Nucleobase-guanidiniocarbonyl-pyrrole conjugates as novel fluorimetric sensors for single stranded RNA

Article abstract of DOI:10.3390/molecules22122213

We demonstrate here for the first time that a guanidiniocarbonyl-pyrrole (GCP) unit can be applied for the fine recognition of single stranded RNA sequences-an intuitively unexpected result since so far binding of the GCP unit to ds-DNA or ds-RNA relied s

Full text of DOI:10.3390/molecules22122213

molecules
Article
Nucleobase–Guanidiniocarbonyl-Pyrrole Conjugates
as Novel Fluorimetric Sensors for Single
Stranded RNA
Željka Ban 1, Biserka Žinic´ , Robert Vianello 1 ID , Carsten Schmuck 2 and Ivo Piantanida 1,
*
1
1
Division of Organic Chemistry and Biochemistry, Ruđer Boškovic´ Institute, Bijenicˇka cesta 54,
10000 Zagreb, Croatia; Zeljka.Ban@irb.hr (Ž.B.); biserka.zinic@irb.hr (B.Ž.); robert.vianello@irb.hr (R.V.)
Institute for Organic Chemistry, University of Duisburg-Essen, Universitätsstrasse 7, 45141 Essen, Germany;
carsten.schmuck@uni-due.de
2
*
Correspondence: Ivo.Piantanida@irb.hr; Tel.: +385-1-4571-326
Received: 1 November 2017; Accepted: 9 December 2017; Published: 13 December 2017
Abstract: We demonstrate here for the first time that a guanidiniocarbonyl-pyrrole (GCP) unit can
be applied for the fine recognition of single stranded RNA sequences—an intuitively unexpected
result since so far binding of the GCP unit to ds-DNA or ds-RNA relied strongly on minor or major
groove interactions, as shown in previous work. Two novel nucleobase–GCP isosteric conjugates
differing in the flexibility of GCP unit revealed a fluorimetric recognition of various single stranded
RNA, which could be additionally regulated by pH. The more rigid conjugate showed a specific
fluorescence increase for poly A only at pH 7, whereby this response could be reversibly switched-off
at pH 5. The more flexible derivative revealed selective fluorescence quenching by poly G at
pH 7 but no change for poly A, whereas its recognition of poly AH+ can be switched-on at pH 5.
The computational analysis confirmed the important role of the GCP fragment and its protonation
states in the sensing of polynucleotides and revealed that it is affected by the intrinsic dynamical
features of conjugates themselves. Both conjugates showed a negligible response to uracil and cytosine
ss-RNA as well as ds-RNA at pH 7, and only weak interactions with ds-DNA. Thus, nucleobase–GCP
conjugates can be considered as novel lead compounds for the design of ss-RNA or ss-DNA selective
fluorimetric probes.
Keywords: pyrrolocytosine; guanidiniocarbonyl-pyrrole; DNA/RNA binding; fluorimetric recognition
1. Introduction
Molecular visualization and recognition of DNA and RNA are an important area of ongoing
research in medicine and molecular biology, in which small molecules can be not only useful dyes but
also effective anticancer, antibiotic and antiviral therapeutic agents [
abundant ds-DNA, many DNA and RNA structures were discovered (i-motif, triplex and G-quadruplex
structures), becoming novel targets for drugs, for instance in cancer chemotherapy [ ]. Most of the
1]. Besides the naturally most
2
studies of small molecules were focused on their interactions with double- or multiple-stranded
DNA or RNA, but not with single stranded DNA/RNA. That was mostly due to a lack of interest
since small molecules generally bind more strongly to multi-stranded nucleic acid structures than
to single-stranded (ss) polymers. Moreover, ss-polynucleotides adopt poorly defined secondary
structures, making ligand design a very challenging task. Nevertheless, single-stranded nucleic acids
such as poly(A) have critical roles in cell biology [3–5], while single stranded G-rich structures can
fold in a variety of G-quadruplexes. Furthermore, some RNA in the genomes of various cardioviruses
and encephalomyocarditis viruses contain stretches composed of more than 75% cytosine, and single
stranded C-polymers can adopt a non-B form structure called i-motif under slightly acidic or neutral
pH [6,7]. Very few small molecules reveal selective binding to single stranded DNA or RNA structures.
Molecules 2017, 22, 2213; doi:10.3390/molecules22122213
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Molecules 2017, 22, 2213
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For instance, some isoquinoline alkaloids bind poly(A) with considerable affinity [
8,
9] as does
the the aminoglycoside antibiotic neomycin [10]. The most effective approach to ss-polynucleotide
recognition by small molecules (MW < 1000) was combining intercalative unit with a nucleobase,
as introduced by Lhomme et al. [11
of phenanthridinium [13 17] and bis-phenanthridinium-nucleobase conjugates [18
DBTAA-adenine conjugates specifically recognised poly dT [20].
,
12], and developed for various ss-polynucleotides by a series
–
,19]. Also, some
Our research on novel moieties for interactions with DNA/RNA revealed intriguing properties
of the guanidiniocarbonyl-pyrrole system (abbreviated GCP), which we studied in detail on various
ds-DNA and ds-RNA. Particularly interesting were aryl-GCP conjugates, which, depending on
their structure, showed specific fluorimetric and CD recognition of different ds-DNA or ds-RNA
structures depending on pH or steric and/or H-bonding properties of conjugates [21–25]. Mostly,
the GCP unit interacted within the DNA or RNA grooves, although there were indications that in
its protonated form (pH 5) GCP could interact also with negatively charged DNA/RNA backbone.
Particularly latter property raised the question whether GCP unit could also be used for interactions
with DNA/RNA targets which do not have a well-defined secondary structure and hence minor or
major grooves, respectively.
Up till now, we did not assay the interactions of aryl-GCP conjugates with single stranded DNA
or RNA. To address this, we relied on our previous research based on condensed-aryl-nucleobase
conjugates [13–20] targeting particular ss-polynucleotide sequences, whereby large aryl moiety ensured
sufficient DNA/RNA affinity and spectroscopic response while an attached nucleobase contributed to
particular ss-sequence recognition by a set of H-bonds. In the research presented here we designed
and prepared a novel fluorescent cytosine-amino acid analogue
peptide coupling to ensure broad applicability (Figure 1). Further on, we attached GCP on lysine at
two different positions and consequently coupled the two different GCP-lysines with . The obtained
1 with C- and N-end available for
1
nucleobase–GCP conjugates are isosteres, differing in GCP position either on the shorter and more
rigid alpha-amino position (2) or on the longer, more flexible side-chain amino position (3).
1
2
R = H
R =
H N
O
H N
H N
R
HN
NH
N H
N H
N
C
O
NH
O
O
O
OCH
2
3
O
N
H N
2
O
H N
O
H N
N
NH
O
N H
N H
3
R =
O
NH
O
NH
O
NH
2
2
3
H N
2
NH
O
2
H N
HN
H N
O
NH
3
2
O
3
HN
Li-1
H N
O
Figure 1. Studied compounds 1–3 and previously studied analogue Li-1 [26].
2. Results and Discussion
2.1. Chemistry
We have designed and synthesized a new type of molecular probes by combining two molecular
fragments Pyrr-C and GCP, both well known for their capability to strongly interact with nucleic acids.
The fluorescent pyrrolocytosine nucleobase (Pyrr-C) [27] preserves the H-bonding pattern of the parent

Molecules 2017, 22, 2213
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nucleobase cytosine [28,29], while the guanidiniocarbonyl-pyrrole (GCP) moiety is known as a highly
efficient DNA minor groove binder [23,25].
We synthesized the fluorescent Pyrr-C-amino acid analogue
1 (Scheme 1) which served as the
starting compound for the preparation of the two novel nucleobase-guanidiniocarbonyl-pyrrole
conjugates
2 and 3. Previous studies have shown that the Sonogashira coupling of terminal
alkynes with 5-iodocytosine derivatives gave the 5-alkynyl products while N4-benzoyl-5-iodocytosine
derivatives lead to the annulated pyrrolocytosine derivatives via domino cross-coupling and
cyclization reaction [30,31].
With these facts in mind, the 5-iodo-N4-benzoylcytosine
5
was synthesized in a very good
yield starting from cytosine (which was previously iodinated by using I2/HIO3 in biphasic
AcOH/H2O/CH2Cl2 yielding 5-iodocytosine 32 33], followed by the reaction with benzoic
4
[ ,
anhydride and the presence of DMAP in acetonitrile as described earlier for the synthesis of
N4-benzoylcytosine (Scheme 1) [34].
As the next step, we planned to introduce N-1 substituent by alkylation of
5 with
N-(2-bromoethyl)phthalimide. This substituent may have multiple roles; (i) it may simplify the
isolation of the subsequent products due to increased lipophilicity; (ii) after removal of the phthalimide
group a free amino group is generated enabling further functionalization; and (iii) the phthalimide
groups itself could contribute to intermolecular interaction by H-bonding and/or aromatic stacking
interactions. The best yield of N-1 alkylated product was obtained by alkylation of
5 with
N-(2-bromoethyl)phthalimide in the presence of K2CO3/DMF under microwave irradiation at 140 ◦C
giving 6 in 51% yield.
Pyrr-C derivative
8
was synthesized by adopting the methods reported in the literature [35].
with protected [36
The one pot sequential Sonogashira cross-coupling reaction and annulation of
6
]
propargylglycine
7
was used. Compound
8
was readily deprotected by CH2Cl2/TFA to afford 1 in a
very good yield (88%).
Bz
O
N
O
HN
Bz
I
O
O
HN
I
NH
N
2
Br
O
I
N
N
N
O
DMAP
CH CN
K CO
DMF3
2
N H
O
N H
O
3
O
6
N
MW, 140 o
35 min
51%
C
82 %
O
5
4
H N
NHBoc
NH
COOMe
C CH
2
HN
HN
Boc
N
N
OCH
OCH
O
O
3
3
7
O
CH2Cl2/TFA
O
N
N
CuI
Pd PPh
72 h
88 %
Et N3
D3MF
4
1
O
O
8
N
N
O
O
24 h, 50 oC
64 %
Scheme 1. Synthesis of novel fluorescent Pyrr-C-amino acid analogue 1.
Having the fluorescent Pyrr-C-amino acid analogue
guanidiniocarbonyl-pyrrole unit (Boc-GCP-OH) [37 13 through a Gly-Lys dipeptide bridge (Scheme 2).
We planned to first attach the fluorescent Pyrr-C unit to Gly-Lys dipeptide and the GCP 13 unit
in the second step (Scheme 2). Starting from the commercially available Z-D-Lys(Boc)-OH and
H-Gly-OMe hydrochloride, the Z-D-Lys(Boc)-Gly-OMe dipeptide was synthesized in excellent yield
by a standard coupling reaction using HBTU/HOBt as coupling reagents and the base, triethylamine
in acetonitrile [38]. Dipeptide was readily deprotected with 2 M NaOH in dioxane/H2O (v/v 3:1)
1 in hand, we decided to link it with the
]
1
9
9

Molecules 2017, 22, 2213
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giving 10
.
The same coupling procedure was applied for coupling of the Pyrr-C amino acid
analogue
1
with Z-D-Lys(Boc)-Gly-OH dipeptide 10; the product 11 is obtained in moderate yield.
The Boc-protecting group was removed with a mixture of CH2Cl2/TFA affording 12 in quantitative
yield. In the next step, 12 was coupled with GCP 13 giving the conjugate 14. Unexpectedly, the
removal of Cbz protecting group in 14 by hydrogenolysis failed and no deprotected compound 15
could be isolated.
NHBoc
O
H C
N H
HCl
H- Gl y- OMe
(i)
3
x
Z-
D
-Lys(Boc)OH
+
HN
O
98 %
Cbz
9
(ii)
55%
O
O
H N
NHBoc
1
R
HO
HN
N
(i)
N H
N H
HN
O
HN
1
O
C
Cbz
Cbz
10
OCH
49 %
O
O
3
N
R1= NHBoc
R1= NH2
11
12
(iii)
100 %
O
N
O
O
O
N H
NH
(i)
O
HN
GCP 13
57%
NEt3
NHBoc
H N
O
H N
H N
NH
HN
N H
N H
N
NHBoc
HN
O
O
OCH
O
C
Cbz
O
O
3
N
14
O
(iv)
N
O
O
H N
H N
H N
O
NH
HN
N H
N H
N
NHBoc
NH
O
O
C
2
OCH
O
O
3
N
O
15
O
N
Scheme 2. Attempted synthesis of 15. Reagents and conditions: (i) HBTU, HOBt, Et3N, CH3CN; (ii) 2 M
NaOH, dioxane/water (v/v 3:1); (iii) TFA/CH2Cl2; (iv) H2, Pd/C, MeOH.
Hence, the synthetic approach was changed in the way that the GCP 13 is firstly attached to
Gly-Lys dipeptide at either of the two (alpha-amino or side-chain amino) positions, and the obtained
products are subsequently coupled with the fluorescent Pyrr-C unit 1 (Scheme 3a,b).
The synthesis of novel nucleobase-guanidiniocarbonyl-pyrrole conjugate
2 started by
hydrogenolysis of Z-D-Lys(Boc)-Gly-OMe dipeptide which afforded MeO-Gly-H-Lys(Boc)-
9
dipeptide 16 in quantitative yield (Scheme 3a). Using a standard coupling reaction with HBTU/HOBt
conjugate 17 was synthesized in moderate yield. The methyl ester was then removed by basic
hydrolysis yielding 18, which was then coupled with Pyrr-C derivative
compound 19. Finally desired conjugate , with the GCP-unit attached to the
of Lys unit, was obtained by deprotection of Boc-protecting groups with CH2Cl2/TFA. The preparation
of nucleobase–GCP conjugate with the GCP-unit attached to the side-chain amino position of Lys
unit is outlined in Scheme 3b. In this case, the starting materials were amino acid Boc-D-Lys(Z)-OH and
1
resulting in hybrid
2
α
-amino position
3
H-Gly-OMe hydrochloride, which were coupled by HBTU/HOBt activation giving the dipeptide 20
After the Cbz deprotection, 21 was obtained in quantitative yield. Then, 21 was coupled with
guanidiniocarbonyl-pyrrole derivative 13 under standard HBTU/HOBt conditions, providing 22
.

Molecules 2017, 22, 2213
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in moderate yield. The methyl ester in the conjugate 22 was easily removed by basic hydrolysis giving
the GCP peptide 23 which was then coupled with the Pyrr-C derivative under standard coupling
1
conditions giving the conjugate 24. Finally, Boc removal from 24 was again carried out with TFA,
providing the Pyrr-C-guanidiniocarbonyl-pyrrole derivative 3 in high yield.
O
O
H N
H N
NH
NH
O
O
2
(ii)
(i)
H C
N H
H C
N H
N H
3
3
9
NHBoc
O
O
O
O
GCP 13
100 %
58 %
16
17
(iii) 75 %
NHBoc
NHBoc
O
O
H N
H N
H N
H N
H N
NH
NH
(ii)
HO
HN
N H
N H
N H
N H
2
NHBoc
O
R
O
O
N
O
OCH
O
O
C
1
O
O
3
50 %
N
18
2
R
NHBoc
O
R2= NHBoc
R2= NH2
19
2
N
(iv)
O
100 %
(a)
O
HN
Cbz
O
HCl
(i)
H- Gl y- OMe
H C
NH
3
+
x
Boc-D
-Lyz(Z)-OH
NHBoc
O
96 %
20
(ii)
100 %
O
O
H N
HN
NH
NH
O
O
2
(i)
H C
NH
H C
NH
N H
3
3
NHBoc
NHBoc
O
NHBoc
O
O
O
O
GCP 13
40 %
21
22
70 %
47 %
(iii)
O
H N
HN
NH
HO
NH
N H
NHBoc
NHBoc
(i)
O
O
23
Pyrr-C 1
O
H N
HN
H N
NH
HN
NH
N H
N
3
R
NHBoc
O
O
O
C
OCH
O
O
3
N
R3= NHBoc
R3= NH2
24
(iv)
95 %
O
3
N
O
(b)
Scheme 3. Synthesis of nucleobase–GCP conjugates
Pd/C, MeOH; (ii) HBTU, HOBt, Et3N, CH3CN; (iii) LiOH, THF/water (v/v 4:1); (iv) CH2Cl2/TFA;
) (i) HBTU, HOBt, Et3N, CH3CN; (ii) H2, Pd/C, MeOH; (iii) LiOH, THF/water (v/v 4:1); (iv)
2 and 3. Reagents and conditions: (a) (i) H2,
(
b
TFA/CH2Cl2.
2.2. Interactions with DNA and RNA
All compounds 1–3 are moderately soluble in water, and for easier application 0.01 M stock
solutions were prepared in DMSO and diluted in buffered solutions prior to use (0.05 M Na cacodylate,
DMSO content of the final solutions <0.01%). The UV/Vis spectra (Figure 2) of buffered solutions of
1
–
3
are proportional to their concentrations up to c = 2
×
10−5 mol dm−3, pointing out that studied
compounds do not aggregate by intermolecular stacking at experimental conditions used. Since it
is known that GCP unit is protonated at weakly acidic conditions (pH 5) [25], UV/Vis spectra were

Molecules 2017, 22, 2213
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collected at pH 7 and pH 5, but only negligible differences were observed. At pH 7 compounds
bear one positive charge, while at pH 5 the GCP group is also protonated [25], so have
charge +2. Absorption maxima and corresponding molar extinction coefficients ( ) are given in Table
S1 (Supplementary Materials). Heating of the aqueous solutions of
up to 90 ◦C did not cause any
1
–3
2, 3
ε
1–3
significant changes in the UV/Vis spectra and reproducibility upon cooling back to room temperature
verified the chemical stability of the compounds.
2.5x104
2.0x104
O
N
N
O
HN
N
O
1.5x104
+
NH3
1.0x104
HN
ε
O
HN
5.0x103
N
O
H
0.0
250
275
300
325
350
375
400
425
λ / nm
Figure 2. UV/Vis spectrum of
2 at pH 7.0, sodium cacodylate buffer, I = 0.05 M, note marked absorption
ranges for two chromophores.
The studied compounds have two fluorophores (GCP and nucleobase) connected by a more
(compound ) or less (compound ) flexible linker. As shown in Figures 2 and 3 (and Figure S12 of
Supplementary Materials for reference compound ), excitation at exc = 299 nm excites all fluorophores,
while excitation at = 355 nm excites only the nucleobase. That allows sequential fluorimetric
3
2
1
λ
λ
exc
studies, whereby fluorimetric response upon excitation at λexc = 299 nm monitors the impact of studied
binding to all chromophores, while excitation at
on nucleobase-phthalimide moiety.
λ
exc
= 355 nm is focused on the changes induced only
2.5x107
emission upon exc. at 299 nm
emmision upon exc. at 355 nm
350
excitation spectrum
2.0x107
300
250
200
150
100
50
1.5x107
1.0x107
Li-1 excitation
Li-1 emission
5.0x106
0
0.0
250
300
350
400
450
500
550
600
650
200
240
280
320
360
400
440
480
520
λ / nm
λ / nm
(a)
(b)
Figure 3.
and emission spectrum (
(
a
) Previously reported analogue GCP [26] fluorescence excitation spectrum (
λ
= 350 nm)
= 299 nm ,
em
λ
exc
= 299 nm); (
b
) excitation (
λ = 447 nm ) and emission (λ
em exc
λ
exc
= 355 nm ) spectra of 2. Done at pH 7.0, sodium cacodylate buffer, I = 0.05 M.

Molecules 2017, 22, 2213
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2.3. DNA/RNA Binding Studies
Interactions of 1–3 with DNA and RNA were studied at both pH 7 and pH 5, due to the difference
in the protonation state of GCP [25]. It should be stressed that poly A and poly C are single stranded
under neutral conditions (pH 7), while at pH 5 they become double stranded (poly AH+-poly AH+
and poly CH+-poly CH+) [19,39].
With representative ds-DNA (calf thymus DNA) as well as with ds-RNA (poly A–poly U) none of
the studied compounds showed any stabilisation in thermal denaturation experiments, at both pH 7
and pH 5 (Figures S46 and S47 of Supplementary Materials). Lack of thermal stabilisation excludes
intercalative binding mode [2].
Fluorimetric titrations of
2 (Figure 4) and 3 (Figure S29 of Supplementary Materials) at pH 7
revealed significant emission increase only upon addition of ct-DNA, but no emission change upon
titration with ds-RNA (poly A–poly U, Figures S19 and S33 of Supplementary Materials). Intriguingly,
at pH 5 also poly A–poly U yielded significant increase of fluorescence (Figure S24 of Supplementary
Materials), but only for
pH 5, could be partially attributed to the protonation of the GCP unit, which as previously shown, can
significantly contribute to polynucleotide binding [22 25]. However, obviously position of GCP unit
in the compound is also important for efficient binding, thus only for but not giving measurable
2. The lack of binding of 2, 3 to ds-RNA at pH 7, but efficient binding of 2 at
–
2
3
fluorimetric changes are observed. Processing of titration data by Scatchard equation [40,41] revealed
similar affinities (log Ks ≈ 4, Table 1) for all ds-DNA and ds-RNA (RNA only at pH 5).
300
260
250
240
220
200
200
180
150
160
140
0.0
3.0x10-4 6.0x10-4 9.0x10-4 1.2x10-3 1.5x10-3
100
50
0
c(ct-DNA) / mol dm-3
400
450
500
550
600
λ / nm
Figure 4. Fluorescence spectra changes of
pH 7.0, sodium cacodylate buffer, I = 0.05 M,
c(DNA) fitted to Scatchard equation [40,41].
2
(c = 2
λ = 355 nm. Inset: emission at 447 nm dependence on
exc
×
10−6 mol dm−3) upon addition of ct-DNA at

Molecules 2017, 22, 2213
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Table 1. Binding constants (log Ks) a calculated from the fluorescence titrations (
with ds- and ss-polynucleotides at pH = 7.0 and pH = 5.0 (buffer sodium cacodylate, I = 0.05 mol dm−3).
λexc = 355 nm) of 2 and 3
2
3
pH 7
log Ks/ c I/I0
pH 5
log Ks/ c I/I0
4.0/1.5
pH 7
log Ks/ c I/I0
pH 5
log Ks/ c I/I0
Polynucleotide
ct-DNA
poly A–poly U
poly A
3.7/1.8
3.7/1.6
4.4/1.5
b
b
b
3.5/1.8
b
b
d
4.1/2.4
4/1.2
b
b
b
b
d
d
poly G
poly C
poly U
5/0.8
4/0.9
b
b
b
b
b
b
a
Processing of titration data by means of Scatchard equation [40,41] gave values of ratio n[bound
2
, 3]/[polynucleotide] = 0.1–0.3, for easier comparison all log Ks values were re-calculated for fixed n = 0.2.
Correlation coefficients were >0.99 for all calculated Ks; b Too small changes for accurate processing by Scatchard eq;
c
I0—starting fluorescence intensity of dye; I—fluorescence intensity of dye/polynucleotide complex calculated by
Scatchard equation; d Small changes allowed only the estimation of log Ks.
In order to gain insight into the changes of polynucleotide properties induced by small molecule
binding, we have chosen CD spectroscopy as a highly sensitive method for conformational changes in
the secondary structure of polynucleotides [42]. In addition, achiral small molecules can eventually
acquire induced CD spectrum (ICD) upon binding to polynucleotides, which could give useful
information about modes of interaction [43].
All studied compounds are chiral but intensity of their CD spectra in the 230–500 nm
range (Figures S42–S45 of Supplementary Materials) is negligible with respect to CD spectra of
polynucleotides, allowing accurate correction of CD titrations. Addition of
2 or 3 to ct-DNA or
ds-RNA did not significantly change the CD spectrum of the polynucleotide at both pH 7 and pH 5,
thus pointing out that no DNA/RNA conformational change happened upon compound binding.
Moreover, no ICD bands >300 nm were observed, thus chromophores of
oriented along polynucleotide chiral axis [43].
2, 3 did not become uniformly
The lack of thermal stabilisation, negligible changes in CD experiments and rather low binding
constants (log Ks 4) suggest that the observed fluorescence changes of are a consequence
≈
2, 3
of random aggregation of compounds along double stranded helix of polynucleotide, based on
non-specific electrostatic interactions of their positive charge with DNA/RNA backbone as well as
hydrophobic interactions.
Single Stranded (ss)-Polynucleotides
Compounds
with ss-polynucleotides. Fluorimetric titrations were performed using sequential excitation properties
of and , whereby = 299 nm excites all fluorophores, while excitation at = 355 nm
2 and 3 contain a fluorescent analogue of cytosine, thus we studied their interactions
2
3
λ
exc
λ
exc
excites only nucleobase-phthalimide. It is important to stress that
did not change emission upon addition of any ss-polynucleotide at
Supplementary Materials).
1
λ
(reference nucleobase unit)
= 355 nm (Figure S40 of
exc
In general, the addition of any ss-polynucleotide to GCP-rigid analogue
2
yielded indiscriminate
at only nucleobase-
= 355 nm) at pH 7 induced significant emission increase of only for poly A
did not change (Figure S25 of Supplementary
Materials), likely due to the formation of poly AH+-poly AH+ [19
39]. Thus, general fluorescence
increase upon exc = 299 nm can be attributed to the interaction of GCP-unit, which non-selectively
binds to all polynucleotides, while highly selective emission upon = 355 nm excitation can be
emission increase upon excitation at = 299 nm. However, excitation of
λ
exc
2
fluorophore (
λ
2
exc
(Figure 5a), while intriguingly at pH 5 emission of
2
,
λ
λ
exc
attributed to the particular interaction of nucleobase-unit of 2 with poly A (observed only at pH 7).

Molecules 2017, 22, 2213
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For GCP-flexible analogue
all ss-RNA. Intriguingly, at pH 7 analogue
emission quenching for poly G in respect to other ss-polynucleotides (Figure 5b), but did not show
poly A recognition as . Only at acidic conditions (pH 5) at which GCP is protonated (and poly a forms
ds-structure poly AH+-poly AH+) [39],
exhibited the poly A-selective response, whereby intriguingly
3, the excitation at
λ
exc
= 299 nm yielded also fluorescence increase for
3
upon excitation at
λ
exc
= 355 nm showed highly selective
2
3
at the same acidic conditions emission of 2 did not change (Figure S35 of Supplementary Materials).
Thus, only 3 yielded emission response upon interacting with poly AH+-poly AH+.
To examine the influence of GCP-unit on interaction with ss-polynucleotides, we tested
interactions of compound 12 (analogue of
Supplementary Materials), which at both, pH 7 and pH 5 showed non-selective fluorimetric results,
similar to the response of free nucleobase (Pyrr-C). These fluorimetric titrations clearly stressed
synergistic effect of GCP conjugation with nucleobase for the ss-polynucleotide recognition by and 3.
2 lacking GCP unit) with poly A (Figure S41 of
1
2
All fluorimetric titrations with sufficiently large emission change were processed by means of Scatchard
eq. to obtain binding constants (Table 1).
In CD experiments addition of
2 or 3 did not change significantly the CD spectra of
ss-polynucleotides, at both pH 7 and pH 5, thus indicating that no polynucleotide conformational
change happened upon compound binding. Moreover, no ICD bands >300 nm were observed, thus
chromophores of
axis [43].
2,
3
upon binding did not become uniformly oriented along polynucleotide chiral
a)
b)
pA
pG
pC
pU
1.10
1.8
1.6
1.4
1.2
1.0
0.8
1.05
1.00
0.95
0.90
pA
pG
pC
pU
0.85
0.80
0.75
0.70
3.0x10-5 6.0x10-5 9.0x10-5 1.2x10-4 1.5x10-4 1.8x10-4
0.0
1.0x10-4
2.0x10-4
3.0x10-4
0.0
c(ss-polynucleotide) / mol dm-3
c(ss-polynucleotide) / mol dm-3
Figure 5. Fluorimetric titrations normalised to starting fluorescence intensity of compound: for 2 (a) and
3 (b), λexc = 355 nm, with all ss-polynucleotides. Done at pH 7.0, sodium cacodylate buffer, I = 0.05 M.
2.4. Molecular Modelling
The ss-RNA do not have well-defined structural features in aqueous solutions and therefore did
not allow accurate modelling approach. However, conjugates
2 and 3, although isosteres, significantly
differ in flexibility of GCP-unit attachment, former being significantly more rigid than latter (Figure 1).
Such difference in structure could be responsible for dramatic differences in ss-RNA recognition.
In order to examine conformational features of
2 and 3 and inspect whether their intrinsic
dynamics in aqueous solution play any role in determining their ability to interact with biological
systems studied here, we performed molecular dynamics (MD) simulations of different protonation
forms of 2 and 3 placed in the explicit water solvation followed by the quantum-mechanical analysis
at the DFT level with implicit solvation.
Initially, we performed the analysis of the acid/base features of
2 and 3 in water to determine
their predominant protonation forms under experimental conditions (pH values of 5 and 7). For that
purpose, we calculated aqueous phase pKa values of fragments III (Scheme 4) that are building
blocks of compounds . In doing so, we assumed that the free amino group originating from lysine
I–
2–3

Molecules 2017, 22, 2213
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will be protonated at both pH values since in lysine its pKa value is 10.5, which is unlikely to change
much or be reduced by 3–4 pKa units in cases studied here [44].
O
*
O
N
O
O
O
*
N
N
N
H
NH2
NH2
HN
N
NH
*
III
= 5.2
I
II
pKa = 3.7
p
Ka
pKa = 2.1
Scheme 4. Constituting fragments of systems
2 and 3 and the pKa values calculated at the MP2/6–
311++G(2df,2pd)//(SMD)/M06–2X/6–31+G(d) level of theory. The most favourable protonation atom
is denoted with an asterisk. * represents the most basic position within a molecule.
The pKa values calculated for I and II are slightly lower than those of succinimide (pKa = 3.9) and
cytosine (pKa = 4.5) used as references for their evaluation. This implies that even at pH = 5 both of
these fragments will predominantly be unprotonated. Interestingly, although guanidine (pKa = 13.6)
and its derivatives provide some of the strongest organic bases know today [45
carbonyl-pyrrole fragment significantly reduces its basicity to pKa(III) = 5.2, being a consequence of an
extended conjugated -system, which depletes the electron density from the guanidine imino nitrogen
–47], combining it to the
π
leading to much-reduced basicity. This suggests that at pH = 7, the fragment III will be neutral, while
at pH = 5 it will assume monoprotonated form III+, thus putting our results in firm agreement with
earlier experimental observations [22], and confirming that this fragment is predominantly responsible
for the biological activity investigated here.
Having this in mind, we prepared the geometries of monocationic 2+ and 3+ (protonated at the
free amino group, corresponding to pH = 7), as well as dicationic 22+ and 32+ (also protonated at
the guanidine moiety, corresponding to pH = 5), and submitted all four systems to MD simulations.
Following the clustering analysis of the obtained trajectories, we optimized a set of representative
structures in each case at the (SMD)/M06–2X/6–31G(d) level of theory and extracted total electronic
energies in search of most stable, thus most dominant structures in solution (Figure 6).
+
2+
+
2+
2 –a
2 –a
3 –a
3 –a
Figure 6. Most stable structures of 2 and 3 in their mono- and di-protonated forms. These are identified
after a set of structures, elucidated with the clustering analysis of the corresponding MD trajectories,
was optimized at the (SMD)/M06–2X/6–31G(d) level of theory. A larger set of representative structures
is placed in the Supplementary Materials (Figures S48–S51). Abbreviations GUA, PYRR, N-Lys, Pyrr-C
and Phth denote the guanidine group, pyrrole moiety, free amino group, nucleobase, and phthalimide
moiety, respectively. Only selected hydrogen atoms are depicted due to clarity.
It is interesting to observe that, at neutral pH, 2 reveals notable interactions only with ss-poly A,
while, surprisingly, at pH = 5, no interactions are detected with either of the ss-polynucleotides. Under

Molecules 2017, 22, 2213
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acidic conditions, the most stable structure 22+–a has the guanidine fragment engaged in three rather
strong hydrogen bonds involving one of its amino groups interacting with the carbonyl moiety on
the nucleobase (1.77 Å), and the other amino group interacting both with the same carbonyl fragment
(2.08 Å) and the carbonyl centre from phthalimide (2.29 Å). This is the only case in all four studied
systems where, in the most dominant structure, the guanidine moiety is so strongly involved in the
intramolecular stabilization, which is a pattern also observed in several other structures subsequently
less stable than 22+–a (Figure S49 of Supplementary Materials).
This clearly diminishes the ability of the guanidine group in 22+ to interact with other systems as
revealed at pH = 5 here. Let us also mention that the most stable 22+ structure having the guanidine
group not involved in any intramolecular hydrogen bonding, 22+–e (Figure S49 of Supplementary
Materials), is 2.9 kcal/mol less stable than 22+–a, which turns out to be too high to be overcome and
provides evidence to no activity of 2 towards any of the ss-polynucleotides at pH =5.
At pH = 7, system 2 .
loses an extra proton on the guanidine moiety and is present as monocationic 2+
In the most stable structure 2+–a the guanidine moiety is not involved in any hydrogen bonding, which
obviously allows 2+ to interact with ss-poly A. The intramolecular stabilization in 2+–a predominantly
occurs through the interaction of the protonated free amino group with (a) the imino nitrogen on the
nucleobase (2.11 Å); (b) the carbonyl group on phthalimide (2.43 Å); and (c) the carbonyl moiety in
the amide fragment (1.92 Å). Interestingly, only 0.6 kcal mol−1 above 2+–a, a structure 2+–b is also
dominated by the intramolecular interactions with the protonated free amino group, but there the
guanidine fragment is found in the stabilizing π–π stacking interactions with phthalimide (Figure S48
of Supplementary Materials), which likely affects its interactions with ss-polynucleotides other than
ss-poly A. The structure with a non-interacting cytosine nucleobase is 9.6 kcal mol−1 less stable than
2+–a, which is clearly too high to have any impact on the potential pairing with ss-poly G.
Under acidic conditions (pH = 5), system 3 is the only molecule that exhibits interactions with
two polynucleotides, namely, poly A and poly G (Table 1). In its most stable structure 32+–a the
guanidine moiety is not involved in any hydrogen bonding, although the nearby pyrrole fragment and
vicinal carbonyl group interact with the neighbouring amide fragments at 2.04 and 1.88 Å, respectively.
The structure where the whole GCP fragment i−s1free of any hydrogen bonds is 32+–b (Figure S51 of
Supplementary Materials), which is 2.4 kcal mol less stable than 32+–a. Taken all together, all of this
allows 32+ to interact with both poly A and poly G, yet relative changes in increasing or decreasing the
fluorescence upon binding are the smallest here (Table 1).
Under physiological conditions (pH = 7, system 3+), the situation with
3
is similar to that with 2+
in a way that it shows notable interactions with only one polynucleotide, namely ss-poly G (Table 1).
The most stable structure 3+–a has the guanidine moiety free of any hydrogen bonding interactions,
yet, as in 32+–a, the interactions of the pyrrole and amide carbonyl of the GCP fragment with the
nucleobase carbonyl (1.95 Å) and the protonated free amino group (1.75 Å), respectively, are evident.
The structure with the whole GCP fragment deprived of intramolecular interactions is here only
1.9 kcal/mol above the most stable structure, which is likely responsible for slightly higher log Ks = 5
with 3+ compared to log Ks = 4 with 32+
In concluding this section, let us mention that the computational results aided in the interpretation
of the relative trends in the observed reactivities of and towards ss-polynucleotides. Since the
.
2
3
conformational analysis revealed that in all cases there is not a single structure below 5–6 kcal mol−1
having nucleobase fragment not interacting intramolecularly with the rest of the system, this leads
us to conclude that in the investigated cases nucleobase moiety is less important for the interaction
with ss-polynucleotides. As such, this analysis underlined the important role of the GCP fragment,
particularly its guanidine moiety, in successful polynucleotide recognition. It turned out that lowering
the pH conditions promotes the interactions with ss-polynucleotides, since protonated guanidine group
interacts better, being in agreement with previous experiments [21
–25]. Yet, in a more rigid system,
this effect is overshadowed by the propensity of 22+ to form strong charge-dipole intramolecular

Molecules 2017, 22, 2213
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interactions with protonated guanidine group, therefore hindering its ability to interact with other
systems. This is why 2+ outnumbers 22+ in intermolecular interactions.
3. Materials and Methods
3.1. General Information
Solvents were distilled from appropriate drying agents shortly before use. TLC was carried out
on DC-plastikfolien Kieselgel 60 F254 and preparative thick layer (2 mm) chromatography was done
on Merck 60 F254 plates (Merck KGaA, Darmstadt, Germany). (Merck, Merck KGaA, Darmstadt,
Germany). NMR spectra were recorded on AV600 and AV300 MHz spectrometers (Bruker BioSpin
GmbH, Rheinstetten, Germany), operating at 150.92 or 75.47 MHz for 13C and 600.13 or 300.13 MHz
1
for H nuclei using DMSO-d6 as the internal standard. Mass spectrometry was performed on the
Agilent 6410 Triple Quad mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and high
resolution mass spectra (HRMS) were obtained using a Q-Tof2 hybrid quadrupole time-of-flight mass
spectrometer (Micromass, Cary, NC, USA). The electronic absorption spectra were obtained on a Cary
100 Bio spectrometer and CD spectra on a JASCO J815 spectrophotometer (JASCO, Oklahoma City,
OK, USA), all in quartz cuvettes (1 cm). The spectroscopic studies were performed in aqueous buffer
solution (pH 7.0 and pH 5.0, sodium cacodylate buffer, I = 0.05 mol dm−3). Under the experimental
conditions absorbance of
dissolved in sodium cacodylate buffer, I = 0.05 mol dm−3, pH = 7. Calf thymus (ct-)DNA was
additionally sonicated and filtered through a 0.45 m filter [48 49]. Polynucleotide concentration
2 and 3 were proportional to its concentration. Polynucleotides were
µ
,
was determined spectroscopically as the concentration of phosphates. Spectroscopic titrations were
performed at pH 7.0 and pH 5.0 (I = 0.05 mol dm−3, sodium cacodylate buffer) by adding portions
of polynucleotide solution into the solution of the studied compound for UV/Vis experiments and
for CD experiments were done by adding portions of compound stock solution into the solution of
polynucleotide. Titration data were processed by Scatchard equation. Values for Ks and n given in
Table 1 all have satisfactory correlation coefficients (>0.999). Thermal melting curves for DNA and their
complexes with studied compounds were determined as previously described [50] by following the
absorption change at 260 nm as a function of temperature. Absorbance of the ligands was subtracted
from every curve, and the absorbance scale was normalized. The Tm values are the midpoints of the
transition curves, determined from the maximum of the first derivative and checked graphically by
the tangent method.
∆
Tm values were calculated subtracting Tm of the free nucleic acid from Tm of
the complex. Every
in ∆Tm is ± 0.5 ◦C.
∆Tm value here reported was the average of at least two measurements, the error
3.2. Synthesis
N4-Benzoyl-5-iodocytosine
5: To a suspension of 5-iodocytosine 4 (2.9 g, 12.24 mmol) and DMAP (196
mg, 1.6 mmol) in anhydrous CH3CN (26.2 mL) at room temperature, benzoic anhydride (3.05 g, 13.46
mmol) was added. The reaction mixture was heated to reflux for 18 h and then EtOH (50 mL) was
added dropwise. After standing at room temperature overnight, the solid was filtered off, washed
with EtOH (3
MHz, DMSO-d6)
8.17 (s, 1H, H-6), 7.61 (m, 1H, Bz), 7.51 (m, 2H, Bz); 13C-NMR (151 MHz, DMSO-d6)
×
20 mL) dried in vacuo to afford
/ppm: 12.87 (s, 1H, NH-4), 11.93 (br. s, 1H, NH-1), 8.24 (d, J = 6.4 Hz, 2H, Bz)
/ppm: 178.1
5
(3.42 g, 82%) as a white solid: 1H-NMR (300
δ
,
δ
(C=O, Bz), 158.3 (C-2), 149.9 (C-6), 148.1 (C-4), 136.6 (Bz), 132.6 (Bz), 129.4 (Bz), 128.3 (Bz), 67.6 (C-5).
ESI-MS: m/z calcd. for C11H7IN3O2 [M − H]− 339.96, found 339.80.
N4-Benzoyl-1-(2-phthalimidoethyl)-5-iodocytosine : N4-benzoyl-5-iodocytosine
6 5 (198 mg, 0.58 mmol),
potassium carbonate (80.1 mg, 0.58 mmol) and N-(2-bromoethyl)phthalimide (294.6 mg, 1.16 mmol)
and dry DMF (12 mL) were put in a 20 mL glass vial equipped with a small magnetic stirring bar.
Then the mixture was put into the cavity of the MW reactor (Monowave 3000, Anton Paar® GmbH,
A-8054 Graz, Austria) and irradiated at 140 ◦C for 35 min. After evaporation of the solvent, CH2Cl2

Molecules 2017, 22, 2213
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and MeOH were added and the resulting solid was filtered off. The filtrate was evaporated and the
residue was purified by preparative chromatography (hexane/ethyl acetate 1:1) to give 152 mg (51%)
1
of yellow powder
6
: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 12.87 (br. s, 1H, NH-4), 8.41 (s, 1H,
H-6), 8.14 (br. s, 2H, Bz), 7.90–7.82 (m, 4H, Pht), 7.58–7.46 (m, 3H, Bz), 4.05–3.95 (br. s, 2H, CH2Pht),
3.94–3.86 (br. s, 2H, CH2Pht); 13C-NMR (75 MHz, DMSO-d6)
δ
/ppm: 173.8 (C=O, Bz), 167.7 (C=O, Pht),
163.9 (C-2), 155.9 (C-4), 151.8 (C-6), 137.9 (Bz), 134.4 (Pht), 134.3 (Bz), 131.6 (Pht), 129.2 (Bz), 128.0 (Bz),
123.1 (Pht), 59.9 (C-5), 47.5 (CH2Pht), 36.6 (CH2Pht). ESI-MS: m/z calcd. for C21H14IN4O4 [M − H]−
513.01, found 512.60.
Pyrr-C Amino acid analogue
0.078 mmol) in anhydrous DMF (546
8
: To a solution of N4-benzoyl-1-(2-phtalimidoethyl)-5-iodocytosine
L), CuI (3 mg, 0.016 mmol), Et3N (22 L, 0.156 mmol), protected
6 (40 mg,
µ
µ
propargylglycine
7 (53.2 mg, 0.234 mmol) and Pd(PPh3)4 (9 mg, 0.008 mmol) were sequentially added.
The reaction mixture was stirred in the dark at 50 ◦C for 24 h after which the solvent was removed
under vacuum. The residue was purified by preparative chromatography (CH2Cl2/CH3OH 9:1) to
1
give the product
8
(25 mg, 64%) as a yellow powder: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 10.99
(s, 1H, NH-7), 8.27 (s, 1H, H-4), 7.81 (s, 4H, Pht), 7.32 (d, J = 8.0 Hz, 1H, NH), 5.85 (s, 1H, H-5), 4.38–4.30
(m, 1H, CHCH2), 4.17–4.11 (m, 2H, CH2Pht), 3.95–3.92 (m, 2H, CH2Pht), 3.60 (s, 3H, OCH3), 2.97–2.73
(m, 2H, CH2CH), 1.38–1.23 (m, 9H, C(CH3)3); 13C-NMR (75 MHz, DMSO-d6)
δ
/ppm: 172.2 (C=O),
167.7 (C=O, Pht), 159.5 (C-2), 155.3 (C=O), 154.7 (C-6), 140.3 (C-4), 137.2 (C-7a), 134.3 (Pht), 131.6
(Pht), 123.1 (Pht), 108.6 (C-4a), 98.1 (C-5), 78.4 (C(CH3)3), 52.5 (CHCH2), 51.9 (OCH3), 49.3 (CH2Pht),
37.0 (CH2Pht), 29.4 (CH2CH), 28.1 (C(CH3)3). ESI-MS: m/z calcd. for C25H28N5O7 [M + H]+ 510.20,
found 510.30.
Pyrr-C Amino acid analogue
1: Compound 8 (160 mg, 0.314 mmol) was dissolved in a 1:1 mixture of
TFA/CH2Cl2 (4 mL) and stirred at room temperature for 72 h. After evaporation of volatiles, the
residue was purified by preparative chromatography (CH2Cl2/MeOH 9:1). Product
1
(144 mg, 88%)
1
was obtained as a yellow foam: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 10.94 (br. s, 1H, NH-7), 8.24
(s, 1H, H-4), 7.82 (s, 4H, Pht), 7.70–7.31 (m, 2H, NH2), 5.84 (s, 1H, H-5), 4.14–4.12 (m, 2H, CH2Pht),
3.95–3.93 (m, 2H, CH2Pht), 3.74–3.65 (m, 1H, CHCH2), 3.58 (s, 3H, OCH3), 2.89–2.69 (m, 2H, CH2CH);
13C-NMR (75 MHz, DMSO-d6)
δ/ppm: 175.1 (C=O), 167.7 (C=O), 159.5 (C-2), 154.8 (C-6), 140.0 (C-4),
138.2 (C-7a), 134.3 (Pht), 131.6 (Pht), 123.1 (Pht), 108.7 (C-4a), 97.7 (C-5), 53.5 (CHCH2), 51.5 (OCH3),
49.3 (CH2Pht), 37.0 (CH2Pht), 33.2 (CH2CH). HRMS (MALDI-TOF/TOF): m/z calcd. for C20H20N5O5
[M]+ 410.1464, found 410.1458.
Z-D-Lys(Boc)-Gly-OMe
(98.6 mg, 0.789 mmol) were dissolved in dry CH3CN (10 mL) under argon and HOBt (105.4 mg,
0.789 mmol), HBTU (295.8 mg, 0.789 mmol) and dry Et3N (435 L, 3.16 mmol) were added. The reaction
mixture was stirred at room temperature overnight. Product
9: Z-D-Lys(Boc)OH (300 mg, 0.789 mmol) and H-Gly-OMe hydrochloride
µ
9
(348 mg, 98%) was isolated by
1
preparative chromatography (CH2Cl2/CH3OH 9:1) as a white powder: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 8.31 (t, J = 5.8 Hz, 1H, NH), 7.40–7.29 (m, 6H, Ph, NH), 6.75 (t, J = 5.1 Hz, 1H, NH), 5.02 (s, 2H,
CH2Ph), 4.02–3.95 (m, 1H, CHCH2), 3.92–3.75 (m, 2H, COCH2NH), 3.62 (s, 3H, OCH3), 2.91–2.84
(m, 2H, CH2NH), 1.68–1.49 (m, 2H, CH2CH), 1.46–1.23 (m, 13H, 2
CH2, C(CH3)3); 13C-NMR
(75 MHz, DMSO-d6) /ppm: 172.6 (C=O), 170.2 (C=O), 156.0 (C=O, Cbz), 155.6 (C=O, Boc), 137.0 (Ph),
128.3 (Ph), 127.8 (Ph), 127.7 (Ph), 77.4 ( (CH3)3), 65.4 (OCH2Ph), 54.5 (CHCH2), 51.7 (OCH3), 40.5
O
×
δ
C
(COCH2NH), 39.8 (CH2NH), 31.6 (CH2CH), 29.2 (CH2), 28.3 (C(CH3)3), 22.7 (CH2). ESI-MS: m/z calcd.
for C22H33N3NaO7 [M + Na]+ 474.22, found 474.22.
Z-D-Lys(Boc)-Gly-OH 10: Compound
9
(170 mg, 0.377 mmol) was dissolved in a 3:1 mixture of
dioxane/water (4 mL), 2M NaOH (470
µL, 0.942 mmol) was added and reaction mixture was stirred at
room temperature for 24 h. The solvent was evaporated, the residue was dissolved in water (~20 mL)
and acidified with 1 M HCl to pH 5. The product was extracted with EtOAc (5
Na2SO4 yielding 10 (90 mg, 55%) as a colourless soil: H-NMR (600 MHz, DMSO-d6)
×
20 mL) dried over
/ppm: 7.55
1
δ

Molecules 2017, 22, 2213
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(d, J = 8.1 Hz, 1H, NH), 7.37–7.23 (m, 6H, Ph, NH), 6.75–6.73 (m, 1H, NH), 5.03 (q, J = 12.6 Hz, 2H,
CH2Ph), 3.90–3.86 (m, 1H, CHCH2), 3.25–3.21 (m, 2H, COCH2NH), 2.89–2.83 (m, 2H, CH2NH),
1.64–1.24 (m, 15H, 3 /ppm: 170.6 (C=O), 170.0
CH2, C(CH3)3); 13C-NMR (75 MHz, DMSO-d6)
(C=O), 156.1 (C=O, Cbz), 155.6 (C=O, Boc), 137.1 (Ph), 128.4 (Ph), 127.7 (Ph), 127.6 (Ph), 77.3 ( (CH3)3),
O
×
δ
C
65.3 (OCH2Ph), 55.1 (CHCH2), 44.1 (COCH2NH), 39.9 (CH2NH), 31.4 (CH2CH), 29.1 (CH2), 28.3
(C(CH3)3), 22.8 (CH2). ESI-MS: m/z calcd. for C21H30N3O7 [M − H]− 436.21, found 436.0.
Pyrr-C Tripeptide 11: Pyrr-C amino acid analogue
0.088 mmol) were dissolved in dry CH3CN (5 mL) under argon and HOBt (11.9 mg, 0.088 mmol),
HBTU (33.3 mg, 0.088 mmol) and dry Et3N (49 L, 0.352 mmol) were added. Reaction was stirred at
1 (46 mg, 0.088 mmol) and dipeptide 10 (38.4 mg,
µ
room temperature overnight. Product 11 (36 mg, 49%) was isolated by preparative chromatography
1
(CH2Cl2/CH3OH 9:1) as a yellow powder: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 11.03 (br. s, 1H,
NH-7), 8.34–8.32 (m, 1H, NH), 8.26 (s, 1H, H-4), 8.17–8.12 (m, 1H, NH), 7.81 (s, 4H, Pht), 7.48–7.42
(m, 1H, NH), 7.35–7.26 (m, 5H, Ph), 6.75 (t, J = 5.6 Hz, 1H, NH), 5.87 (s, 1H, H-5), 5.05–4.95 (m, 2H,
O
CH2Ph), 4.62 (br. s, 1H, CHCH2), 4.20–4.08 (m, 2H, CH2 Pht), 4.00–3.89 (m, 3H, CHCH2, CH2Pht),
3.74–3.65 (m, 2H, COCH2NH), 3.58 (s, 3H, OCH3) 2.97–2.85 (m, 4H, CH2CH, CH2NH), 1.62–1.49
(m, 2H, CH2CH), 1.44–1.16 (m, 15H, 3 /ppm:
CH2, C(CH3)3); 13C-NMR (151 MHz, DMSO-d6)
×
δ
172.3 (C=O), 171.4 (C=O), 168.8 (C=O), 167.7 (C=O), 159.5 (C-2), 156.1 (C=O, Cbz), 155.6 (C=O, Boc),
154.8 (C-6), 140.3 (C-4), 139.0 (C-7a), 136.8 (Ph), 134.3 (Pht), 131.6 (Pht), 128.3 (Ph), 127.8 (Ph), 127.7 (Ph),
123.1 (Pht), 108.6 (C-4a), 98.1 (C-5), 77.3 (C(CH3)3), 65.4 (OCH2Ph), 54.7 (CHCH2), 52.0 (OCH3), 51.2
(
CHCH2), 49.3 (CH2Pht), 41.6 (COCH2NH), 39.8 (CH2NH), 37.0 (CH2Pht), 31.4 (CH2CH), 29.6 (CH2),
29.2 (CH2), 28.3 (C(CH3)3), 22.8 (CH2). ESI-MS: m/z calcd. for C41H48N8NaO11[M + Na]+ 851.33,
found 851.50.
Pyrr-C Tripeptide 12: Compound 11 (160 mg, 0.193 mmol) was dissolved in 1:1 mixture of TFA/CH2Cl2
(4 mL) and stirred at room temperature for 20 h. After removal of remaining TFA under reduced
1
pressure, product 12 (162.7 mg, 100%) was obtained as a yellow foam: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 11.19 (br. s, 1H, NH-7), 8.38–8.35 (m, 2H, NH, H-4), 8.19–8.14 (m, 1H, NH), 7.81 (s, 4H, Pht),
7.63 (br. s, 3H, NH3), 7.50–7.46 (m, 1H, NH), 7.36–7.31 (m, 5H, Ph), 5.92 (s, 1H, H-5), 5.04–4.97 (m, 2H,
CH2Ph), 4.64–4.61 (m, 1H, CHCH2), 4.20–3.59 (m, 10H, 2 × CH2Pht, CHCH2, COCH2NH, OCH3
+ H2O), 3.02–2.86 (m, 2H, CH2CH), 2.74 (br. s, 2H, CH2), 1.62–1.17 (m, 6H, 3
CH2); 13C-NMR
O
×
(151 MHz, DMSO-d6)
158.4, 158.3, 157.8 (4
δ
/ppm: 172.5 (C=O), 172.4 (C=O), 172.4 (C=O), 168.0 (C=O), 159.8 (C-2), 158.5,
CF3C=O), 156.4 (C=O, Cbz), 154.5 (C-6), 140.8 (C-4), 139.9 (C-7a), 137.3 (Ph),
×
134.6 (Pht), 131.7 (Pht), 128.6 (Ph), 127.9 (Ph), 123.3 (Pht), 119.7 (CF3C=O), 109.2 (C-4a), 98.9 (C-5),
66.0 (OCH2Ph), 52.4 (CHCH2), 51.53 (OCH3), 51.51 (CHCH2), 49.7 (CH2Pht), 40.2 (COCH2NH), 38.9
(
CH2NH), 37.2 (CH2Pht), 31.2 (CH2CH), 29.9 (CH2), 26.7 (CH2), 22.4 (CH2). ESI-MS: m/z calcd. for
C36H41N8O9 [M]+ 729.3, found 729.3.
Pyrr-C Tripeptide GCP conjugate 14: Compound 12 (30 mg, 0.035 mmol) and Boc-GCP-OH 13 (14.1 mg,
0.035 mmol) were dissolved in dry CH3CN (3 mL) under argon and HOBt (4.7 mg, 0.035 mmol),
HBTU (13.3 mg, 0.035 mmol) and dry Et3N (19 µL, 0.14 mmol) were added. Reaction was stirred at
room temperature overnight. Product 14 (20 mg, 57%) was isolated by preparative chromatography
1
(CH2Cl2/CH3OH 9:1) as a yellow powder: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 11.03 (m, 2H,
NH-7, NH), 9.27 (br. s, 2H, 2
×
NH), 8.59–8.42 (m, 1H, NH), 8.40–8.29 (m, 1H, NH), 8.27 (s, 1H,
H-4), 8.18 (m, 1H, NH), 7.80 (s, 4H, Pht), 7.50 (m, 1H, NH), 7.34–7.20 (m, 6H, Ph, NH), 6.79–6.67
(m, 2H, CH-Pyrr), 5.87 (s, 1H, H-5), 5.03–4.96 (m, 2H, OCH2Ph), 4.63–4.59 (m, 1H, CHCH2), 4.18–4.09
(m, 2H, CH2Pht), 4.01–3.92 (m, 3H, CHCH2, CH2Pht), 3.77–3.67 (m, 2H, COCH2NH), 3.58 (s, 3H,
OCH3), 3.19–3.16 (m, 2H, CH2NH), 3.00–2.86 (m, 1H, CH2CH), 1.63–1.17 (m, 15H, 3
×
CH2, C(CH3)3);
13C-NMR (151 MHz, DMSO-d6)
δ
/ppm: 172.9 (C=O), 172.1 (C=O), 171.8 (C=O), 170.1 (C=O), 169.4
(C=O), 168.2 (C=O), 160.2, 159.7 (C-2), 158.8, 156.7 (C=O, Cbz), 155.3 (C=O, Boc), 154.4 (C=O, C-6),
140.8 (C-4), 137.3 (C-7a), 134.8 (Pht), 131.8 (Pht), 128.7 (Ph), 127.8 (Ph), 123.5 (Pht), 121.0 (Pyrr), 112.2
(CH-Pyrr), 109.3 (C-4a), 98.7 (C-5), 78.1 (C(CH3)3), 65.9 (OCH2Ph), 55.1 (CHCH2), 52.5 (OCH3), 51.6

Molecules 2017, 22, 2213
15 of 22
(
CHCH2), 49.7 (CH2Pht), 42.0 (COCH2NH), 38.9 (CH2NH), 37.4 (CH2Pht), 31.6 (CH2CH), 29.9 (CH2),
29.0 (CH2), 28.1 (C(CH3)3), 23.2 (CH2). ESI-MS: m/z calcd. for C48H53N12O13 [M
found 1004.9.
−
H]− 1005.39,
Lys(Boc)-Gly-OMe 16: Z-D-Lys(Boc)-Gly-OMe 9 (430 mg, 0.95 mmol) was dissolved in methanol (50 mL)
and 10% Pd/C (43 mg) was added. The reaction mixture was stirred under a hydrogen atmosphere
for 24 h at room temperature, after which time the catalyst was filtered off through a Celite pad and
washed several times with methanol. The solvent was evaporated under reduced pressure to provide
1
the product 16 (302 mg, 100%) as a colourless oil: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 8.50–8.46
(m, 1H, NH), 7.64–7.56 (m, 1H, NH), 6.79–6.73 (m, 2H, NH2), 4.09 (br. s, 1H, CHCH2), 3.96–3.82
(m, 2H, COCH2NH), 3.63 (s, 3H, OCH3), 2.96–2.82 (m, CH2NH), 1.69–1.56 (m, 2H, CH2CH), 1.42–1.33
(m, 13H, 2
(C=O), 155.6 (C=O), 77.4 (
33.3 (CH2CH), 29.3 (CH2), 28.3 (C(CH3)3), 22.0 (CH2). ESI-MS: m/z calcd. for C14H28N3O5 [M + H]+
×
CH2, C(CH3)3); 13C-NMR (75 MHz, DMSO-d6)
δ/ppm: 170.2 (C=O), 168.0(C=O), 166.1
C
(CH3)3), 54.1 (CHCH2), 51.7 (OCH3), 44.3 (COCH2NH), 40.5 (CH2NH),
318.2, found 318.3.
GCP Dipeptide 17: Dipeptide 16 (61 mg, 0.192 mmol) and Boc-GCP-OH 13 (76.2 mg, 0.192 mmol)
were dissolved in dry CH3CN (3 mL) under argon and HOBt (26 mg, 0.192 mmol), HBTU (73 mg,
0.192 mmol) and dry Et3N (107 µL, 0.768 mmol) were added. Reaction was stirred at room temperature
overnight. Product 17 (67 mg, 58%) was isolated by preparative chromatography (CH2Cl2/CH3OH
1
9:1) as a yellow powder: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 11.62 (br. s, 1H, NH), 10.88 (br. s, 1H,
NH), 6.83–6.80 (m, 2H, CH-Pyrr), 6.75 (t,
J = 5.0 Hz, 1H, NH), 4.47–4.40 (m, 1H, CHCH2), 3.85–3.80 (m, 2H, COCH2NH), 3.62 (s, 3H, OCH3),
2.90–2.88 (m, 2H, CH2NH), 1.75–1.64 (m, 2H, CH2CH), 1.61–1.23 (m, 22H, 2
CH2, 2 × C(CH3)3);
13C-NMR (75 MHz, DMSO-d6)
/ppm: 172.4 (C=O), 170.2 (C=O), 159.4, 158.4, 155.5 (C=O, Boc),
113.6 (CH-Pyrr), 112 (CH-Pyrr), 77.3 ( (CH3)3), 52.5 (CHCH2), 51.6 (OCH3), 40.5 (COCH2NH), 39.8
NH), 9.33 (s, 1H, NH), 8.57 (s, 1H, NH), 8.46–8.41 (m, 2H, 2
×
×
δ
C
(
CH2NH), 31.6 (CH2CH), 29.2 (CH2), 28.2 (C(CH3)3), 27.8 (C(CH3)3), 22.9 (CH2). ESI-MS: m/z calcd.
for C26H42N7O9 [M + H]+ 596.3, found 596.3.
GCP Dipeptide 18: To a solution of dipeptide 17 (90 mg, 0.147 mmol) in a 4:1 mixture of THF/water
(25 mL), lithium hydroxide (37.8 mg, 0.882 mmol) was added and reaction mixture was stirred at room
temperature overnight. The solvent was evaporated and a concentrated citric acid solution was added
to the water phase (pH ~5). White precipitate which formed was filtered and washed several times
1
with water yielding product 18 (64 mg, 75%) as a white soil: H-NMR (300 MHz, DMSO-d6)
δ
/ppm:
11.64 (br. s, 3H, 2
×
NH, COOH), 9.32 (s, 1H, NH), 8.57 (s, 1H, NH), 8.45 (d, J = 8.1 Hz, 1H, NH), 8.24
(t, J = 5.7 Hz, 1H, NH), 6.83 (s, 2H, CH-Pyrr), 6.74 (t, J = 5.5 Hz, 1H, NH), 4.45–4.41 (m, 1H, CHCH2),
3.80–3.66 (m, 2H, COCH2NH), 2.89–2.87 (m, 2H, CH2NH), 1.83–1.63 (m, 2H, CH2CH), 1.60–1.24 (m,
22H, 2
×
CH2, 2
×
C(CH3)3); 13C-NMR (75 MHz, DMSO-d6)
δ/ppm: 172.1 (C=O), 171.1 (C=O), 159.4,
158.4, 155.5 (C=O, Boc), 129.4 (Pyrr), 113.7 (CH-Pyrr), 112.9 (CH-Pyrr), 77.3 (C(CH3)3), 52.5 (CHCH2)
40.4 (COCH2NH), 39.8 (CH2NH), 31.6 (CH2CH), 29.2 (CH2), 28.2 (C(CH3)3), 27.8 (C(CH3)3), 22.9 (CH2).
ESI-MS: m/z calcd. for C25H40N7O9 [M + H]+ 582.29, found 582.4.
Pyrr-C Tripeptide GCP conjugate 19: Compound
0.052 mmol) were dissolved in dry CH3CN (5 mL) under argon and HOBt (7 mg, 0.052 mmol),
HBTU (19.6 mg, 0.052 mmol) and dry Et3N (29 L, 0.208 mmol) were added. Reaction was stirred at
1 (27 mg, 0.052 mmol) and dipeptide 18 (30 mg,
µ
room temperature overnight. Product 19 (25 mg, 50%) was isolated by preparative chromatography
1
(CH2Cl2/CH3OH 9:1) as a yellow powder: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 11.01 (br. s, 2H,
NH, H-4), 7.81 (s, 4H, Pht), 6.80–6.73
CH-Pyrr), 5.87 (d, J = 12.2 Hz, 1H, H-5), 4.64–4.57 (m, 1H, CHCH2), 4.33 (br. s,
NH, NH-7), 9.29 (br. s, 2H, 2
(m, 3H, NH, 2
×
NH), 8.61–8.23 (m, 5H, 4
×
×
1H, CHCH2), 4.20–4.07 (m, 2H, CH2Pht), 3.97–3.88 (m, 2H, CH2Pht), 3.74–3.65 (m, 2H, COCH2NH),
3.57 (m, 3H, OCH3), 3.03–2.87 (m, 4H, CH2CH, CH2NH), 1.80–1.56 (m, 2H, CH2CH), 1.49–1.23 (m,
22H, 2 × CH2, 2 × C(CH3)3); 13C-NMR (75 MHz, DMSO-d6) δ/ppm: 172.1 (C=O), 171.4 (C=O), 169.9

Molecules 2017, 22, 2213
16 of 22
(C=O), 168.9 (C=O), 167.7 (C=O), 167.0, 159.5 (C-2), 158.5, 155.5 (C=O, Boc), 154.7 (C-6), 140.3 (C-4),
136.8 (C-7a), 134.3 (Pht), 131.6 (Pht), 123.1 (Pht), 113.1 (CH-Pyrr), 111.9 (CH-Pyrr), 108.6 (C-4a), 98.1
(C-5), 77.3 (C(CH3)3), 68.3 (OCH2Ph), 53.1 (CHCH2), 52.0 (OCH3), 51.1 (CHCH2), 49.3 (CH2Pht), 41.6
(COCH2NH), 39.7 (CH2NH), 37.0 (CH2Pht), 31.3 (CH2CH), 29.6 (CH2), 29.2 (CH2), 28.2 (C(CH3)3),
27.8 (C(CH3)3), 22.9 (CH2). ESI-MS: m/z calcd. for C45H55N12O13 [M − H]− 971.4, found 971.0.
Conjugate Pyrr-C Tripeptide
TFA/CH2Cl2 (4 mL) and stirred at room temperature for 20 h. After removal of remaining TFA under
reduced pressure, product
(15 mg, 100%) was obtained as a yellow foam: 1H-NMR (300 MHz,
2: Compound 19 (14 mg, 0.014 mmol) was dissolved in 1:1 mixture of
2
DMSO-d6)
(m, 8H, 4
δ
/ppm: 12.57–12.28 (m, 1H, NH), 11.37 (br. s, 1H, NH), 10.98–10.92 (m, 1H, NH-7), 8.59–8.26
NH, NH3, H-4), 7.81–7.70 (m, 7H, Pht, NH3), 7.12 (s, 1H, CH-Pyrr), 6.89 (s, 1H, CH-Pyrr),
×
5.91–5.88 (m, 1H, H-5), 4.63–4.61 (m, 1H, CHCH2), 4.43 (s, 1H, CHCH2), 4.18–4.13 (m, 2H, CH2Pht), 3.94
(m, 2H, CH2Pht), 3.76–3.67 (m, 2H, COCH2NH), 3.59 (s, 3H, OCH3), 3.01–2.77 (m, 4H, CH2CH, CH2),
1.76–1.23 (m, 6H, 3
×
CH2); 13C-NMR (151 MHz, DMSO-d6)
δ
/ppm: 171.8 (C=O), 171.4 (C=O), 168.8
CF3C=O), 154.8 (C-6), 140.4 (C-4),
(C=O), 167.7 (C=O), 159.4 (C-2), 159.1, 158.4, 158.2, 158.0, 157.8 (4
×
136.9 (C-7a), 134.3 (Pht), 131.6 (Pht), 123.1 (Pht), 118.2 (CF3C=O), 114.9 (CH-Pyrr), 113.6 (CH-Pyrr),
108.7 (C-4a), 98.3 (C-5), 68.3 (OCH2Ph), 52.7 (CHCH2), 52.0 (CHCH2), 51.3 (OCH3), 49.3 (CH2Pht), 41.5
(COCH2NH), 38.7 (CH2NH), 37.0 (CH2Pht), 31.2 (CH2CH), 29.6 (CH2), 26.6 (CH2), 22.4 (CH2). HRMS
(MALDI-TOF/TOF): m/z calcd. for C35H40N12O9 [M + H]+ 773.3119; found 773.3137.
Boc-D-Lys(Z)-Gly-OMe 20: Boc-D-Lyz(Z)-OH (400 mg, 1.051 mmol) and H-Gly-OMe hydrochloride
(131.5 mg, 1.051 mmol) were dissolved in dry CH3CN (10 mL) under argon and HOBt (141.9 mg,
1.051 mmol), HBTU (398.2 mg, 1.051 mmol) and dry Et3N (585 µL, 4.2 mmol) were added. Reaction
was stirred at room temperature overnight. Product 20 (455 mg, 96%) was isolated by preparative
1
chromatography (CH2Cl2/CH3OH 9:1) as a colorless oil: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 8.21
(t, J = 5.8 Hz, 1H, NH), 7.39–7.32 (m, 5H, Ph), 7.22 (t, J = 5.3 Hz, 1H, NH), 6.83 (d, J = 8.1 Hz, 1H, NH),
5.00 (s, 2H, OCH2Ph), 3.92–3.74 (m, 3H, CHCH2, COCH2NH), 3.61 (s, 3H, OCH3), 3.00–2.96 (m, 2H,
CH2NH), 1.47–1.24 (m, 15H, 3
170.3 (C=O), 156.1 (C=O, Cbz), 155.3 (C=O, Boc), 137.3 (Ph), 128.4 (Ph), 127.7 (Ph), 78.0 (
×
CH2, C(CH3)3); 13C-NMR (75 MHz, DMSO-d6)
δ
/ppm: 172.8 (C=O),
(CH3)3), 65.1
C
(OCH2Ph), 54.1 (CHCH2), 51.6 (OCH3), 40.5 (COCH2NH), 40.1 (CH2NH), 31.6 (CH2CH), 29.1 (CH2),
28.2 (C(CH3)3), 22.7 (CH2). ESI-MS: m/z calcd. for C22H33N3NaO7 [M + Na]+ 474.22, found 474.22.
Boc-D-Lys-Gly-OMe 21: Cbz-protected compound 20 (380 mg, 0.842 mmol) was dissolved in methanol
(50 mL) and 10% Pd/C (38 mg) was added. The reaction mixture was stirred under the hydrogen
for 16 h at room temperature, after which time the catalyst was filtered off through a Celite pad
and washed several times with methanol. The solvent was evaporated under reduced pressure to
1
provide the product 21 (267 mg, 100%) as a colourless oil: H-NMR (300 MHz, DMSO-d6)
δ
/ppm:
8.29 (t, J = 5.8 Hz, 1H, NH), 6.89 (d, J = 8.1 Hz, 1H, NH), 4.50 (br. s, 2H, NH2), 3.96–3.74 (m, 3H,
CHCH2, COCH2NH), 3.62 (s, 3H, OCH3), 2.60 (t, J = 6.9 Hz, 2H, CH2NH2), 1.66–1.16 (m, 15H, 3 × CH2
C(CH3)3); 13C-NMR (75 MHz, DMSO-d6)
/ppm: 172.5 (C=O), 167.0 (C=O), 155.0 (C=O, Boc), 77.7
,
δ
(C(CH3)3), 53.7 (CHCH2), 51.4 (OCH3), 40.5 (COCH2NH), 39.8 (CH2NH), 31.2 (CH2CH), 29.3 (CH2),
27.9 (C(CH3)3), 22.2 (CH2). ESI-MS: m/z calcd. for C14H28N3O5 [M + H]+ 318.2, found 318.2.
GCP Dipeptide 22: Compound 21 (103 mg, 0.325 mmol) and Boc-GCP-OH 13 (128.9 mg, 0.325 mmol)
were dissolved in dry CH3CN (8 mL) under argon and HOBt (43.9 mg, 0.325 mmol), HBTU
(123.2 mg, 0.325 mmol) and dry Et3N (181 µL, 1.3 mmol) were added. Reaction was stirred at room
temperature overnight. Product 22 (78 mg, 40%) was isolated by preparative chromatography (eluent:
1
CH2Cl2/CH3OH 9:1) as a slightly yellow oil: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 11.30 (br. s, 2H,
NH), 6.86 (d, J = 8.2 Hz, 1H, NH), 6.75 (d, J = 3.6 Hz,
1H, CH-Pyrr), 6.66 (d, J = 3.6 Hz, 1H, CH-Pyrr), 3.96–3.74 (m, 3H, CHCH2, COCH2NH), 3.61 (s, 3H,
OCH3), 3.22–3.16 (m, 2H, CH2NH), 1.50–1.29 (m, 24H, 3 CH2, 2
C(CH3)3); 13C-NMR (75 MHz,
DMSO-d6) /ppm: 172.8 (C=O), 170.2 (C=O), 158.5, 155.2 (C=O, Boc), 113.6 (CH-Pyrr), 111.9 (CH-Pyrr),
2
×
NH), 9.27 (s, 1H, NH), 8.53–8.22 (m, 3H, 3
×
×
×
δ

Molecules 2017, 22, 2213
17 of 22
77.9 (C(CH3)3), 54.0 (CHCH2), 51.6 (OCH3), 40.5 (COCH2NH), 38.3 (CH2NH), 31.6 (CH2CH), 29.0
(CH2), 28.2 (C(CH3)3), 27.8 (C(CH3)3), 22.9 (CH2). ESI-MS: m/z calcd. for C26H42N7O9 [M + H]+ 596.3,
found 596.3.
GCP Dipeptide 23: To a solution of 22 (69 mg, 0.116 mmol) in a 4:1 mixture of THF/water (25 mL), lithium
hydroxide (29.2 mg, 0.696 mmol) was added and reaction mixture was stirred at room temperature
overnight. The solvent was evaporated and a concentrated citric acid solution was added to the water
phase (pH ~5). The product was extracted with EtOAc (5
23 (47 mg, 70%) as a white foam: H-NMR (300 MHz, DMSO-d6)
×
20 mL), dried over Na2SO4 yielding
/ppm: 12.07–11.84 (m, 2H, NH,
1
δ
COOH), 11.32 (s, 1H, NH), 9.32 (s, 1H, NH), 8.57 (s, 1H, NH), 8.30 (t, J = 5.3 Hz, 1H, NH), 8.06 (t,
J = 5.4 Hz, 1H, NH), 6.84–6.72 (m, 3H, NH, 2 CH-Pyrr), 3.97–3.89 (m, 1H, CHCH2), 3.84–3.65 (m,
2H, COCH2NH), 3.24–3.17 (m, 2H, CH2NH), 1.68–1.23 (m, 24H, 3 CH2, 2
C(CH3)3); 13C-NMR
(75 MHz, DMSO-d6) /ppm: 172.5 (C=O), 171.1 (C=O), 159.4, 159.2, 158.4, 155.3 (C=O, Boc), 129.7, 125.2,
112.3 (CH-Pyrr), 111.6 (CH-Pyrr), 77.9 ( (CH3)3), 54.3 (CHCH2), 40.6 (COCH2NH), 38.5 (CH2NH), 31.7
×
×
×
δ
C
(
CH2CH), 28.8 (CH2), 28.2 (C(CH3)3), 27.8 (C(CH3)3), 22.9 (CH2). ESI-MS: m/z calcd. for C25H38N7O9
[M − H]− 580.27, found 580.1.
Pyrr-C Tripeptide GCP conjugate 24: Compound
0.061 mmol) were dissolved in dry CH3CN (7 mL) under argon and HOBt (8.27 mg, 0.061 mmol),
HBTU (23.20 mg, 0.061 mmol) and dry Et3N (34 L, 0.245 mmol) were added. Reaction was stirred at
1 (32 mg, 0.061 mmol) and GCP-dipeptide 23 (35.55 mg,
µ
room temperature overnight. Product 24 (28 mg, 47%) was isolated by preparative chromatography
1
(CH2Cl2/CH3OH 9:1) as a white powder: H-NMR (300 MHz, DMSO-d6)
δ
/ppm: 11.03 (br. s, 2H,
NH), 8.34–8.26 (m,
2H, NH, H-4), 8.04 (br. s, 1H, NH), 7.81 (s, 4H, Pht), 6.95–6.73 (m, 3H, CH-Pyrr, 2 NH), 6.45 (d,
NH, NH-7), 9.59 (br. s, 1H, NH), 9.29 (br. S, 1H, NH), 8.58–8.50 (m, 2H, 2
×
×
J = 3.7 Hz, 1H, CH-Pyrr), 5.87 (s, 1H, H-5), 4.64–4.57 (m, 1H, CHCH2), 4.20–4.08 (m, 3H, CH2Pht,
CHCH2), 3.95–3.89 (m, 2H, CH2Pht), 3.71–3.67 (m, 2H, COCH2NH), 3.58 (s, 3H, OCH3), 3.03–2.69
(m, 4H, CH2CH, CH2NH), 1.66–1.23 (m, 24H, 3
/ppm: 172.4 (C=O), 171.4 (C=O), 168.9 (C=O), 168.8, 167.7 (C=O), 159.5, 158.9 (C-2), 158.6, 158.5, 155.4
(C=O, Boc), 154.8 (C-6), 140.4 (C-4), 136.8 (C-7a), 134.3 (Pht), 131.6 (Pht), 123.1 (Pht), 113.6 (CH-Pyrr),
111.7 (CH-Pyrr), 108.6 (C-4a), 98.2 (C-5), 78.1 ( (CH3)3), 63.8 (CH2), 54.3 (CHCH2), 52.0 (OCH3), 51.2
×
CH2, 2
×
C(CH3)3); 13C-NMR (75 MHz, DMSO-d6)
δ
C
(COCH2NH), 49.3 (CH2Pht), 41.6 (COCH2NH), 40.6 (CH2NH), 37.0 (CH2Pht), 31.5 (CH2CH), 29.6
(CH2), 29.0 (CH2), 28.2 (C(CH3)3), 27.7 (C(CH3)3), 22.8 (CH2). ESI-MS: m/z calcd. for C45H57N12O13
[M + H]+ 973.42, found 973.4.
Pyrr-C Tripeptide GCP conjugate 3: Compound 24 (19 mg, 0.02 mmol) was dissolved in 1:1 mixture
of TFA/CH2Cl2 (4 mL) and stirred at room temperature for 20 h. After removal of remaining TFA
under reduced pressure, the residue was purified by preparative chromatography (CH2Cl2/MeOH 9:1)
and product
3
(19 mg, 95%) was obtained as a yellow foam; 1H-NMR (300 MHz, DMSO-d6)
δ
/ppm:
NH,
12.20 (br. s, 1H, NH), 11.18–10.97 (m, 2H, NH-7, NH), 8.66 (br. s, 1H, NH), 8.55–8.11 (m, 9H, 2
×
2 × NH3, H-4), 7.81 (s, 4H, Pht), 7.54 (s, 1H, NH), 7.02 (br. s, 1H, CH-Pyrr), 6.53 (s, 1H, CH-Pyrr), 5.88
(s, 1H, H-5), 4.63 (br. s, 1H, CHCH2), 4.17–4.14 (m, 3H, CH2Pht, CHCH2), 3.95–3.91 (m, 2H, CH2Pht),
3.81–3.79 (m, 2H, COCH2NH), 3.59 (m, 3H, OCH3), 2.95–2.82 (m, 4H, CH2CH, CH2NH), 1.73–1.24
(m, 6H, 3
×
CH2); 13C-NMR (75 MHz, DMSO-d6)
δ/ppm: 169.7 (C=O), 168.9, 168.2, 167.7, 158.1,
157.6, 154.7 (C-6), 140.4 (C-4), 136.8 (C-7a), 134.3 (Pht), 131.6 (Pht), 123.0 (Pht), 113.8 (CH-Pyrr), 113.0
(CH-Pyrr), 108.6 (C-4a), 98.2 (C-5), 62.6 (CH2), 52.3 (CHCH2), 52.1 (OCH3), 51.2 (COCH2NH), 49.3
(
CH2Pht), 41.5 (COCH2NH), 38.4 (CH2NH), 37.0 (CH2Pht), 30.8 (CH2CH), 28.9 (CH2), 27.2 (CH2), 21.4
(CH2). HRMS (MALDI-TOF/TOF): m/z calcd. for C35H40N12O9 [M + H]+ 773.3114; found 773.3102.
3.3. Computational Details
In order to sample the conformational flexibility of investigated systems and probe their
intrinsic dynamical features in the aqueous solution, classical molecular dynamics (MD) simulations
were performed employing standard generalized AMBER force fields (ff14SB and GAFF) [51] as

Molecules 2017, 22, 2213
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implemented within the AMBER16 program package [52]. All structures were subsequently solvated
in a truncated octahedral box of TIP3P water molecules spanning a 10 Å thick buffer of solvent
molecules around each system. Upon gradual heating from 0 K, MD simulations were performed at
400 K for a period of 100 ns, maintaining the temperature constant using the Langevin thermostat
with a collision frequency of 1 ps−1. Following the clustering analysis of the structures in the
obtained MD trajectories, around 20–25 different structures in each case were subjected to geometry
re-optimization in Gaussian09 [53] at the M06–2X/6–31G(d) level of theory with aqueous solution
being modelled through the implicit SMD solvation. The idea behind this computational strategy
was to investigate whether intrinsic dynamical features of investigated conjugates both affect and can
explain their tendency to interact with ss-polynucleotides, which avoids difficulties and inaccuracies
associated with the computational prediction of the structure of single-stranded polynucleotides, as
very recently emphasized by Jeddi and Saiz [54]. pKa values were calculated in a relative fashion with
the MP2/6–311++G(2df,2pd)//(SMD)/M06–2X/6–31+G(d) model, using AH+ + BREF
equation, and employing the following reference bases (BREF): succinimide (pKa = 3.9)
O-protonation in , cytosine (pKa = 4.5) [55] for the N-protonation in II, and guanidine (pKa = 13.6)
→
A + BREFH+
55] for the
55
[
I
[
]
for the N-protonation in III. The choice of such computational setup was prompted by its recent
success in evaluating solution-phase geometries, reactivities and pKa values of a large variety of
systems [56–59].
4. Conclusions
Two structural isomers of fluorescent cytosine derivative conjugated with guanidiniocarbonyl-
pyrrole (GCP) were prepared, whereby they differ in the position of GCP:
2
being more rigid due to
the proximity of GCP to the peptide backbone, while
of a long aliphatic chain of lysine.
3 is more flexible with GCP attached at the end
In comparison with previously studied GCP derivatives [21–25], here studied 2, 3 did not
induce thermal stabilisation of ds-DNA/RNA nor showed ICD bands of GCP about 300 nm, which
supported random aggregation of compounds along double stranded helix of polynucleotide, based on
non-specific electrostatic interactions of positive charge with DNA/RNA backbone and hydrophobic
interactions. However, even such non-defined binding was highly sensitive to ds-polynucleotide
secondary structure, at pH 7 giving fluorescence increase only for ds-DNA, but not ds-RNA.
Intriguingly,
/poly G order of magnitude higher affinity than with ds-DNA/RNA. With ss-polynucleotides
fluorescence emission of nucleobase-phthalimide fluorophore in and became very selective: at
pH 7 revealed highly selective emission increase only for poly A, while emission of was selectively
quenched only by addition of poly G. Observed selectivities can only be attributed to the positioning
of the GCP unit, whereby more flexible combination in prefers poly G as a Watson-Crick partner for
cytosine, while more rigid responds only to the mismatched poly A. Molecular modelling confirmed
the important role of the GCP fragment in sensing of ss-polynucleotides in solution and showed
that these interactions can be rationalized by intrinsic dynamical features of and themselves.
2 and 3 interacted with single stranded (ss) RNA with similar affinity or in case
of
3
2
3
2
3
3
2
2
3
Additionally, the evidence is provided that successive recognition of ss-polynucleotides is influenced
by a fine interplay between two factors: (a) the protonation state of the GCP moiety; and (b) the ability
of the GCP unit to form hydrogen bonds with the rest of the molecule. In general, protonated GCP
groups are slightly more susceptible towards ss-polynucleotides (as in 32+) unless these monocationic
units are stabilized intramolecularly, which then diminishes their sensing ability (as in 22+).
At pH 5 the fluorimetric response of the cytosine-phthalimide fluorophore changed significantly.
The poly A selectivity of
of the polynucleotide into the ds-form poly AH+-poly AH+ or according to a molecular modelling
study (Figure 6), to the stronger intramolecular packing of , in which GCP unit is strongly involved.
However, same poly AH+-poly AH+ induced the fluorescence increase of more flexible
, which has
2 was completely lost, which could be attributed either to the rearrangement
2
3

Molecules 2017, 22, 2213
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less intramolecularly blocked GCP unit and therefore was able to adapt to a new binding motif within
ds-RNA and direct nucleobase fluorophore in interactions causing strong fluorescence change.
The general conclusion is that due to the fine differences in intramolecular interactions of 2 or 3
and their interactions with ss-RNA at different pH it is possible to finely tune recognition of adenine
versus guanine sequences, while cytosine and uracil did not cause measurable fluorimetric changes.
Here presented results demonstrated for the first time that the intriguing GCP unit can be applied
for the recognition of single stranded RNA sequences—an intuitively unexpected result since so far
binding of GCP unit to ds-DNA or ds-RNA relied strongly on minor or major groove interactions.
Since ss-polynucleotides do not have structurally defined grooves, clearly GCP unit can take part
in H-bonding and/or electrostatic interactions in a selective manner, thus expanding its uses in the
design of novel DNA or RNA targeting small molecules. Thus, nucleobase–GCP conjugates can be
considered as novel lead compounds for the design of ss-RNA or ss-DNA selective fluorimetric probes.
We have performed the preliminary screening of in vitro activity of 2 and 3 against the human
tumor cell lines “HeLa Kyoto” and “HEK293T” by standard MTT test. Cytotoxicity of compounds
was tested in the c = 10−4–10−6 M range and no effect on cell proliferation was observed nor cell
morphology was changed. These results indicate negligible cytotoxicity of studied compounds and
encourage further studies of compounds as in vitro markers.
Supplementary Materials: Supplementary Materials are available online, Additional characterization data,
DNA/RNA binding data.
Acknowledgments: Financial support from Croatian Science Foundation (Grant HRZZ-1477) is gratefully
acknowledged.
Author Contributions: I.P. and C.S. designed the research concept and wrote the paper; Ž.B. and B.Ž. synthesized
new compounds; Ž.B. performed titration experiments; R.V. performed molecular modeling.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–3 are available from the authors.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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