) in positive ion mode. For both MALDI-TOF MS and MS/MS analysis, CHCA
matrix was used. Dried purified samples (both intact and derivatized) were diluted in 5 µL of matrix,
1 µL of mixture was spotted onto the MALDI plate and allowed to dry.
3.2. General Procedure for the Preparation of Compounds 1 and 2
Ac-Fca-Pro-OMe (1) and Boc-Fca-Pro-OMe (2). Ac-Fca-OH (200 mg, 0.76 mmol) and Boc-Fca-OH
(262 mg, 0.76 mmol), prepared by hydrolysis of the fully protected Fca [25], were activated using
EDC (162 mg, 0.84 mmol) and HOBt (114 mg, 0.84 mmol) in CH2Cl2, respectively. Pro-OMe
(obtained from HCl·Pro-OMe (254 mg, 1.53 mmol) by treatment with Et3N in CH2Cl2, pH ~ 8) was
added and the mixtures were stirred at room temperature until complete consumption of the ferrocene
starting material, as monitored by TLC. The standard work-up (washing with a saturated aqueous
solution of NaHCO3, 10% aqueous solution of citric acid and brine, drying over Na2SO4 and
evaporation in vacuo) followed with TLC-purification of the crude products [CH2Cl2:EtOAc = 5:1 (1)
and CH2Cl2:EtOAc = 10:1 (2)] gave orange resin of 1 (213 mg, 70%) and orange crystals of 2 (274 mg,
79%), respectively.
Ac-Fca-Pro-OMe (1). IR (Nujol) νmax 3434 w (NHfree), 3351, 3231 m (NHassoc.), 1741 s (C=OCOOMe),
1676 s (C=OAc), 1604 s (C=OCONH), 1535 s (amide II) cm−1; Rf = 0.30 (CH2Cl2:EtOAc = 5:1);
1H- NMR (CDCl3, 600 MHz) δ 8.38 (s, 0.09H, NHcis), 8.23 (s, 0.91H, NHtrans), 4.80-4.78 (m, 1H,
CHFn), 4.62 (dt, 1H, J1 = 2.9 Hz, J2 = 1.5 Hz, CHFn), 4.60 (m, 1H, CHαPro), 4.60–4.58 (m, 1H, CHFn),
4.54 (dt, 1H, J1 = 2.7 Hz, J2 = 1.4 Hz, CHFn), 4.40–4.37 (m, 2H, CHFn), 4.07–4.05 (m, 1H, CHFn),
4.03–4.01 (m, 1H, CHFn), 3.81 (s, 3H, CH3COOMe), 3.79–3.73 (m, 2H, CHδPro), 2.33–2.28 (m, 1H,
CHβPro), 2.11–2.08 (m, 1H, CHγPro), 2.06 (s, 3H, CH3Ac), 1.99–1.93 (m, 2H, CHβPro+CHγPro); 13C-NMR
(CDCl3, 150 MHz) δ 173.78 (COCOOMe), 169.94 (COCO-N), 169.69 (COAc), 94.20 (CqFn), 79.92 (CqFn),
73.22, 71.49, 71.08, 70.46, 66.71, 66.54, 66.18, 64.71 (CHFn trans), 70.46, 70.18, 70.06, 66.71, 66.42
(CHFn cis), 60.90 (CHαPro cis), 60.15 (CHαPro trans), 52.67 (CH3COOMe cis), 52.53 (CH3COOMe trans), 49.18
(CH2δPro trans), 47.52 (CH2δPro cis), 29.82 (CH2βPro cis), 29.03 (CH2βPro trans), 25.83 (CH2γPro trans), 23.83
(CH3Ac), 22.36 (CH2γPro cis); HRMS (MALDI) m/z 398.0939 (calcd for C19H22N2O4Fe, 398.0923).
Boc-Fca-Pro-OMe (2): mp 125–128 °C. IR (Nujol) νmax 3430 m (NHfree), 3356 sh, 3315 m (NHassoc.)
,
1741 sh (C=OCOOMe), 1718 s (C=OBoc), 1604 s (C=OCONH), 1532 s (amide II) cm−1; Rf = 0.50
(CH2Cl2:EtOAc = 10:1); 1H-NMR (CDCl3, 600 MHz) δ 7.12 (br. s, 0.93H, NHtrans), 6.80 (br. s, 0.07H,
NHcis), 4.88–4.84 (m, 1H, CHFn), 4.65 (dt, 1H, J1 = 2.7 Hz, J2 = 1.5 Hz, CHFn), 4.62 (dd, 1H,
J1 = 8.8 Hz, J2 = 6.6 Hz, CHαPro), 4.42–4.37 (m, 2H, CHFn), 4.37–4.34 (m, 1H, CHFn), 4.07–4.03 (m,
1H, CHFn), 3.98–3.95 (m, 1H, CHFn), 3.87–3.80 (m, 1H, CHδPro), 3.81 (s, 3H, CH3COOMe), 3.77–3.73
(m, 1H, CHδPro), 2.28–2.24 (m, 1H, CHβPro), 2.16–2.14 (m, 1H, CHγPro), 2.04–1.95 (m, 2H, CHβPro+CHγPro),
1.49 (s, 9H, CH3Boc); 13C-NMR (CDCl3, 150 MHz) δ 174.01 (COCOOMe), 169.77 (COCO-N), 153.91
(COBoc), 96.79 (CqFn), 79.80 (CqFn), 76.36 (CqBoc), 72.77, 71.86, 71.22, 70.96, 66.09, 65.45, 64.40,
62.75 (CHFn trans), 70.64, 70.49, 69.98 (CHFn cis), 60.64 (CHαPro cis), 60.36 (CHαPro trans), 52.57
(CH3COOMe), 48.77 (CH2δPro trans), 47.50 (CH2δPro cis), 32.03 (CH2βPro cis), 28.58 (CH2βPro trans), 28.57

Molecules 2014, 19
12871
(CH3Boc), 25.98 (CH2γPro trans), 22.17 (CH2γPro cis). HRMS (MALDI) m/z 456.1353 (calcd for
C22H28N2O5Fe, 456.1342).
3.3. General Procedure for the Preparation of Compounds 3 and 4
Boc-Pro-Fca-OMe (3) and Ac-Pro-Fca-OMe (4). HCl·Fca-OMe, obtained from the fully protected Fca
(500 mg, 1.4 mmol) by action of gaseous HCl in CH2Cl2, was treated with NEt3 to give unstable free
base [22] which was coupled with Boc-Pro-OH (602 mg, 2.8 mmol) using standard EDC/HOBt
method. After standard work-up and TLC purification (CH2Cl2-EtOAc = 5:1), yellow crystals of
Boc-Pro-Fca-OMe (3, 1060 mg, 83%) were obtained. The transformation of Boc-peptide 3 (400 mg,
0.89 mmol) to Ac-analogue 4 was carried out by action of acetyl chloride (380 μL, 5.34 mmol): the
free base, obtained via hydrochloride salt provided by deprotection of 3 in the above described manner,
was cooled at 0 °C and acetyl chloride was dropwise added. After stirring in an ice bath for 15 min,
TLC indicated no residual starting material remained and the reaction mixture was poured into water
and extracted with CH2Cl2. The combined organic phases were washed with a brine, dried over
Na2SO4 and evaporated to dryness in vacuo. The resulting crude product was purified by TLC on
silicagel (EtOAc) to give orange solid of 4 (325 mg, 92%).
Boc-Pro-Fca-OMe (3): mp 128–132 °C; IR IR (Nujol) νmax 3419 sh, 3405 m (NHfree), 3290 m, 3234 w
(NHassoc.), 1706 s (C=OCOOMe), 1694 s (C=OBoc), 1655 s (C=OCONH), 1532 s (amide II) cm−1;
1
Rf = 0.33 (CH2Cl2:EtOAc = 5:1); H-NMR* (CDCl3, 600 MHz) δ 8.78 (s, 0.66H, NHtrans), 7.53
(s, 0.33H, NHcis), 4.80–4.77 (m, 0.67H, CHFn trans), 4.76 (dt, 0.66H, J1 = 2.6 Hz, J2 = 1.3 Hz, CHFn
trans), 4.74 (dt, 0.66H, J1 = 2.7 Hz, J2 = 1.3 Hz, CHFn trans), 4.70 (dt, 0.32H, J1 = 2.5 Hz, J2 = 1.3 Hz,
CHFn cis), 4.69 (dt, 0.33H, J1 = 2.4 Hz, J2 = 1.2 Hz, CHFn cis), 4.64 (dt, 0.32H, J1 = 2.7 Hz, J2 = 1.4 Hz,
CHFn cis), 4.49 (dt, 0.34H, J1 = 2.7 Hz, J2 = 1.4 Hz, CHFn cis), 4.48–4.45 (m, 0.64H, CHFn trans),
4.44–4.42 (m, 0.34H, CHFn cis), 4.40–4.38 (m, 0.33H, CHFn cis), 4.38–4.37 (m, 0.65H, CHFn trans),
4.36–4.33 (m, 1.4H, CHαPro trans+CHFn trans), 4.26 (dd, 0.33H, J1 = 8.7 Hz, J2 = 3.4 Hz, CHαPro cis),
4.11–4.09 (m, 0.32H, CHFn cis), 4.09–4.07 (m, 0.32H, CHFn cis), 4.02–3.99 (m, 1.32H, CHFn trans), 3.79
(s, 0.98H, CH3COOMe cis), 3.78 (s, 1.94H, CH3COOMe trans), 3.62 (ddd, 0.35H, J1 = 10.9 Hz, J2 = 7.4 Hz,
J3 = 3.6 Hz, CHδPro cis), 3.52-3.46 (m, 0.36H, CHδPro cis), 3.44 (ddd, 0.70H, J1 = 10.3 Hz, J2 = 7.2 Hz,
J3 = 2.8 Hz, CHδPro trans), 3.28 (td, 0.7H, J1 = 10.2 Hz, J2 = 7.2 Hz, CHδPro trans), 2.43–2.38 (m, 0.70H,
CHβPro trans), 2.29–2.21 (m, 0.31H, CHβPro cis), 2.21–2.15 (m, 0.34H, CHβPro cis), 2.00–1.90 (m, 2.07H,
CHγPro trans + CHγPro cis), 1.90-1.83 (m, 0.63H, CHβPro trans), 1.48 (s, 5.97H, CH3Boc trans), 1.45 (s, 3.08H,
CH3Boc cis). 13C-NMR* (CDCl3, 150 MHz) δ 172.39 (COCOOMe cis), 172.21 (COCOOMe trans), 171.52
(COCO-NH cis), 170.24 (COCO-NH trans), 156.34 (COBoc trans), 155.06 (COBoc cis), 95.30 (CqFn trans), 93.94
(CqFn cis), 81.19 (CqFn), 80.91 (CqBoc trans), 73.04, 71.10, 70.69, 66.54, 66.33, 62.54, 62.40, (CHFn trans),
72.74, 71.44, 70.95, 66.84, 66.82, 63.77, 62.98 (CHFn cis), 72.90 (CHFn), 61.59 (CHαPro cis), 59.83
(CHαPro trans), 52.25 (CH3COOMe cis), 52.08 (CH3COOMe trans), 47.17 (CH2δPro), 31.21 (CH2βPro cis), 28.33
(CH3Boc), 27.21 (CH2βPro trans), 24.71 (CH2γPro trans), 23.77 (CH2γPro cis); HRMS (MALDI) m/z 456.136
(calcd for C22H28N2O5Fe, 456.1342). * Due to the poor signal resolution at the higher temperatures,
NMR spectra were recorded at 228 K.

Molecules 2014, 19
12872
Ac-Pro-Fca-OMe (4): mp 136–138 °C; IR IR (Nujol) νmax 3416 w (NHfree), 3272 m, 3229 m (NHassoc.),
1709 s (C=OCOOMe), 1690 s (C=OAc), 1623 s (C=OCONH), 1563 s (amide II) cm−1; Rf = 0.22
(CH2Cl2:EtOAc = 5:1); 1H-NMR (CDCl3, 600 MHz) δ 8.81 (s, 0.92H, NHtrans), 7.29 (s, 0.08H, NHcis),
4.75–4.72 (m, 2H, CHFn), 4.67–4.65 (m, 2H, CHFn+CHαPro), 4.63 (m, 0.06H, CHFn cis), 4.56 (dt, 0.93H,
J1 = 2.7 Hz, J2 = 1.4 Hz, CHFn trans), 4.54–4.53 (m, 0.06H, CHFn cis), 4.40–4.39 (m, 0.16H, CHFn cis),
4.38–4.36 (m, 1.87H, CHFn trans), 4.10–4.08 (m, 0.14H, CHFn cis), 4.01–3.99 (m, 1.86H, CHFn trans), 3.79
(s, 3H, CH3COOMe), 3.63 (ddd, 1H, J1= 10.3 Hz, J2 = 9.1 Hz, J3 = 3.0 Hz, CHδPro), 3.45 (td, 1H,
J1 = 9.9 Hz, J2 = 7.0 Hz, CHδPro), 2.59–2.56 (m, 1H, CHβPro), 2.20–2.15 (m, 1H, CHγPro), 2.18 (s, 3H,
CH3Ac), 2.06–2.01 (m, 1H, CHγPro), 1.87–1.81 (m, 1H, CHβPro); 13C-NMR (CDCl3, 150 MHz) δ 171.77
(COCOOMe), 171.67 (COCO-NH), 169.30 (COAc), 95.78 (CqFn), 72.29 (CqFn), 72.55, 72.53, 71.41, 71.22,
66.33, 66.27, 63.11, 62.99, (CHFn trans), 71.73, 71.35, 66.85, 66.72, 64.08, 63.65 (CHFn cis), 60.15
(CHαPro), 51.94 (CH3COOMe cis), 51.68 (CH3COOMe trans), 48.51 (CH2δPro trans), 47.11 (CH2δPro cis), 31.36
(CH2βPro cis), 26.81 (CH2βPro trans), 25.23 (CH3Ac), 23.22 (CH2γPro trans), 22.71 (CH2γPro cis); HRMS
(MALDI) m/z 398.0936 (calcd for C19H22N2O4Fe, 398.0923).
3.4. Crystallography
Crystals suitable for single-crystal X-ray diffraction analysis were grown from CDCl3 by slow
evaporation of the solvent at low temperature (277 K). Single crystal measurements were performed on
an Oxford Diffraction Xcalibur Nova R (microfocus Cu tube) at room temperature [293(2) K].
Program package CrysAlis PRO [76] was used for data reduction. The structures were solved using
SHELXS97 [77] and refined with SHELXL97 [77]. The models were refined using the full-matrix
least squares refinement; all non-hydrogen atoms were refined anisotropically. Hydrogen atoms bound
to C atoms were modelled as riding entities using the AFIX command. Molecular geometry
calculations were performed by PLATON [78], and molecular graphics were prepared using
ORTEP-3 [79], and CCDC-Mercury [80]. Crystallographic and refinement data for the structures
reported in this paper are shown in Table 7.
Table 7. Crystallographic, data collection and structure refinement details.
Compound
Boc-Pro-Fca-OMe (3)
Empirical formula
Formula wt./g·mol−1
C22H28FeN2O5
456.31
Crystal dimensions/mm
0.08 × 0.06 × 0.03
P 21
Space group
a/Å
b/Å
c/Å
α/°
β/°
γ/°
Z
10.951(5)
8.571(5)
11.944(5)
90
107.315(5)
90
2

Molecules 2014, 19
12873
Table 7. Cont.
Compound
V/Å3
Dcalc/g cm−3
µ/mm−1
Boc-Pro-Fca-OMe (3)
1070.3(9)
1.416
5.954
Θ range/°
3.88–75.86
293(2)
T/K
Radiation vawelength
Diffractometer type
1.54179 (CuKα)
Xcalibur Nova
−13 < h < 13;
−10 < k < 10;
−11 < l < 14
5054
Range of h, k, l
Reflections collected
Independent reflections
Observed reflections
(I ≥ 2σ)
3430
3108
Absorption correction
Rint
Multi-scan
0.0507
R (F)
Rw (F2)
0.1024
0.2806
Goodness of fit
H atom treatment
No. of parameters
No. of restraints
Δρmax, Δρmin (eÅ−3)
1.268
Constrained
271
1
1.3091; −1.272
CCDC 1011219 contains the supplementary crystallographic data for this paper. This supplementary
crystallographic data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.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).
3.5. Biological Evaluation
3.5.1. Cell Culture and Stock Solutions
MCF-7 cell line derived from human breast adenocarcinoma (ATCC No. HTB-22) and HeLa cell
line derived from the human cervical adenocarcinoma (ATCC No. CCL-2) were obtained from the
Ruđer Bošković Institute, Zagreb, Croatia. Both cell lines were maintained in Dulbecco’s modified
Eagle’s medium (DMEM, Gibco, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine
serum (FBS, Gibco) in the incubator at 310 K in humidified atmosphere with 5% CO2. Stock solutions
of tested compounds 1–4 were prepared as 10 mM solutions in ethanol (EtOH) and stored at 277 K.
The stock solutions were further diluted with culture medium prior to use in cytotoxicity assay.

Molecules 2014, 19
12874
3.5.2. Cytotoxicity Assay
The effect of the synthesised compounds 1–4 on cell proliferation was examined by the WST-1 assay
(Roche, Mannheim, Germany), modification of classical MTT test [81]. MCF-7 and HeLa cells from the
exponential growth phase were trypsinized and plated out in 96-well plates at a density of 5 × 104 cells/well
in 100 µL of media. After overnight cell growth, the media was replaced with fresh one containing
different concentrations of individual compound (1–500 µM). The appropriate volume of EtOH was
added to control cells, but the final concentration of EtOH in the control and treated cells was less than
0.2% and had no interference with the biological activities tested. Following exposure for 72 h, 10 µL
of tetrazolium salt WST-1 {4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate) was added to each well and cells were incubated for the further 4 h. The absorbance was
measured at 450 nm on the microplate reader (Tecan, Mannedorf, Switzerland). The experiments
were performed three times with four parallels for each concentration and data were expressed as the
means ± S.D. Cell viability was presented as percentage of treated cells vs. control cells by setting the
viability of control cell as 100%. The IC50 values, defined as the concentration of tested compound that
resulted with 50% growth inhibition, were calculated from the dose-response curves using equations of
best-fitted trend-lines.
3.5.3. Staining with Crystal-Violet
Light microscopy was used to observe the morphological changes during the treatment. MCF-7
cells (1 × 105 cells/mL) were seeded in the plate with 6 wells and after overnight cell growth exposed
to the highest concentration of tested compounds (500 µM) for 72 h. The cells were then washed with
PBS and stained with crystal-violet dye solution (Sigma–Aldrich, St. Louis, MO, USA). Images of
control and treated MCF-7 cells were taken using an inverted microscope (Carl Zeiss, Göttingen,
Germany) and Dino-Eye digital camera.
4. Conclusions
Four novel conjugates 1–4 of Fca and proline have been prepared. Their conformations in solution
have been elucidated using IR, NMR and CD spectroscopic methods. In addition, the crystal structure
of peptide 3 has been resolved.
Due to lack of the amide proton at the lower Cp ring of 1 and 2, their stabilization by intramolecular
hydrogen bonding has been provided through interchain interaction of NHFca and OCPro (9-membered
IHB ring) which corresponds to IHB pattern of the type A presented in their alanine analogues III.
Hence, the conformation B founded in III has not been realized. The alteration of peptides 1 and 2 into
3 and 4, respectively, notably influenced their conformational settings. The same donor (NHFca) and
acceptor (OCPro) were engaged in hydrogen bonding, but in a different manner, i.e., an intrachain
IHBs have been founded. With respect to their alanine analogues IV, stabilized by three alternative
patterns of hydrogen bonding, peptides 3 and 4 have been adopted 7-membered IHB ring (γ-turn)
correlated to IVA conformation. Although it has been shown that hydrogen bonding is somewhat
difficult in the presence of the bulky Boc group, its replacement with an Ac group did not cause
significant conformational changes.

Molecules 2014, 19
12875
The biological evaluation of peptides 1–4, performed with regard to antiproliferative effect on
MCF-7 and HeLa cell line, has been revealed no or rather modest cytotoxic effects only at the maximal
tested concentration in the high micromolar range. Those results are not satisfactory if we consider the
tested compounds as promising cytotoxic agents candidates, but are valuable as a guideline to direct
the synthesis of the corresponding analogues with improved biological properties. The goal of the
presented biological evaluation was to find lead structures as a starting point for future search of
optimized drug candidates what will surely be our further scientific interest.
Supplementary Materials
Supplementary materials (HRMS, 1H and 13C NMR spectra of novel compounds 1–4,
1H NMR titration of compounds 1–4 with DMSO in CDCl3, Variable-temperature
1H NMR spectra of 1–4 and checkCIF_PLATON report of compound 3) can be accessed at:
http://www.mdpi.com/1420-3049/19/8/12852/s1.
Acknowledgments
This research was supported by the Ministry of Science, Education and Sports of the Republic of
Croatia (Grant Numbers 058-1191344-3122, 058-0582184-2414, 098-1191344-2943) and by
University of Zagreb, Croatia (Financial Support 2).
Author Contributions
Lidija Barišić suggested the research work and discussed the experimental data; Monika Kovačević
and Alan Čače finalized the experimental work; Kristina Radošević and Visnja Gaurina Srček
preformed biological evaluation on human cancer cell lines; Sunčica Roca measured NMR spectra and
assisted in spectral assignment; Krešimir Molčanov was concerned with crystallographic work; Lidija
Barišić and Kristina Radošević wrote the paper, edited and revised manuscript. All authors read and
approved the final manuscript.
Conflicts of Interest
All authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–4 are available from the authors.
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