Synthesis of new riboflavin modified ODNs: Effect of riboflavin moiety on the G-quadruplex arrangement and stability

Article abstract of DOI:10.1016/j.bioorg.2020.104213

In the panorama of modified G-quadruplexes (G4s) with interesting proprieties, here, it has been reported the synthesis of new modified d(TGGGAG) sequences forming G-quadruplexes, with the insertion of a riboflavin unit (Rf, vitamin B₂). Exploi

Full text of DOI:10.1016/j.bioorg.2020.104213

BioorganicChemistry104(2020)104213
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Bioorganic Chemistry
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Short communication
Synthesis of new riboflavin modified ODNs: Effect of riboflavin moiety on
the G-quadruplex arrangement and stability
⁎
Valeria Romanuccia, ,1, Rosario Olivaa,b,1, Luigi Petracconea, Sandra Claesc, Dominique Scholsc,
⁎
Armando Zarrellia, Giovanni Di Fabioa,
a Department of Chemical Sciences, University of Napoli Federico II, Complesso di Monte Sant’Angelo, Via Cintia 4, I-80126 Napoli, Italy
b Physical Chemistry I – Biophysical Chemistry, TU Dortmund University, Otto-Hahn Strasse 4a, D-44227 Dortmund, Germany
c Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Herestraat 49, 3000 Leuven, Belgium
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the panorama of modified G-quadruplexes (G4s) with interesting proprieties, here, it has been reported the
synthesis of new modified d(TGGGAG) sequences forming G-quadruplexes, with the insertion of a riboflavin unit
(Rf, vitamin B2). Exploiting the flavin similarity with the hydrogen bond pattern of guanine and aiming at mimic
a typical nucleoside scaffold, the synthesis of the riboflavin building block 3 it has been efficiently carried out.
The effect of insertion of riboflavin mimic nucleoside on the G-quadruplex properties has been here, for the first
time investigated. A biophysical characterization of Rf-modified sequences (A-D) has been carried out by circular
dichroism (CD), fluorescence spectroscopy, differential scanning calorimetry (DSC) and native gel electro-
phoresis. CD and electrophoresis data have suggested that Rf-modified sequences are able to form parallel tet-
ramolecular G4 structures similar to that of the unmodified sequence. Analysis of the DSC thermograms has
revealed that all modified G-quadruplexes have a higher thermal stability compared with the natural sequence,
particularly the stabilisation is higher when the Rf residue is introduced at the 3′-end. Further, DSC analysis has
revealed that the Rf residues introduced at the 3′-end are able to form additional stabilising interactions, en-
ergetically almost comparable to the enthalpic contribution of a G-tetrad. Fluorescence measurement are con-
sistent with this result showing that the Rf residues introduced at 3′-end are able to form stacking interactions
with the adjacent bases within the G-quadruplex structure. The whole of data suggested that the introduction of
Rf unit can stabilize G-quadruplex structures and can be a promising candidate for future theranostic applica-
tions.
Modified ODNs
G-quadruplex
Riboflavin
Chemical modification
DSC
CD
1. Introduction
[1,2]. Several reports deserved particular attention to the elevated
fascinating polymorphism of G-quadruplex structures that seems to be
G-quadruplexes (G4s) are non-canonical DNA structures which are
formed and stabilized by stacked G-tetrads. Each G-tetrad contains four
guanines associated through hydrogen bonding between Hoogsteen
face (O6 and N7) in order to form a planar structure also called G-
quartet stabilized by monovalent cations as K+, Na+ and NH4+ which
interact with the lone pairs on the O6 atoms surrounding the central
core. Over the last years, G-rich oligonucleotides able to form G4, have
attracted considerable interest in a large variety of fields including
medicinal chemistry, supramolecular chemistry, and nanotechnology
strongly implicated in their biological activity [3,4]. Moreover, the
understanding of the marked polymorphism of nucleic acids is essential
in the drug design and for the study of complex biological mechanisms
[5]. Many reports in the last years have investigated how chemical
modifications to the bases or the backbone of oligonucleotides forming
G-quadruplexes effect the features of these higher-order structures
[6–11]. G-quadruplexes have been recognized as a highly appealing
DNA structures implicated in several diseases as cancer [12,13], neu-
rodegeneration [14] and HIV infection [15–16]. In this regard, some G-
Abbreviations: Rf, Riboflavin; G4, G-quadruplex; CD, Circular dichroism; DSC, Differential scanning calorimetry; PAGE, Polyacrylamide gel electrophoresis; ODNs,
Oligodeoxynucleotides; DMT, 4,4′-dimethoxytrityl; DMF, Dimethylformamide; DIPEA, N,N-Diisopropylethylamine; DCCI, N,N′-Dicyclohexylcarbodiimide; CPG,
Controlled Pore Glass; RP-HPLC, Reverse Phase-High Performance Liquid Chromatography; HIV, Human Immunodeficiency Virus; TBE buffer, Tris-borate-EDTA
buffer; ACN, Acetonitrile; DCM, Dichloromethane; TEAA, Triethylammonium acetate
⁎ Corresponding authors.
E-mail addresses: valeria.romanucci@unina.it (V. Romanucci), difabio@unina.it (G. Di Fabio).
1 These authors contributed equally.
https://doi.org/10.1016/j.bioorg.2020.104213
Received 31 May 2020; Received in revised form 31 July 2020; Accepted 24 August 2020
Availableonline01September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

V. Romanucci, et al.
BioorganicChemistry104(2020)104213
quadruplex aptamers have been found to be active at different stages of
HIV infection [17]. Among G4 discovered to be potent anti-HIV apta-
mers, many reports described the activity of the Hotoda’s sequence.
This short d(TGGGAG) sequence forms a very stable parallel tetra-
molecular G4 and showed significant anti-HIV activity [18]. Taking
advantage on our extensive knowledge about d(TGGGAG) sequence
[19–21], we synthesized new modified d(TGGGAG) sequences with the
insertion of a riboflavin moiety in different position of the sequence.
Riboflavin (Rf) is an essential water-soluble vitamin, precursor of
flavin-based redox cofactors FMN and FAD. Rf presents interesting
biological and physicochemical properties such as transporter specific
cell internalization, implication in redox reactions and photosensitizer
[22]. Rf and its derivatives FMN and FAD may serve as highly versatile
building blocks for the construction of complex systems [23]. Over the
years, many reports described the synthesis of a variety of Rf deriva-
tives including polymers [24], proteins [25] and nucleic acids [26].
Regarding the latter case, the Rf ligand ability has been studied for the
selective binding into DNA-duplex aptamers containing abasic site and
also as DNA G-quadruplex ligand for biosensor applications [27]. In
1995, Riboflavin was also found to specifically bind RNA aptamer
containing G-quartet structure [28]. Very recently, it has been shown
that the Rf fluorescence is quenched when bound to G-quadruplex
structure respect to single stranded form [29,27]. Further, several stu-
dies have shown the Rf ability to form non-covalent bonds such as p-p
stacking [24], hydrogen [30], or electrostatic interactions [31].
In this work, we report the synthesis, biophysical and biological
characterization of new Rf-modified sequences. The d(TGGGAG) se-
quence has been used as a model of parallel tetramolecular G4 struc-
ture. The insertion of riboflavin into lead sequence has been inspired by
its similarity with the Hoogsteen hydrogen bond pattern of guanine
combined with its excellent fluorescence properties that may be a useful
tool for theranostic applications which involve the combination of
therapy and diagnostic imaging into an unique system.
has been obtained by succinylation of the free hydroxyl group (3′-OH)
of intermediate 3 and subsequent solid phase reaction of 5 with a CPG
support. New modified oligonucleotides (ODNs) were purified by RP-
HPLC and finally analyzed by MALDI-TOF. The stability of benzylidene
protection of the final Rf-modified ODNs was evaluated by HPLC ana-
lysis; the results confirmed its high stability, even during the acid
treatment carried out in the ODN assembly. None cleavage it was ob-
served.
2.2. Native gel electrophoresis of Rf-modified sequences defined the
molecularity of complexes
In order to determine the stoichiometry of resulting structures
formed by Rf-modified ODNs, non-denaturing polyacrylamide gel
electrophoresis (PAGE) experiment has been performed. Aiming at
observing the mobility of all complexes, [d(TG4T)]4 and d(T6) were
loaded as G4 and single stranded references, natural d(TGGGAG) and
modified sequences (A − D) were loaded on the gel in single stranded
(ss) conditions (no potassium added, indicated by ‘‘−”) and G4 con-
ditions (10 mM phosphate buffer + 100 mM KCl, indicated by ‘‘+’’, see
paragraph 4.5.1). In absence of potassium, as expected, all the se-
quences show a single well defined band corresponding to the single-
stranded form (Fig. 1).
The addition of potassium is central to induce the G-quadruplex
folding, in fact in presence of K+, this band disappears and a single
band at lower mobility was observed for A, C and D. This behavior is
similar to the one observed for the d(TGGGAG) and is consistent with
the formation of G-quadruplex structures. The variation in the elec-
trophoretic mobility of both ss and G4 forms of modified A-D sequences
respect to the natural ones, is due to the contribute of Rf moiety and its
interaction within the sequence depending on its position in it [34,21].
Only the sequence B presents a smearing retarded band (Fig. 1) sug-
gesting the not complete formation of the tetramolecular complex and/
or the formation of additional lower molecularity complexes.
To explore how riboflavin unit affects the G quadruplex structure
and if is able to additionally stabilize the G-quartet stacking, new Rf-
modified sequences (A-D) have been investigated through different
techniques in a combined approach (CD, DSC, native electrophoresis
and fluorescence). Moreover, the anti-HIV activity of all sequences has
been evaluated.
2.3. CD profiles of Rf-modified sequences confirmed the formation of
parallel G quadruplex structures
The sequences A and D presented an additional Rf unit at 5′and 3′-
end of d(TGGGAG) sequence, instead in B and C sequences, the 5′and 3′
terminal bases were replaced with the Rf unit.
2. Results and discussion
All secondary structures formed by Rf-modified sequences (A-D)
have been characterized by CD in order to evaluate how the insertion of
riboflavin moiety into d(TGGGAG) sequence influences its G-quad-
ruplex arrangement. CD experiments have been performed in potassium
buffer (10 mM KH2PO4/K2HPO4 + 100 mM KCl, see paragraph 4.5.1).
As shown in the CD spectra reported in Fig. 2, the G4 arrangements of
A-D sequences except B, are similar to that adopted by natural d
(TGGGAG) sequence with a typical profile of a parallel G4 structure
characterized by a positive band at 263 nm and a negative band at
243 nm [35]. On the other hand, the CD spectrum of B appears less
intense compared to the other sequences. This behavior could be the
result of a not complete G-quadruplex formation as already observed by
gel electrophoresis. Small differences in the A-D CD profiles indicate
slight structural or conformational variations in the modified G-quad-
ruplexes due to not identical ODN sequences and also for the con-
tribution of the riboflavin mimic nucleoside [9].
2.1. Synthesis of riboflavin-building blocks and insertion into the lead
sequence
The synthetic effort has been devoted for the synthesis of nucleo-
side-riboflavin building block 3, in particular aiming to make riboflavin
moiety similar to a nucleoside unit, the ribityl chain has been rigidified
with 2′,4′-O-benzylidene protection. This synthetic procedure has been
inspired by a previous study described by Schwogler et al. that reported
the incorporation of Rf moiety into duplex DNA sequence by H-phos-
phonate chemistry [32]. Interestingly, even if we followed the same
experimental conditions to obtain a selective benzylidene protection,
we did not observe similar yield of the desired diastereoisomer 2 (22%
respect to 50% previously reported).
After benzylidene protection, the primary hydroxyl group of ribityl
chain has been protected with DMT group, indeed the 3′-OH group has
been functionalized with phosphoramidite (intermediate 4) [33]. The
synthetic approach takes advantage from the different reactivity of
primary and secondary hydroxyl groups following the classic nucleo-
side/tide chemistry. All intermediates were gained in very good yields
(Scheme 1), only the intermediate 2 was obtained with a yield not
greater than 22%. An efficient post-assembling conjugation to lead se-
quences provided to the Rf-modified sequences A and B. Conversely, in
order to synthesize the 3′-modified sequences C and D, it has been again
useful the key intermediate 3. The riboflavin functionalized support 6
2.4. DSC characterization of modified G-quadruplexes unfolding
The thermal unfolding of the studied complexes was followed by
means of differential scanning calorimetry. In Fig. 3 the heating and
cooling DSC profiles for the modified sequences are shown. For all
samples the formation of the G-quadruplexes (an esothermic peak) was
not observed in the cooling profiles, this indicates that the formation/
dissociation of the complexes is not at thermodynamic equilibrium
2

V. Romanucci, et al.
BioorganicChemistry104(2020)104213
Scheme 1. Synthetic scheme of riboflavin building blocks 3 and 5 and solid phase synthesis of Rf-modified A-D sequences.
during the heating and cooling experiments [36]. This result, observed
also for the natural sequence (Fig. S1), is in agreement with the well-
known slow kinetics of tetramolecular G-quadruplexes folding [36,37].
On the other hand, successive heating scans of the modified se-
quences were superimposable indicating that, at ODN concentrations of
DSC experiments, few minutes at low temperature (before each tem-
perature scan the sample was equilibrated at 5 °C for 15 min) were
enough to allow the formation of these G-quadruplexes (Fig. S2). In-
terestingly, this result reveals that the introduction of the Rf residue
increases the G4 formation kinetics in comparison with the unmodified
sequence.
(i.e. the G-quadruplex dissociation) and we preferred not to further
discuss the enthalpy change for this sequence. Inspection of the en-
thalpy changes for the other sequences reveals that when the Rf is in-
troduced at the 3′-end (sequences C and D), there is and enthalpy in-
crease (in comparison with the reference sequence) for the G-
quadruplex dissociation of ΔΔH̴ 40 kJ/mol and ̴ 30 kJ/mol for C and D,
respectively. This enthalpy increase can be assigned to the contribution
of the Rf residue in the sequence C in which the Rf residue is added to
the unmodified d(TGGGAG) sequence. However, in the sequence D, the
Rf residue replaces a G residue of the reference sequence suggesting
that its enthalpic contribution to the G-quadruplex stabilization could
be even higher than the observed ΔΔH. Taken together these data re-
veal that the four Rf residues introduced at the 3′-end are able to form
additional stabilizing interactions, energetically almost comparable to
the enthalpic contribution of a G-tetrad.
In Table 1, the melting temperatures and the enthalpy changes re-
lative to the G-quadruplex dissociation process were reported. Inspec-
tion of Table 1 reveals that all the modified G-quadruplexes have a
higher thermal stability (Tm) compared with the natural sequence,
particularly the stabilization is higher when the Rf residue is introduced
at the 3′-end.
2.5. Quenching of riboflavin fluorescence depending on G4 or ss
arrangements formed by Rf-modified sequences
However, differently from the other sequences, the DSC profile of
the sequence B shows at least two well resolved DSC peaks and an
anomalous low total enthalpy change, this result, in agreement with
electrophoresis and CD results, suggests by the presence of multiple
species and a not complete formation of a single well defined tetra-
molecular complex. For this reason, it is not possible to assign the
corresponding total enthalpy change to a single well defined process
In order to verify if the Rf residue is able to form stacking interac-
tion with the adjacent bases within the G-quadruplex structures, we
exploit the properties of the Rf fluorescence in both ss and G4 forms. In
this frame, it has been reported that Rf fluorescence is more quenched
when stacked in a G-quadruplex structure than in the unstructured
3

V. Romanucci, et al.
BioorganicChemistry104(2020)104213
2.6. Anti-HIV activity of Rf-modified sequences
The anti-HIV activity and cellular cytotoxicity of the Rf-modified
oligodeoxynucleotides were evaluated against HIV-1 NL4.3 and HIV-2
ROD replication in a CD4+ T cell line MT-4 cell cultures using the 3-
(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT)
method as described previously by Pauwels et al. [38]. The Rf-modified
sequences A and B exhibited moderate HIV-1 and HIV-2 activity as
reported by the IC50 in the Table 2. The sequences C and D modified at
3′-end did not show anti-HIV activity.
3. Conclusions
In conclusion, we report here, the synthesis of a riboflavin mimic
nucleoside and its insertion into a model sequence forming tetra-
molecular G-quadruplex structure. All secondary structures formed by
Rf-modified sequences have been characterized by native PAGE, CD,
DSC and fluorescence analyses. Also the anti-HIV activity of all rf-
modified sequences has been evaluated. CD and native PAGE data have
reported similar behavior to the parallel G-quadruplex structure for
almost all modified sequences. The melting temperature (Tm) of all Rf-
modified sequences results to be higher than that of natural one, in
particular the insertion of Rf unit at 3′-end of the d(TGGGAG) sequence
(C and D sequences) has more effect on the stability of G-quadruplex
than modification at the 5′-end (A and B sequences).
Fig. 1. Native gel electrophoresis of natural sequence d(TGGGAG) and Rf-
modified sequences A − D sequences loaded at 50 µM (ss concentration) in two
conditions (“−”, no potassium added; “+” 100 mM KCl); 15% polyacrylamide
gel supplemented with 10 mM KCl. The gel was run at 26 °C at constant voltage
(90 V) for 2.0 h.
Consistent with these results, DSC and fluorescence analyses have
revealed the ability of Rf units inserted at 3′-end of the lead sequence to
form additional stabilizing interaction energetically similar to that of a
G-tetrad. In particular DSC experiments highlighted the higher enthalpy
of G4 dissociation of sequences C and D respect to the natural d
(TGGGAG) one due to the stacking interactions of Rf-moiety with the
adjacent bases within the G-quadruplex structure. These preliminary
findings reveal the ability of Rf residue to establish significant stacking
interactions with the adjacent tetrads of G4 structures.
200000
TG3AG
A
B
C
D
150000
100000
50000
0
Future studies will be address to the characterization of these in-
teractions and, in turn, to assess the potential formation of Rf-tetrad in a
large variety of relevant G-quadruplexes.
4. Materials and methods
4.1. General methods
-50000
Riboflavin (1) was purchased from Sigma-Aldrich – Italy. HPLC-
grade CH3CN was purchased from Carlo Erba Reagents and Sigma-
Aldrich, respectively. Unless otherwise indicated, other chemicals were
obtained from Sigma-Aldrich.
220
240
260
280
300
320
wavelength / nm
Fig. 2. CD spectra of natural sequence d(TGGGAG) and Rf-modified A-D se-
quences (1x10-4M ss concentration) registered in 10 mM phosphate buffer
(pH = 7.0) containing 100 mM KCl.
Reactions were monitored by TLC (precoated silica gel plates F254,
Merck) and column chromatography (Merck Kieselgel 60, 70–230
mesh). Analysis and purification was performed on a Shimadzu LC-8A
PLC system equipped with a Shimadzu SCL-10A VP System control and
a Shimadzu SPD-10A VP UV–vis detector. HPLC purifications were
carried out on Phenomenex RP18 column (Gemini, 10 µm C18(2),
250 mm × 21.2 mm i.d.) using a linear gradient of CH3CN in 0.1 M
TEAA in H2O (pH 7.0) from 20% to 100% over 30 min at a flow rate of
7 mL/min with detection at 260 and 360 nm. MALDI-TOF mass spec-
trometric analyses were performed on AB SCIEX TOF/TOF 5800.
Desalting was conducted using gel filtration chromatography on a
Sephadex G10 column, eluted with H2O/EtOH 4:1 (v/v).
single strand form [29,27]. Hence, the fluorescence emission spectra of
all Rf-modified sequences were collected before denaturation of sam-
ples and after fast cooling at room temperature. The experiments were
conducted at low G-quadruplex concentrations (̴ 1 μM). In these con-
ditions, we can assume that, after denaturation, the G-quadruplexes
were not formed upon cooling. A significant quenching of riboflavin
fluorescence (λ = 530 nm) has been found for the sequences C and D
when structured in G4 respect to their dissociated ss form, whereas, a
smaller quenching was observed for the G4 of A and none for B (Fig. 4).
These findings reveal that in the C and D sequences, the Rf residue is
able to establish significant stacking interactions with the adjacent
tetrads of G4 structures as confirmed by the higher enthalpy of dis-
sociation measured by DSC experiments.
4.2. Synthesis of riboflavin building blocks
4.2.1. Synthesis of 2′,4′-O-benzylidene-riboflavin (2)
Riboflavin (1) (2 g, 5.31 mmol) was dissolved in dry DMF (25 mL),
(+)-camphor-10- sulfonic acid (CSA) (62 mg, 0.26 mmol) and 796 μL of
benzaldehyde dimethyl acetal (5.31 mmol) were added. The reaction
was taken at 80 °C in the dark for a night. The reaction was monitorated
4

V. Romanucci, et al.
BioorganicChemistry104(2020)104213
Fig. 3. DSC first heating (black lines) and cooling (red lines) profiles of (A) Rf-TGGGAG, (B) Rf-GGGAG, (C) TGGGAG-Rf and (D) TGGGA-Rf. All the experiments were
carried out at 2x10-4M of ss ODNs in 10 mM potassium phosphate buffer + 100 mM KCl (pH 7.0). (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
82.5; 78.8; 63.8; 61.7; 46.8; 21.0 and 19.2 ppm.
Table 1
Thermodynamic parameters for the G-quadruplex dissociation.
Sequences
ΔHd (kJ mol−1 of G-quadruplex)
Tm
4.2.2. Synthesis of 5′-O-DMT-2′,4′-O-benzylidene-riboflavin (3)
Compound 2 (426 mg, 0.92 mmol) was dissolved in dry pyridine
(3 mL), 4,4′- dimethoxytrityl chloride (279 mg, 0.82 mmol) was added
at room temperature. After 2 h, the reaction was quenched by addition
of methanol (3 mL). Solvents were evaporated, and residual pyridine
was coevaporated with toluene. Dichloromethane was added to the
residue and washed two times with water. The combined organic ex-
tracts were dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified on silica gel in dichloromethane-
methanol (97:30 to 95:50 + 1% triethylamine). The 5′-O-DMT-2′,4′-O-
benzylidene-riboflavin 3 was obtained in good yield (667 mg,
0,87 mmol, 95%).
TGGGAG
327
324
250
365
355
67.7
(A) RfTGGGAG
(B) Rf-GGGAG
(C) TGGGAG-Rf
(D) TGGGA-Rf
85.6
81.7–88
95.3
99.3
Errors on enthalpy changes are within 5%, errors on Tm are within 0.2 °C.
by TLC (dichloromethane-methanol, 9:1, v:v) and quenched at room
temperature by addition of triethylamine until neutrality. After eva-
poration of solvents, the residue concentrated under reduced pressure
and subsequently purified on silica gel with dichloromethane-methanol
(95:5 to 90:10) to yield compound 2 (537 mg, 1.16 mmol, 22%).
Compound 2: 1H NMR (DMSO‑d6, 400 MHz, r.t.) 8.02 ppm (1H, s,
aromatic-Rf); 7.82 ppm (1H, s, aromatic-Rf); 5.52 ppm (1H, s, H6′); 5.20
and 4.85 ppm (2H, m, H1′); 4.18 ppm (1H, m, H2′); 3.80–3.73 ppm (1H,
m, H5′); 3.65 ppm (1H, m, H4′); 3.60–3.41 ppm (2H, overlapped signals,
Compound 3: 1H NMR (CDCl3, 400 MHz): 8.07 ppm (1H, s, aro-
matic-Rf), 7.95 ppm (1H, s, aromatic-Rf); 7.37–7.10 ppm (9H, m, aro-
matic-DMT), 6.82–6.79 ppm (4H, m, Hm DMT), 5.68 ppm (1H, s,H6′);
5.21–4.83 ppm (2H, m, H1′); 4.28 ppm (1H, m, H2′); 3.97 ppm (1H, m,
H4′); 3.80 ppm (6H, s, OCH3-DMT); 3.53 ppm (1H, m, H3′); 3.52 ppm
(1H, m, H5′) 3.41 ppm (1H, m, H5′); 2.46 ppm (3H, s, CH3-Rf); 2.43 ppm
(3H, s, CH3-Rf).13C NMR (CDCl3, 100 MHz): δ = 159.2, 158.4, 154. 4,
148.1, 144.9, 137.5, 137.4, 136.1, 136.0, 135.5, 135.3, 132.3, 132.2,
130.2, 130.1, 128.8, 128.2, 128.0, 127.7, 126.7, 125.9, 118.2, 113.0,
H5′ and H3′); 2.39 ppm (3H, s, CH3-Rf); 2.45 ppm (3H, s, CH3-Rf). 13
C
NMR (DMSO- d6, 100 MHz, r.t.): δ = 160.3; 155.8; 151.0; 146.0; 138.3;
137.5; 136.2; 134.1; 132.4; 130.9; 128.9; 128.1; 126.5; 118.7; 100.0;
5

V. Romanucci, et al.
BioorganicChemistry104(2020)104213
Fig. 4. Fluorescence emission spectra of (A) Rf-TGGGAG, (B) Rf-GGGAG, (C) TGGGAG-Rf and (D) TGGGA-Rf at the temperature of 25 °C (black lines) and after
heating at 100 °C and fast re-cooling at 25 °C (red lines). The spectra were recorded after excitation at 450 nm, by using a 1-cm path length quartz cuvette. All the
spectra were recorded at 40x10-5 M of ss ODNs in 10 mM potassium phosphate buffer + 100 mM KCl (pH 7.0). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
with ethyl acetate-hexane (9:1 with 3% trimethylamine) afforded the
5′-O-DMT-2′,4′-O-benzylidene-riboflavin-phosphoramidite 4 (146 mg,
0.15 mmol, 80%). The characterization of the intermediate phosphor-
amidite 4 was carried out by 31P NMR and ESI-MS analyses. 31P NMR
(161.98 MHz, CDCl3, r. t., mixture of diastereoisomers) δ = 149.91;
Table 2
IC50 and CC50 values of Rf-modified oligodeoxynucleotides.
Compound
Unit
IC50 HIV-1 (NL4.3)
IC50 HIV-2 (ROD)
CC50
A
µM
2.59
8.15
20
4.47
> 20
> 20
> 20
> 20
> 1000
B
µM
8.77
149.41
ppm;
ESI-MS
(positive
ion)
m/z
calcd
for
C
µM
> 20
> 20
17.92
C
54H59N6O9P = 966,41, found 967,41 [M−H]+.
D
µM
> 20
12.26
AMD3100
ng/ml
4.2.4. Synthesis of 3′-O-succinyl-5′-O-DMT-2′,4′-O-benzylidene-riboflavin
(5)
IC50: 50% Inhibitory concentration or compound concentration required to
inhibit HIV-induced cytopathogenic effect evaluated in the CD4+ MT-4T cell
line. CC50: 50% Cytotoxic concentration of the compounds in parallel evaluated
in this cell line.
5′-O-DMT-2′,4′-O-benzylidene-riboflavin 3 (290 mg, 0.37 mmol)
was dissolved in dry pyridine (2 mL), triethylamine (TEA) (260 µL,
1.89 mmol) and 150 mg of succinyl anhydride (1.48 mmol) were
added. The reaction was stirred at 50 °C for 2 h. After 2 h, the mixture
was extracted two times with dichloromethane and water. The com-
bined organic extracts were dried over Na2SO4, filtered and con-
centrated under reduced pressure. The residue was purified on silica gel
in dichloromethane-methanol (97:30 to 95:50 + 1% triethylamine).
The 3′-O-succinyl-5′-O-DMT-2′,4′-O-benzylidene-riboflavin 5 was ob-
tained in good yield (0.28 mmol, 76%).
113.4, 100.4, 86.0, 80.4, 80.2, 63.8, 55.2, 47.5, 21.3 and 19.5 ppm.
4.2.3. Synthesis
of
5′-O-DMT-2′,4′-O-benzylidene-riboflavin-
phosphoramidite (4)
To 5′-O-DMT-2′,4′-O-benzylidene-riboflavin 3 (145 mg , 0.19 mmol)
dissolved in anhydrous dichloromethane (4 mL), DIPEA (131 μL,
0.76 mmol) and 2-cyanoethyl-N,N-diisopropylamino-chloropho-
sphoramidite (116 μL, 0.28 mmol) were added. After 1 h, the solution
was concentrated in vacuo and purified on column chromatography
Compound 5: 1H NMR (CDCl3, 400 MHz): 8.03 ppm (1H, s, aro-
matic-Rf); 7.87 (1H, s, aromatic-Rf); 7.50–7.11 ppm (9H, m, aromatic-
6

V. Romanucci, et al.
BioorganicChemistry104(2020)104213
DMT), 6.84 ppm (4H, d, J = 8.7 Hz, Hm DMT); 5.45 ppm (1H, s, H6′);
5.37–5.28 ppm (2H, overlapped signals, H3′and H1′); 4.58–4.42 ppm
(2H, overlapped signals, H2′and H1′); 4.07 ppm (1H, m, H4′); 3.82 ppm
(6H, s, OCH3-DMT); 3.38 ppm (1H, m, H5′); 3.22 ppm (1H, m, H5′);
2.83–2.51 ppm (4H, m, CH2 succinyl); 2.46 ppm (6H, s, CH3-Rf). 13C
NMR (CDCl3, 100 MHz): δ = 175.3, 172.0, 159.2, 158.4, 154. 4, 148.1,
144.9, 137.5, 137.4, 136.1, 136.0, 135.5, 135.3, 132.3, 132.2, 130.2,
130.1, 128.8, 128.2, 128.0, 127.7, 126.7, 125.9, 118.2, 113.0, 113.4,
99.5, 86.0, 78.0, 75.9, 65.5, 62.8, 54.7, 47.9, 29.4, 30.4, 21.2 and
19.4 ppm.
measurements.
4.5.2. Native gel electrophoresis
All stock solutions of (A-D) were loaded on the gels after dilution to
50 µM (ss concentration) and with the addition of 25% of glycerol. The
oligonucleotide d(T6) was used as a single-stranded 6-mer marker and d
(TG4T) as a tetramolecular G-quadruplex one, [d(TG4T)]4. Native gel
electrophoresis was performed on 15% polyacrylamide gel (acryla-
mide/bi-acrylamide 19:1) and the running buffer (TBE 0.5X) was sup-
ported by 10 mM KCl in order to preserve the native conditions. The gel
was run at 26 °C at constant voltage (100 V) for 2.5 h. The bands were
visualized by UV shadowing after stained with Stains All (Sigma-
Aldrich) according to the manufacturer’s instructions.
4.3. Synthesis of Rf-functionalized solid support (6)
In a typical experiment to 150 mg of HL CPG-NH2 (108 umol/g,
0.09 mmol) suspended in 1.0 mL of dry pyridine and 50 uL of DIPEA
(0.27 mmol), a mixture of 5 (0.27 mmol), DCCI (56 mg, 0.27 mmol),
DIPEA (50 µL, 0.27 mmol), and dry pyridine (500 uL) was added and
shaken at room temperature for 72 h. After exhaustive washings with
pyridine, DMF, ACN, DCM and Et2O, the support was dried under re-
duced pressure. DMT tests performed on dried and weighed samples of
resulting support 6 allowed to determine the incorporation yields of
derivative 5, onto the resin, which resulted to be in the range of
0.04–0.05 mmol/g. After treatment with 1.0 mL of acetic anhydride/
pyridine (1:1, v/v) at r.t. for 1 h (capping), the resin was washed ex-
haustively with DCM, CH3OH and Et2O, dried under reduced pressure
and used for automated syntheses of sequences C and D.
4.5.3. Circular dichroism (CD)
CD spectra were recorded on a Jasco J-715 spectropolarimeter
equipped with a Peltier-type temperature control system (model PTC-
348WI), using a quartz cuvette with a path length of 0.1 cm. CD
parameters for spectra recording were the following: spectral window
220–320 nm, data pitch 1 nm, band width 2 nm, response 4 s, scanning
speed 40 nm/min, temperature 10 °C, 3 accumulations. All sequences
were analysed at 1x10-4 M single stranded concentration in 10 mM
KH2PO4/K2HPO4 + 100 mM KCl buffer.
4.5.4. Differential scanning calorimetry (DSC)
DSC measurements were performed by means of NanoDSC from TA
Instruments (New Castle, DE, USA). The excess molar heat capacity
function (< ΔCp >) was obtained after the subtraction of the baseline.
A buffer–buffer scan was subtracted from the sample scan. Briefly a
volume of 300 µL of Rf-modified G-quadruplex sample at concentra-
tions ranging from 35 µM to 135 µM (concentration per mol of G-
quadruplex) was placed in the calorimetry vessel. Successive heating
scans were performed in the range 25–110 °C at scan rate of 1 °C/min to
check the reversibility of the melting process. The obtained DSC data
were analyzed by means of the NanoAnalyze software supplied with the
instrument and plotted using the Origin software package (OriginLab,
Northampton, MA, USA). The transition enthalpies, ΔH°, were obtained
by integrating the area under the heat capacity vs temperature curves.
The enthalpy change values are the averages of at least three different
heating experiments.
4.4. Synthesis, HPLC purification and MALDI-TOF of Rf-modified ODN
sequences
Oligomers A and B were synthesised starting with functionalised
CPG support with 0.1 meq/g initial loading, on which the sequences d
(TGGGAG) and d(GGGAG) were assembled in a standard manner. An
additional coupling with phosphoramidite building block 4 was then
performed. Oligomers C and D were synthesised starting from the
support 6 in a standard manner.
Oligomers A–D were detached from the solid support and depro-
tected by treatment with trimethylamine /pyridine (1:1, v/v) at 50 °C
for 1 h, followed by treatment with conc. aq. ammonia at 50 °C for 5 h.
The combined filtrates and washings were concentrated under reduced
pressure, re-dissolved in water, analysed and purified by HPLC.
Purification of the crude modified oligonucleotides A–D was carried out
on Phenomenex RP18 column (Gemini, 10 µm C18(2),
250 mm × 21.2 mm) using a linear gradient of CH3CN in 0.1 M TEAA
in H2O, pH 7.0 from 20 to 100% over 30 min at flow rate of 7 mL/min
with detection at 260 and 360 nm. The A–D ODNs were desalted on a
Sephadex G10 column eluted with H2O/EtOH 4:1 (v/v). The modified
ODNs were obtained with an average yield of 25–31%. MS (MALDI-
TOF) m/z calcd. for A [d(Rf-TGGGAG)] and C [d(TGGGAG-Rf]:
[M]calc. = 2398.69 m/z; [M−H]-exp = 2397.34 m/z.
4.5.5. Fluorescence spectroscopy
Fluorescence emission spectra of Rf-modified sequences were ac-
quired by means of Fluoromax-4 fluorimeter from Horiba Scientific
(Edison, NJ, USA) by using a 1-cm path length quartz cuvette. The
sample concentrations were around 4 µM single strand concentration.
The samples were excited at the wavelength of 450 nm, and emission
spectra were acquired in the range 470–750 nm. The excitation and
emission slits of the monochromators were set to 2 nm and 6 nm, re-
spectively. The temperature was controlled by means of a Peltier system
that ensures an accuracy of 0.1 °C. During the acquisition, the sam-
ples were under gentle stirring. For each sample two spectra were re-
corded: the first one at the temperature of 25 °C and the second one
after heating the sample at 100 °C and fast cooling at 25 °C.
MS
(MALDI-TOF)
m/z
calcd.
for
B
[d(Rf-GGGAG)]:
[M]calc = 2094.50 m/z; [M−H]-exp = 2093.99 m/z. MS (MALDI-TOF)
m/z calcd. for
D
[d(TGGGA-Rf)]: [M]calc
=
2069.49 m/z;
[M−H]-exp = 2068.73 m/z.
4.5. Characterization of Rf-modified G-quadruplexes
4.5.1. G-quadruplex preparation
4.5.6. Anti-HIV assay
The anti-HIV activity and cellular cytotoxicity of the Rf-modified
oligodeoxynucleotides were evaluated against HIV-1 NL4.3 and HIV-2
ROD replication in a CD4+ T cell line MT-4 cell cultures using the 3-
(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT)
method as described previously by Pauwels et al.[38]
Stock ODNs solutions were prepared at single stranded concentra-
tion of 1 mM in 10 mM KH2PO4/K2HPO4 + 100 mM KCl buffer.
Annealing experiments were performed at 80 °C heating for 5 min and
then a fast cooling to room temperature was carried out. After an-
nealing, the G-rich oligonucleotides arrange in G-quadruplex structure
stabilized by potassium coordination. The solutions were stored at 8 °C
for 14 days before all experiments. The concentration of the dissolved
oligonucleotides A-D and natural one was determined by UV
Declaration of Competing Interest
The authors declare that they have no known competing financial
7

V. Romanucci, et al.
BioorganicChemistry104(2020)104213
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
12352–12366, https://doi.org/10.1093/nar/gku999.
[18] V. Romanucci, A. Zarrelli, S. Liekens, S. Noppen, C. Pannecouque, G. Di Fabio, New
findings on the d(TGGGAG) sequence: Surprising anti-HIV-1 activity, Eur. J. Med.
Chem. 145 (2018) 425–430, https://doi.org/10.1016/j.ejmech.2018.01.005.
[19] V. Romanucci, D. Milardi, T. Campagna, M. Gaglione, A. Messere, A. D’Urso,
E. Crisafi, C. La Rosa, A. Zarrelli, J. Balzarini, G. Di Fabio, Synthesis, biophysical
characterization and anti-HIV activity of d(TG3AG) Quadruplexes bearing hydro-
phobic tails at the 5’-end, Bioorg. Med. Chem. 22 (2014) 960–966, https://doi.org/
10.1016/j.bmc.2013.12.051.
Acknowledgements
We acknowledge AIPRAS Onlus (Associazione Italiana per la
Promozione delle Ricerche sull’Ambiente e la Saluta umana) and the
KULeuven (grant no. PF/10/018) and the Fonds voor Wetenschappelijk
Onderzoek (FWO, grant no. G.485.08).for grants in support of this in-
vestigation.
[20] V. Romanucci, M. Gaglione, A. Messere, N. Potenza, A. Zarrelli, S. Noppen,
S. Liekens, J. Balzarini, G. Di Fabio, Hairpin oligonucleotides forming G-quad-
ruplexes: New aptamers with anti-HIV activity, Eur. J. Med. Chem. 89 (2015)
51–58, https://doi.org/10.1016/j.ejmech.2014.10.030.
[21] V. Romanucci, A. Marchand, O. Mendoza, D. D’Alonzo, A. Zarrelli, V. Gabelica,
G. Di Fabio, Kinetic ESI-MS Studies of Potent Anti-HIV Aptamers Based on the G-
Quadruplex Forming Sequence d(TGGGAG), ACS Med. Chem. Lett. 7 (2016)
256–260, https://doi.org/10.1021/acsmedchemlett.5b00408.
Appendix A. Supplementary data
[22] N. Beztsinna, M. Solé, N. Taib, I. Bestel, Bioengineered riboflavin in nano-
technology, Biomaterials. 80 (2016) 121–133, https://doi.org/10.1016/j.
biomaterials.2015.11.050.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104213.
[23] N. Beztsinna, Y. Tsvetkova, M. Bartneck, T. Lammers, F. Kiessling, I. Bestel,
Amphiphilic Phospholipid-Based Riboflavin Derivatives for Tumor Targeting
Nanomedicines, Bioconjug. Chem. 27 (2016) 2048–2061, https://doi.org/10.1021/
acs.bioconjchem.6b00317.
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