Thioctic acid derivatives as building blocks to incorporate DNA oligonucleotides onto gold nanoparticles

Article abstract of DOI:10.3390/molecules190710495

Oligonucleotide gold nanoparticle conjugates are being used as diagnostic tools and gene silencing experiments. Thiol-chemistry is mostly used to functionalize gold nanoparticles with oligonucleotides and to incorporate DNA or RNA molecules onto gold surfaces. However, the stability of such nucleic acid-gold nanoparticle conjugates in certain conditions may be a limitation due to premature break of the thiol-gold bonds followed by aggregation processes. Here, we describe a straightforward synthesis of oligonucleotides carrying thioctic acid moiety based on the use of several thioctic acid-L- Threoninol derivatives containing different spacers, including triglycine, short polyethyleneglycol, or aliphatic spacers. The novel thioctic-oligonucleotides were used for the functionalization of gold nanoparticles and the surface coverage and stability of the resulting thioctic-oligonucleotide gold nanoparticles were assessed. In all cases gold nanoparticles functionalized with thioctic-oligonucleotides had higher loadings and higher stability in the presence of thiols than gold nanoparticles prepared with commercially available thiol-oligonucleotides. Furthermore, the thioctic derivative carrying the triglycine linker is sensitive to cathepsin B present in endosomes. In this way this derivative may be interesting for the cellular delivery of therapeutic oligonucleotides as these results provides the basis for a potential endosomal escape.

Full text of DOI:10.3390/molecules190710495

Molecules 2014, 19, 10495-10523; doi:10.3390/molecules190710495
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Thioctic Acid Derivatives as Building Blocks to Incorporate
DNA Oligonucleotides onto Gold Nanoparticles
Sónia Pérez-Rentero 1,2,†, Santiago Grijalvo 1,2,†, Guillem Peñuelas 1, Carme Fàbrega 1,2 and
Ramon Eritja 1,2,
*
1
Institute for Advanced Chemistry of Catalonia (IQAC), Consejo Superior de Investigaciones
Científicas (CSIC), Jordi Girona 18-26, E-08034 Barcelona, Spain;
E-Mails: sonia.perez@iqac.csic.es (S.P.-R.); santiago.grijalvo@iqac.csic.es (S.G.);
guillemph90@gmail.com (G.P.); carme.fabrega@iqac.csic.es (C.F.)
Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN),
Barcelona, 08034, Spain
2
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: recgma@cid.csic.es;
Tel.: +34-93-400-6145; Fax: +34-93-204-5904.
Received: 11 June 2014; in revised form: 4 July 2014 / Accepted: 4 July 2014 /
Published: 18 July 2014
Abstract: Oligonucleotide gold nanoparticle conjugates are being used as diagnostic tools
and gene silencing experiments. Thiol-chemistry is mostly used to functionalize gold
nanoparticles with oligonucleotides and to incorporate DNA or RNA molecules onto gold
surfaces. However, the stability of such nucleic acid–gold nanoparticle conjugates in certain
conditions may be a limitation due to premature break of the thiol-gold bonds followed by
aggregation processes. Here, we describe a straightforward synthesis of oligonucleotides
carrying thioctic acid moiety based on the use of several thioctic acid-L-threoninol derivatives
containing different spacers, including triglycine, short polyethyleneglycol, or aliphatic
spacers. The novel thioctic-oligonucleotides were used for the functionalization of gold
nanoparticles and the surface coverage and stability of the resulting thioctic-oligonucleotide
gold nanoparticles were assessed. In all cases gold nanoparticles functionalized with
thioctic-oligonucleotides had higher loadings and higher stability in the presence of thiols
than gold nanoparticles prepared with commercially available thiol-oligonucleotides.
Furthermore, the thioctic derivative carrying the triglycine linker is sensitive to cathepsin B
present in endosomes. In this way this derivative may be interesting for the cellular

Molecules 2014, 19
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delivery of therapeutic oligonucleotides as these results provides the basis for a potential
endosomal escape.
Keywords: thioctic acid; threoninol; DNA; DNA-gold nanoparticle conjugates; stability;
surface coverage; cathepsin
1. Introduction
During the past decade colloidal gold nanoparticles (AuNPs) have become attractive materials due
to an increasing interest in their potential applications in biotechnology, molecular diagnostics and
biomedicine [1–6]. Because of their unique properties such as size and shape-dependent optical and
electronic features, good biocompatibility and the ability to bind ligands containing thiols, phosphines
and amines, they have emerged as useful tools for the design of biosensors [3], the development of
methods for cancer detection [7,8] and potential vehicles for drug delivery [9]. The use of AuNPs
requires an appropriate colloidal stability. The immobilization of organic molecules or biomolecules
onto AuNps surface significantly promotes their dispersion in aqueous solutions and improves the
stability of the colloidal suspensions [10].
There are numerous examples of biomolecules that have been immobilized onto AuNPs such as
peptides [11], proteins [12,13] or nucleic acids [14]. Specifically, there are many examples reported in
the literature on the use of nucleic acid-functionalized gold nanoparticles. For instance, they have been
applied as a scaffold to construct higher ordered materials [15] or in the field of biodiagnostics [6,15]
and bioimaging [16] and as well used for therapeutic applications such as a potent cell transfection and
gene regulation material [6].
The conjugation of AuNps with biomolecules such as oligonucleotides, proteins and antibodies can
be carried out through chemisorption of the biomolecule to the particle surface or electrostatic
interactions between the biomolecule. The use of heterobifunctional ligands allows the functionalization
of AuNps with biomolecules through covalent attachments, or specific biomacromolecular interactions
such as streptavidin/biotin [17–20].
Due to the strong affinity of thiolated species for gold surfaces and the ease of modification of
nucleic acids with thiolated derivatives, thiolated oligonucleotides have been extensively used in the
preparation of DNA-functionalized AuNps since the pioneering publication of Mirkin and co-workers
in 1996 [21].
Although such oligonucleotide-AuNP conjugates have been demonstrated to be very stable under
most DNA detection conditions, limited stability has been observed in certain biological media due to
a partial desorption of thiolated oligonucleotides by ligand exchange in the presence of some additives
(e.g., dithiothreitol (DTT), mercaptoethanol or biogenic thiols) [22–24].
Many efforts have been carried out in order to increase the colloidal stability by designing surface
ligands that keep AuNPs from aggregating. For instance, Taton et al. functionalized AuNPs with
thiolated systems like poly(L-lysine)-graft-poly(ethyleneglycol (PLL-g-PEG graft copolymers).
The conjugates were stable under extreme conditions, making this polymer a promising ligand to use
in biotechnological applications [24]. There are also several examples reported in the literature to

Molecules 2014, 19
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enhance the stability in systems containing oligonucleotide-AuNP conjugates. Mirkin et al. described a
few years ago the use of modified steroid cyclic disulfide or a novel trithiol-capped oligonucleotide
that exhibited higher stabilities than the conventional acyclic monothiol-capped oligonucleotide-AuNp
conjugates toward DTT treatment. These stable conjugates were studied as useful tools for diagnostic
probes in colorimetric assays and detection methods [21].
Thioctic acid (TA) has been extensively used for the functionalization of gold surfaces [18].
In most cases, TA has been conjugated with polyethyleneglycol (PEG) units giving decorated
TA-appended-polyethyleneglycol functionalized AuNPs, thereby showing better stabilities than their
acyclic disulfide and single thiol counterparts [18, 25-27]. The conjugation of oligonucleotides with
TA has been recently reported by Graham and co-workers using two different methodologies [28,29].
These include: (I) the use of a TA H-phosphonate derivative to introduce the resultant disulfide unit at
5'-termini of oligonucleotides [28] and (II) the use of the N-hydroxy-succimidyl ester of TA to
incorporate the TA molecule in a solid support having an amino-linker followed by the assembly of the
oligonucleotide by the stepwise approach [29].
Another important factor to consider AuNPs as promising vehicles for drug delivery, apart from
their systemic stability, is the selective cargo release at the target tissue [30,31]. In these terms, the
conjugation of oligonucleotides with TA derivatives could facilitate the incorporation of spacers with
potential breaking points that would allow oligonucleotides to be released at the cellular target site.
It is known that several classes of proteinases are overexpressed by many tumor cells, including cathepsins.
In particular, cathepsin B plays a critical role in tumor progression [32]. In addition, several works has
reported that polypeptides linker has been used for the endosomal escape of several drugs [33–36] because
of the lability to cathepsin. Thus, we thought the possibility to consider the incorporation of linkers
susceptible to cathepsin B in our oligonucleotide-TA conjugates in order to release the therapeutic
oligonucleotide by the action of this protease.
In this work, we investigate the effect of introducing several spacers between the oligonucleotide
and the TA moiety (Figure 1). It has been reported that the introduction of spacers between the AuNPs
surface and biomolecules not only leads to an increased immobilization efficiency [37,38], but also to
functional immobilized biomolecules [38,39]. Moreover, spacers have been employed to protect the
gold surface from thiolated compounds by providing a dense and ordered coating layer [27,40–42].
Then, the contribution of a spacer, in addition of moving the recognition tracts of oligonucleotides
from the particle surface, may be crucial to the stability of the resulting AuNP conjugates.
Here, L-threoninol was selected as a building block to incorporate the TA residue tethered with
different spacers into DNA (Figure 1). We synthesized the thioctic-DNA analogue (TA_I) without any
spacer, which was used as a reference and four different thioctic-DNA analogues containing a spacer.
In particular, TA_II conjugate containing an ethylene glycol-like (hexaethylene glycol) unit between
the oligonucleotide and the threoninol moiety was obtained via phosphate linkages. Finally, the
TA_III series containing an oligo(ethylene glycol)-like molecule, a triglycine peptide and an alkyl
group as spacers. These three spacers were introduced via amide groups in order to separate the
threoninol unit from the TA residue. The resulting oligonucleotides were then successfully loaded onto
AuNPs. The ability of these TA-terminated oligonucleotides to improve the stability of the
corresponding DNA-AuNP conjugates in aqueous solution in the presence of DTT was investigated.
For comparison purposes, the same experiments were performed with AuNP conjugates prepared with

Molecules 2014, 19
10498
oligonucleotides modified with commercially available thiol- and acyclic disulfide-oligonucleotides.
Consequently, this synthetic strategy may be useful to prepare TA-terminated oligonucleotides
modified at the 3'-end by the incorporation of several spacers with different size and properties. To the
best of our knowledge, the use of this strategy in synthesizing such modified oligonucleotides to
functionalize AuNPs has not been reported yet. In addition, the effect promoted by some proteases like
Cathepsin B on our TA-terminated oligonucleotides was evaluated. These preliminary results could
offer a strategy in the search of new drug delivery vehicles containing oligonucleotides.
Figure 1. Preparation of gold nanoparticles functionalized with TA-terminated oligonucleotides.
AuNp
AuNp
AuNp
Citrate stabilized
10 nm AuNp
TA_I-AuNp conjugate
TA_II-AuNp and
TA_III(a-c)-AuNp conjugates
O
3'
5'
HN
O
S
Oligonucleotide
DNA analogue: TA_I
S
=
OH
O
NH2
3'
5'
HN
O
P
HO
Spacer
O
S
O
O
Oligonucleotide
DNA analogue: TA_II
S
O
OH
OH
L-Threoninol
=
S
S
O
H
Spacer
N
3'
5'
HN
O
Oligonucleootiddee
O
OH
DNA analogue: TA_III(a-c)
O
H
N
H
N
O
N
=
Spacer
O
3
H
5
O
O
III(b)
III(c)
II
III(a)
2. Results and Discussion
2.1. Synthesis of Solid Supports Functionalized with TA Derivatives
In order to connect TA to the oligonucleotides we used the readily available aminodiol L-threoninol
(Figure 1). The amino group of L-threoninol allows the selective addition of the TA leaving the
primary and secondary alcohol functions for the preparation of DMT building blocks needed for
oligonucleotide synthesis. L-Threoninol has been extensively used to introduce all kinds of interesting
molecules containing a carboxylic acid function into oligonucleotides [43] including a thiol group
protected with the S-tert-butylsulfanyl group [44]. It was envisaged that the threoninol unit would

Molecules 2014, 19
10499
allow us to directly connect the TA molecule with the oligonucleotide chain (TA_I, Figure 1) or after a
commercially available hexaethylene glycol spacer (TA_II, Figure 1). In addition, the threoninol
linker will allow the introduction of several spacers between the threoninol molecule and the
oligonucleotide. We selected the following molecules as spacers: (i) an oligo(ethylene glycol)
molecule (TA_III(a), Figure 1); (ii) a short peptide (triglycine, TA_III(b), Figure 1) and (iii) an
aliphatic chain (TA_III(c), Figure 1).
The preparation of the appropriate solid supports carrying the TA derivatives was undertaken in
order to obtain 3'-TA oligonucleotides (TA_I, TA_II and TA_III respectively). First, the TA
threoninol derivative (compound 1, Scheme 1) was prepared. L-threoninol was reacted with TA via
amide bond formation by activating the carboxylic acid with HOBt and EDCI. Next, the primary
alcohol was protected with the 4,4'-dimethoxytrityl (DMT) group to give the compound 2 and
subsequently, reacting the secondary alcohol with succinic anhydride yielded the derivative 3.
This compound was used to functionalize the amino-controlled pore glass support (long chain amino
alkyl-controlled pore glass, LCAA-CPG) to yield the CPG solid support 4. This solid support was used
to prepare oligonucleotides labeled as TA_I (see below) and TA_II with the hexaethylene glycol
spacer using the commercially available phosphoramidite (Figure 1).
Scheme 1. Synthesis of solid support 4 and phosphoramidite 12 used for the preparation of
3'-and 5'-TA oligonucleotides TA_I, TA_II and 5'TA_I, respectively.
O
O
NH2
HN
(i)
HN
(ii)
HO
S
DMTO
1
S
2
S
HO
S
OH
OH
L-Threoninol
OH
(iii)
(v)
O
O
O
HN
HN
HN
(iv)
S
DMTO
S
S
DMTO
S
4
S
DMTO
S
O
O
O
3
12
O
P
CN
O
OH
CPG
N
O
Reagents and conditions: (i) Thioctic acid, EDCI, HOBt, DIEA, DMF; (ii) dimethoxytrityl chloride, pyr;
(iii) succinic anhydride, DMAP, pyr; LCAA CPG (long chain aminoalkyl controlled pore glass), TBTU,
TEA, ACN; (v) 2-cyanoethyl-N,N-diisopropylaminohlorophosphine, DIEA, DCM.
Next, three different TA-terminated oligonucleotides (TA_III(a), TA_III(b) and TA_III(c)),
containing different spacers such as an oligo(ethylene glycol)-like molecule, a triglycine peptide and
an alkyl group respectively were prepared. The synthesis of the solid supports 11a-11c required for the
preparation of the corresponding DNA analogues is depicted in Scheme 2. The corresponding
carboxylic acids N-Boc-N'-succinyl-4,7,10-trioxa-1,13-tridecanediamine, Boc-Gly-Gly-Gly-OH and
N-Boc-8-aminooctanoic acid were first transformed into their 4-nitrophenyl esters (5a–5c) and
subsequently reacted with L-threoninol to obtain the corresponding N-Boc-compounds 7a, 7b and 7c in
good yield (69%, 78% and 96%, respectively). Then, the Boc group was removed under acidic
conditions yielding the corresponding amines as trifluoroacetate salts, which were used in the next step

Molecules 2014, 19
10500
without further purification. Next, the reaction of the resulting free amines analogues with TA,
using the same “active-ester” methodology described before but this time using the 4-nitrophenyl ester
6 yielded compounds 8a, 8b and 8c. Finally, the primary alcohol was protected with a DMT group and
the corresponding 3'-hemisuccinate derivatives were synthesized to prepare the corresponding
functionalized solid supports 11a–11c as described above.
Scheme 2. Synthesis of the solid supports needed for the preparation of 3'-TA- terminated
oligonucleotides TA_III(a), TA_III(b) and TA_III(c).
O
O
(i)
Spacer NHBoc
5a-5c
Boc
O2N
O
Spaceerr
N
H
HO
Spacer=III(a)-III(c)
S
S
S
S
(i)
O2N
O
HO
6
O
Thioctic acid
O
S
S
O
O
H
N
Boc
Spacer
Spacer
N
HN
NH2
H
HN
(ii)
(iii), (iv)
O
HO
HO
HO
7a-7c
8a-8c
OH
OH
L-Threoninol
OH
(v)-(vii)
S
.
S
O
H
Spacer
N
HN
O
DMTO
11a-11c
O
CPG
Reagents and conditions: (i) Carboxylic acids III(a)-III(c) or thioctic acid, DCC, p-nitrophenol, pyr.;
(ii) 5a–5c, DMF; (iii) TFA 10% DCM; (iv) 6, DMF. (v) dimethoxytrityl chloride, pyr.; (vi) succinic
anhydride, DMAP, pyr.; (vii) LCAA CPG (long chain aminoalkyl controlled pore glass), TBTU, TEA, ACN.
Furthermore, as a proof of concept about the versatility of the present study, we also derivatized the
DMT-protected alcohol 2 to synthesize the corresponding phosphoramidite 12 (Scheme 1), which was
obtained in good yield (77%) after purification by flash chromatography.
2.2. Synthesis of Novel TA Terminated Oligonucleotides and Alkylthiolated Modified Oligonucleotides
Prior to the synthesis of modified oligonucleotides to functionalize AuNps, we prepared the
dodecathymidine oligonucleotide sequence in order to study their stability under synthesis and
cleavage conditions. Then we used the solid supports 4 and 11a–11c to synthesize oligonucleotides
(T12)TA_I, and (T12)TA_III(a)–(c) (5'TTT TTT TTT TTT-X3', X being the position of the TA
residue). Each oligonucleotide was treated with an ammonia solution under three different cleavage
conditions: (i) 4 h at room temperature; (ii) 1 h at 55 °C and (iii) overnight at 55 °C. The resulting
samples were analyzed by HPLC. Examples of the HPLC profiles of (T12)TA_I and (T12)TA_III(a)
synthesis crudes are depicted in Figure 2A,B. HPLC profiles of the rest of the samples are shown in
the SI (Figure S1). We observed that treatments (i) and (ii) lead to similar HPLC profiles in all cases
and the amount of side compound was increased with the prolonged treatment with ammonia at 55 °C
(treatment iii). We observed a small increase of side compounds (14% to 17%) in the case of

Molecules 2014, 19
10501
(T12)TA_I, however, with the other oligonucleotides we detected higher increases of side compounds
(25% to 41% for (T12)TA_III(a), 33% to 51% for (T12)TA_III(b) and 24% to 36% for
(T12)TA_III(c)) (Supplementary Information, Table S2). In each case, the main peaks were collected
and analyzed by mass spectrometry (Supplementary Information, Table S1). The peak with the highest
retention time (tR) corresponded with the TA-terminated oligonucleotide. In all cases we observed a
compound eluting at tR = 3.2 min. According to the mass analysis this compound was the result of a β-
elimination process for samples (T12)TA_I, (T12)TA_III(a) and (T12)TA_III(c) (Figure S1, SI). In
contrast, for (T12)TA_III(b) this same side compound resulted from the amide bond-cleavage. In addition
to the hydrolysis products we also observed small amounts of side products eluting between tR = 3.2 min
and the main peak. Mass spectrometry analyses of these side compounds (Supplementary Information,
Table S1) indicate that they may correspond to different oxidation products of the disulfide group.
Figure 2. HPLC profiles of model oligonucleotides (XBridge OST C18 column).
(A) (T12)TA_I and (B) (T12)TA_III(a) after different treatments with aqueous concentrated
ammonia. Example of HPLC profiles of purified oligonucleotides; (C) TA_III(a) and
(D) TA_III(c). ▼ stands for TA-terminated oligonucleotides and ■ stands for a β-elimination
side product.
A)
C)
2.5
2.0
1.5
1.0
0.5
0.0
r.t._ 4h
(T12)TA_I
TA_III(a)
0.8
55ºC_ 1h
55ºC_ o.n.
0.6
0.4
0.2
0.0
0
2
4
6
8
10
12
14
16
0
2
4
6
8
10
12
14
t (min)
t (min)
B)
D)
r.t._ 4h
0.5
1.0
(T12)TA_III(a)
55ºC_ 1h
TA_III(c)
0.4
0.3
0.2
0.1
0.0
55ºC_ o.n.
0.8
0.6
0.4
0.2
0.0
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
16
t (min)
t (min)
Next, we prepared the TA-oligonucleotides required for functionalized AuNPs taking some
precautions with TA_III(a)–(c) analogues. We used the dimethylformamidino group (dmf) to protect
guanine in order to minimize the formation of side compounds due to prolonged treatments with

Molecules 2014, 19
10502
ammonia. The DNA analogues TA_I, TA_II and TA_III(a)–(c) (5'CGG AGG TAC ATT CGA CTT
GAT-X3', X stands for 3'-modifications containing the TA residue) (Figure 1) were prepared using the
appropriate solid supports (Schemes 1 and 2). Finally, compound 12 incorporated into the same
sequence at the 5'-end. The coupling efficiency was >90% proving the efficiency of these
phosphoramidite (Supplementary Information, Table S3).
After HPLC analysis, all modified oligonucleotides presented a major peak with some impurities,
that were around 12%–14% for TA_I and TA_II and 25%–31% for TA_III(a)–(c). The main products
were analyzed by mass spectrometry giving the expected mass (Supplementary Information,
Table S3). For the AuNPs surface coverage studies of AuNPs a fluorescein was introduced at the 5'-
termini of the same sequence obtaining the corresponding fluorescently labeled oligonucleotides
(F)TA_I, (F)TA_II and (F)TA_III(a)–(c). All products were purified by HPLC obtaining the desired
oligonucleotide as main product (Supplementary Information, Table S3).
We also prepared two additional 3'-thiolated DNA derivatives ALK_DS and ALK_SH, (5'-CGG
AGG TAC ATT CGA CTT GA-Y-3', being Y 3'-linear alkyldisulfide or alkylthiol modifications),
which were used as controls and their corresponding fluorescently labeled versions (F)ALK_DS and
(F)ALK_SH. A commercially available solid support, containing the linear alkyldisulfide
modification (3'-Thiol modifier C3 S-S CPG; Link Technologies) was used to prepare the
oligonucleotide carrying an acyclic disulfide unit at 3'-termini (ALK_DS). The fluorescent dye was
attached at the 5'-end following the procedure described previously to obtain (F)ALK_DS. After HPLC
analysis, we observed in both cases a single peak that had the expected mass (Supplementary
Information, Table S3). Both oligonucleotides were desalted and used in the next step without further
purification. The alkylthiol oligonucleotides ALK_SH and (F)ALK_SH were obtained from the
corresponding alkyldisulfide oligonucleotides by reduction of the disulfide moiety to thiol in the
presence of TCEP·HCl following a procedure described in the literature [44,45].
2.3. Surface Coverage Quantification and Stability Tests of Oligonucleotide-Gold Nanoparticle Conjugates
In order to study the effect caused by the previously synthesized TA-terminated oligonucleotides on
the coverage and stability of gold nanoparticles, all modified oligonucleotides at their 3'-termini along
with their fluorescent labeled versions were loaded onto colloidal gold nanoparticles (9.7 nm) by a
slow increase of the NaCl concentration and sonication after each saline incremental addition to reach
a total salt concentration of 0.15 M (10 mM phosphate, pH 7.04, 0.15 M NaCl, 0.01% NaN3) [37,46].
Previously the use of higher salt concentrations (0.3 M NaCl) was described [28,29]. In our hands
these protocols are fine with regular alkylthiol group but are not suitable for oligonucleotides carrying
lipoic acid moieties as the resulting nanoparticles have a strong tendency to aggregate. Then the
number of oligonucleotides loaded on each AuNp was calculated by displacement with a 1 M DTT
solution of the corresponding fluorescently labeled oligonucleotides [29,47,48]. By measuring the
concentration of oligonucleotides and gold nanoparticle conjugates of each sample, an average number
of oligonucleotides per particle was calculated. Then, the average area occupied by an oligonucleotide
(footprint) was calculated by dividing the calculated surface area (nm2) by the average number of
oligonucleotides per particle. Results are summarized in Table 1.

Molecules 2014, 19
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Table 1. Surface coverage and half-lives data for oligonucleotide-gold nanoparticle conjugates.
Conjugate
Strands/Particle Footprint (nm2) t1/2 (min)
AuNP-TA_I
94 ± 7
93 ± 13
141 ± 10
103 ± 13
102 ± 12
40 ± 2
3.2 ± 0.2
3.2 ± 0.4
2.1 ± 0.3
2.9 ± 0.4
2.8 ± 0.3
7.4 ± 0.1
3.0 ± 0.2
344 ± 12
282 ± 11
444 ± 8
354 ± 10
450 ± 15
4 ± 1
AuNP-TA_II
AuNP-TA_III(a)
AuNP-TA_III(b)
AuNP-TA_III(c)
AuNP-ALK_DS
AuNP_ALK_SH
97 ± 7
12 ± 2
We found similar surface coverage results with AuNP-TA_I without any spacer (94 strands/particle)
and our control ALK_SH (97 strands/particle). However, the functionalization was less efficient for
AuNP-ALK_DS (40 strands/particle). A possible explanation of this result could be the larger
anchoring group present in ALK_DS oligonucleotide compared to ALK_SH. Then, the ability of the
dialkyldisulfide group to form dense layers would be lower than the alkylthiol group. In addition, it is
described that the adsorption of a dialkyldisulfide is possible, but thermodinamically not stable [49].
The process implies the cleavage of the disulfide bond, which generates two monoalkylthiolated
branches, that do not adsorb adjacent to each other [50]. Maybe the monolayer obtained in this case is
not so uniform compared with the monolayer formed with ALK_SH oligonucleotide in terms of
thiolated species adsorbed onto the surface explaining the reduction in the coverage efficiency.
We observed different trends depending on the linker nature and how it has been incorporated.
AuNP-TA_I and AuNP-TA_II showed similar surface coverage results. In this case, the introduction
of a hexaethylene glycol spacer did not lead to a higher coverage. Maybe, the threoninol and the
phosphate groups, which are placed very close to the surface, are preventing to achieve a higher
loading due to steric and electrostatic effects. In the case of the TA_III series, a slight increase in the
ligand density was observed when a spacer similar in length (alkyl or a triglycine peptide) was
introduced. A higher density of ligand was observed when an oligo(ethylene glycol) was introduced
between the TA group and the oligonucleotide (141 strands/particle), that is an increase of around 50%
on DNA loading compared with AuNP-TA_I conjugates. This result indicates the beneficial effect of
the spacer and is in good agreement with other results published in the literature [37], where thiolated
oligonucleotides with and without an oligo(ethylene glycol) spacer were used to functionalize AuNP.
The authors reasoned that the spacer moves the DNA tract away from the gold surface thus reducing
the interstrand repulsion between negatively charged phosphate/sugar backbones and as well the
tendency of DNA nucleobases to interact with the gold surface. An important characteristic to take into
account regarding monolayers formed on the surface of AuNPs is the radius of curvature.
A consequence of this curvature is a decrease in the chain density moving away from the surface of the
core. The decreasing density translates into an enhanced mobility of the terminal tracts of a ligand
chain [51]. For the TA-terminated DNA oligonucleotides the decreasing density seems to minimize the
interstrand repulsions of the negatively charged tract. It could be a plausible explanation for the higher
density of packing obtained with all three AuNp-TA_III conjugates.
Next, we studied the stability of our AuNP-conjugates against ligand exchange in the presence of
DTT. At high concentration, DTT can displace thiolated ligands, resulting in progressive aggregation

Molecules 2014, 19
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of the AuNPs. Then, the ability of the spacers to provide additional shielding of the inorganic core was
evaluated by assessing the aggregate formation in the presence of DTT (100 mM) at 40 °C. Figure 3A
shows the UV-Vis spectra taken at different times for AuNP-TA_I. During time evolution of the UV-Vis
spectra, a decay of the plasmon peak of colloidal gold (522 nm) was observed, while the intensity
between 600 and 700 nm increased. Then, the displacement of oligonucleotides from AuNPs was
monitored by measuring the changes in signal intensity at 675 nm with time (Figure 3B). The time required
to reach half of the maximum intensity (t1/2) was used to compare the stability of each AuNP-conjugate.
Table 1 summarizes the results obtained in each case. Comparing the conjugates formed with the TA series
with the conjugates formed with oligonucleotides containing an alkylthiol or an acyclic alkyldisulfide a
great difference in the stability was observed. The complete aggregation occurred at 15 min and 30 min for
AuNp-ALK_DS and AuNp_ALK_SH respectively. This is in good agreement with other results
published in the literature [22,29,52], where oligonucleotides were modified with cyclic disulfides to
functionalize AuNPs. In these cases a large increase in the stability was observed in comparison with
AuNPs functionalized with monoalkylthiol- or acyclic disulfide-oligonucleotides, suggesting that those
differences might be due to different binding affinities of the different thiolated groups to gold surfaces.
Figure 3. (A) Time evolution of UV-Vis spectra of AuNp-TA_I in 100 mM DTT;
(B) Displacement of modified TA-appended oligonucleotides from AuNPs by measuring the
changes in signal intensity at 675 nm with time upon treatment with a 100 mM DTT solution.
B)
A)
1.0
0.8
0.6
0.4
0.2
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 min.
AuNp-TA_I
66 min. (1h 6 min.)
130 min. (2h 10 min.)
197 min. (3h 17 min.)
261 min. (4h 21 min.)
330 min. (5h 30 min.)
396 min. (6h 36 min.)
463 min. (7h 43 min.)
530 min. (8h 50 min.)
AuNp-TA_I
AuNp-TA_II
AuNp-TA_III(a)
AuNp-TA_III(b)
AuNp-TA_III(c)
AuNp-ALK_SH
AuNp-ALK_DS
0
100
200
300
400
500
600
700
400
500
600
700
800
t (minutes)
t (min)
(nm)
1.0
0.8
0.6
0.4
0.2
TA analogues
ALK_DS
ALK_SH
0
10
20
t (minutes)
30
40
t (min)
Focusing on the results obtained with conjugates formed with TA-terminated oligonucleotides, we
found that conjugate formed with TA_I (without a spacer) showed a high stabilization in the presence
of DTT (t1/2 344 min.). The conjugates formed with TA_III(a) and TA_III(c), (containing an

Molecules 2014, 19
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oligo(ethylene glycol)-like molecule and an alkyl group as spacer respectively) were very similar and
showed a clear increase of about 100 min in t1/2 compared with conjugates formed with TA_I. On the
other hand, when the spacer was a triglycine peptide (TA_III(b)), the t1/2 value obtained was very
similar to conjugates formed with TA_I. Finally, the conjugates formed with TA_II containing
hexaethylene glycol as a spacer) showed a clear decrease (62 min) in t1/2 when compared with TA_I.
The replacement rate of thiolated ligands depends on a number of parameters such as the packing
density, the order degree or the intermolecular interactions between the ligands [53]. In our case, a
high packing density not necessarily provides an enhanced stability towards thiol replacement.
Interestingly, AuNP-TA_I and AuNP_TA_III(b) showed comparable t1/2 values whereas AuNP-TA_I
resulted to be more stable than AuNP-TA_II. The results obtained could be rationalized as follows:
the stability of the AuNP conjugates functionalized with TA-terminated oligonucleotides towards thiol
replacement is driven by the thermodynamic stability of the anchoring group and by the shielding that
the peripheral groups provide to the gold core. In the case of AuNP-TA_I the negatively charged and
bulky oligonucleotide chain lays very close to the cluster surface, thus providing steric hindrance to the
approaching thiol. When a spacer is added, the oligonucleotide chain is moved further away, then
reducing the interstrand repulsions due to the radial distribution of the molecules attached to AuNPs.
Then the shielding efficiency towards ligand exchange relies on the packing quality of the spacer.
A characteristic of AuNP cores is that they have faceted surfaces, which generate a radial structure
for the covering monolayer [51]. These authors functionalized AuNPs with different alkylthiols
containing an amide group at different positions. They found that the interactions between amide groups
were highly dependent on the distance to the AuNP core [54]. In light of our results, AuNp-TA_III(c)
and AuNp-TA_III(a) conjugates gave the highest rates of half-lives compared to the rest of the series,
showing practically the same stability pattern. It can be reasoned that the amide bond could play an
important role in this higher stability by providing an increase in monolayer order through hydrogen
bonding interactions that leads t1/2 values to enhanced interactions between the neighboring alkyl or
ethylene glycol chains (Figure 4). In the case of the AuNP-TA_III(b) conjugate, we observed similar
t1/2 values compared with AuNP-TA_I. The effect on the stability of this spacer could be explained as
the ability of amide groups to participate in hydrogen-bonding interactions is decreased when the
distance to the surface increases. Finally, the lower stability observed with the AuNP-TA_II could be
attributed to the bulky groups near the amide bond (threoninol group and phosphate linkage) (Figure 4).
These groups may prevent effective hydrogen bonding interactions due to steric and electrostatic
factors that could affect the packing quality of the spacer leading to a decrease in the interactions
between the oligo(ethylene glycol) chains.
2.4. Stability of the Thioctic Acid Derivatives to Cathepsin
AuNPs are potential vehicles for drug delivery [55]. One of the potential applications of the
oligonucleotide-AuNPs described in this work is their use in gene silencing. In addition of a dense and
stable functionalization of the nanoparticles, it will be important to be able to break the linker between
oligonucleotide and nanoparticle to release the cargo inside the cells. It is reported that the triglycine
linker has been used for the endosomal escape of several drugs such as DOTA, doxorubicin and

Molecules 2014, 19
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T-2513 [33–35] because of the lability to cathepsin. Then, we decided to analyze if the same lability
was present in the TA-oligonucleotides described in this work.
Figure 4. Scheme showing (A) favourable interactions (in green) between amide groups and
(B) disfavourable interactions (in red) between bulky or negatively charged substituents.
(A)
(B)
O
O
O
O
O
OH
O
OH
O
OH
HN
HN
O
HN
O
O
O
O
O
O
O
P
P
O
O
O
O
P
OH
O
OH
O
O
OH
HN
HN
HN
HN
O
O
HN
O
O
HN
O
S
S
S
S
S
S
S
S
S
S
S
S
Our study was focused in the determination the specificity of cathepsin B for the triglycine TA
spacer (TA_III) versus the other TA modified oligonucleotides (TA_I and TA_II). The TA
conjugates were incubated with Cathepsin B for 72 h and analyzed by HPLC to elucidate the
enzymatic response (Figures 5A and Supplementary Information, S5). The HPLC profiles for the
TA_III conjugates showed the appearance of a peak with shorter retention time, which corresponded
to the cleavage product once analyzed by mass spectrometry (Figure 5A blue line, Supplementary
Information, Table S4), whereas in the case of the TA_I and TA_II this peak was absent
(Supplementary Information, Figure S5). These results confirmed that cathepsin B was able to
cleavage the amide bonds oligonucleotides from the TA_III conjugates, however it was unable to
liberate the oligonucleotide from TA_I and TA_II conjugates. That indicates that the activity of the
enzyme is affected by the topology around the amide bond. To consolidate these results we
investigated the effect of the proteinase over time with the TA_III(b). Figure 5B shows the increase of
product release over time until a plateau was reached at 72 h as expected. Moreover, we could observe
a different cleavage efficiency of cathepsin B between the three spacers present in the TA_III series
(Figure 5C). We obtained the higher cleavage efficiency with the triglycine spacer (25%) followed by
the alkyl chain and the lower cleavage was for the oligo(ethylene glycol) molecule. These results are
consistent with the fact that the triglycine spacer has a peptide-like nature compared with the other
two linkers.

Molecules 2014, 19
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Figure 5. (A) HPLC profiles obtained with TA_III(b) after 72 h in the presence (blue line)
and in the absence (black line) of cathepsin B (CTB). ▼ stands for TA_III(b) and ■ stands for
the product resulting from the amide-bond cleavage; (B) % amide-bond cleavage of TA_III(b)
with time; (C) % amide-bond cleavage after 72 h of different TA-terminated oligonucleotides.
A)
0.20
TA_III(b)
Cathepsin B
0.15
0.10
0.05
0.00
TA_III(b) + CTB
O
O
H
N
H
N
S
S
HN
N
H
O
O
O
OH
0
5
10
15
20
25
t (min)
C)
B)
40
20
0
25
20
15
10
5
0
0
10
20
30
40
500
6600
7700
8800
TA_III(b) TA_III(c) TA_III(a)
TA_II
TA_I
time (h)
3. Experimental Section
General Methods and Materials
All reagents were purchased from Sigma-Aldrich Spain (Tres cantos, Spain) or Fluka (Sigma-
Aldrich Química S.A., Spain) and were used without further purification. Dry solvents were purchased
as well from Sigma-Aldrich or Fluka and used as supplied. All reactions were carried out under inert
atmosphere of argon. All the standard phosphoramidites and ancillary reagents used for
oligonucleotide synthesis were purchased from Applied Biosystems (Foster City, CA, USA) or Link
Technologies (Glasgow, Scotland, UK). The benzoyl (Bz) group was used for the protection of the
amino group of C and A. For the protection of G we used either the isobutyryl (iBu) group in the case
of TA_I and TA_II and the dimethylformamidino group (dmf) in the case of TA_III(a)–(c). The
hexaethylene glycol phosphoramidite (spacer phosphoramidite C18) was purchased from Glen
Research (Sterling, VA, USA). The 6'-Fluorescein-CE-phosphoramidite and the 3'-thiol modifier C3 S-
S CPG were purchased from Link Technologies. The oligonucleotides were desalted with Sephadex G-
25 (NAP-10 or NAP-5 column, from GE Healthcare Spain (Barcelona, Spain). Cathepsin B was
purchased from Sigma-Aldrich. Aqueous solution of citrate stabilized gold nanoparticles (9.7 nm)
were purchased from BBI Life Sciences (Cardiff, UK) and used as received. Flash column

Molecules 2014, 19
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1
chromatography was carried out on silica gel SDS 0.063–0.2 mm/70–230 mesh. H- and 13C-NMR
spectra were recorded at 25 °C on a Varian Mercury 400 MHz spectrometer (Agilent Tehcnologies,
31
Santa Clara, CA, USA). P-NMR spectra were recorded at room temperature on a Varian Mercury
400 MHz, operating at 162 MHz, using an internal reference using deuterated solvents.
Tetramethylsilane (TMS) was used as an internal reference (0 ppm) for 1H spectra recorded in CDCl3
and the residual signal of the solvent (77.16 ppm) for 13C spectra. For CD3OD or d6-DMSO the
residual signal of the solvent was used as a reference. Chemical shifts are reported in part per million
(ppm) in the δ scale, coupling constants (J) in Hz and multiplicity as follows: s (singlet), d (doublet), t
(triplet), q (quadruplet), quint (quintuplet), m (multiplet) and br (broad signal). IR Spectra were
recorded on a BOMEM MB-120 spectrometer (ABB, Frankfurt am Main, Germany). HPLC analysis
and separations were performed using a Waters 2695 Separations Module (Milford, MA, USA) equipped
with a Waters 2998 Photodiode Array Detector. The HPLC solvent used where: A: 5% ACN in 100 mM
triethylammonium acetate (TEAA) (pH = 7) and solvent B: 70% ACN in 100 mM TEAA (pH = 7).
Oligonucleotides synthesis was done in a DNA/RNA synthesizer (Applied Biosystems, Foster City,
CA, USA) and characterized by mass spectrometry (MALDI-TOF). UV analyses were performed
using a Jasco V-650 instrument (Easton, MD, USA) equipped with a thermoregulated cell holder.
Fluorescent measures were recorded on a Jasco FP-6200 spectrofluorometer equipped as well with a
thermoregulated cell holder. Electrospray ionization mass spectra (ESI-MS) were recorded on a
Micromass ZQ instrument (Milford, MA, USA) with single quadrupole detector coupled to an HPLC,
and high-resolution (HR) ESI-MS on an Agilent 1100 LC/MS-TOF instrument (Santa Clara, CA, USA).
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra were recorded on
a Voyager-DETMRP spectrometer (Applied Biosystems) in negative mode (2,4,6-trihidroxyacetophenone
matrix with ammonium citrate as an additive).
3.1. 4-(Dithiolan-3-yl)-N-[(1R,2R)-2-hydroxy-1-(hydroxymethyl)propyl]butanamide (1)
The compound was obtained as a mixture of two diastereoisomers. A solution of (±)-α-lipoic acid
(344 mg, 1.67 mmol), (2R,3R)-2-aminobutane-1,3-diol (175 mg, 1.67 mmol), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI, 447 mg, 2.50 mmol), hydroxybenzotriazole (HOBt,
339 mg; 2.50 mmol) and N,N-diisopropylethylamine (DIEA, 433 µL, 2.50 mmol) in anhydrous
dimethylformamide (DMF, 15 mL) was prepared under argon. After stirring the reaction mixture at
room temperature overnight, the reaction was complete as judged by TLC and then concentrated to
dryness under reduced pressure. In order to remove the remaining DMF the residue was dissolved in
toluene and concentrated to dryness under reduced pressure (×3). The residue was dissolved in 100 mL
of dichloromethane (DCM) and the organic phase was washed with 50 mL 10% NaHCO3 aqueous
solution (×2) and 50 mL of a saturated NaCl aqueous solution. The organic layer was dried over
anhydrous magnesium sulfate and concentrated to dryness under reduced pressure. The product was
purified by flash chromatography using as eluent pure DCM to DCM with 4% MeOH to give
1
compound 1 (371 mg, 76% yield). IR (film): 3320, 2927, 1643, 1539 cm−1. H-NMR (400 MHz,
CDCl3): δH = 6.21 (br, 1H, NH), 4.19 (qd, J = 6.3 Hz, 1.57 Hz; 1H, OH-CH-CH3), 3.84 (m, 2H,
CH-CH2-OH), 3.57 (quint, J = 6.5 Hz, 1H, N-CH-CH2), 3.14 (m, 2H, S-CH2-CH2-), 2.46 (m, 1H,
S-CH-CH2-), 2.28 (t, J = 7.4 Hz, 2H, CO-CH2-), 1.92 (m, 2H, -CH2-CH2-), 1.70 (m, 4H, -CH2-CH2-),

Molecules 2014, 19
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1.49 (m, 2H, CH-CH2-CH2-), 1.21 (d, J = 7.1 Hz, 3H, CH3-CH) ppm.13C-NMR (125 MHz, CDCl3): δC
= 173.92 (NH-CO-), 69.41 (CH-O-), 65.62 (CH2-O), 56.61 (N-CH-CH2-), 54.63 (CH2-CO), 40.47
(CH2-S), 38.70 (CH2-CH-S), 36.71, 34.82, 29.01, 25.63 (alkyl chain), 20.94 (CH3-CH) ppm. HRMS
(ESI+): m/z calcd for C12H24NO3S2 ([M + H]+) 294.1199 found 294.1192; m/z calcd for
C24H46N2NaO6S4 ([2M + Na]+) 609.2138 found 609.2131.
3.2. General Procedure for the Synthesis of Amides 7a–7c
The corresponding carboxylic acid (2.11 mmol) along with 4-nitrophenol (353 mg, 2.54 mmol)
were dissolved in anhydrous pyridine (15 mL) under argon and the solution was cooled at 0 °C.
Then, 524 mg (2.54 mmol) of 1,3-dicyclohexylcarbodiimide (DCC) were added under argon and the
reaction was stirred at 0 °C for 30 min and at room temperature overnight. The precipitate was filtered
out and the solvent was removed under reduced pressure. The residue was dissolved in toluene and
concentrated to dryness under reduced pressure (×3). The resulting p-nitrophenyl esters 5a–5c were
used in the next step without further purification. Then, the activated acids along with (2R,3R)-2-
aminobutane-1,3-diol (L-threoninol) (244 mg, 2.32 mmol) were dissolved in anhydrous DMF (15 mL)
under argon and allowed to react at room temperature for 3 h. The solvent was removed under reduced
pressure and the residue was dissolved in toluene and concentrated to dryness (×3). Crude compounds
7a and 7c were dissolved in 100 mL of DCM and the organic phase was washed with 50 mL 10%
NaHCO3 aqueous solution (×2) and 50 mL of a saturated NaCl aqueous solution. The organic layers
were dried with anhydrous MgSO4 and the corresponding crudes were purified by flash
chromatography on silica gel using as solvent pure DCM to DCM with 4% MeOH. Crude of
compound 7b once dried with toluene was directly purified by flash chromatography on silica gel
(crude adsorbed onto the silica, DCM/MeOH 100:5→100:15).
3.2.1. tert-Butyl-N-[3-[2-[2-[3-[[4-[[(1R,2R)-2-hydroxy-1-(hydroxymethyl)propyl]amino]-4-oxo-
butanoyl]amino]propoxy]ethoxy]ethoxy]propyl] carbamate (7a)
1
Yield: 738 mg (69%). IR (film): 3325, 2930, 2871, 1690, 1654, 1365, 1170, 1103 cm−1. H-NMR
(400 MHz, CDCl3): δH= 6.75 (br, 1H, NH), 5.07 (br, 1H, OH), 4.14 (dq, J = 6.3 Hz, 2.5 Hz; 1H,
OH-CH-CH3), 3.84 (dd, J = 11.5 Hz, 3.5 Hz, 1H, CH-CH-OH), 3.75 (m, 2H, CH-CH-OH), 3.65
(m, 4H, O-CH2-CH2-O), 3.59 (m, 4H, O-CH2-CH2-O), 3.55–3.47 (m, 4H, CH2-CH2-O and O-CH2-CH2),
3.35 (m, 2H, NH-CH2-), 3.21 (m, 2H, NH-CH2), 2.61–2.54 (m, 4H, NH-CH2-CH2-CH2-O), 1.75 (2t,
4H, CO-CH2-CH2-CO), 1.43 (s, 9H, (CH3)3-C), 1.19 (d, J = 6.4 Hz, 3H, CH3-CH) ppm.13C-NMR
(125 MHz, CDCl3): δC = 173.54 (CO), 172.72 (CO), 172.62 (CO), 77.43 (CH3)3-C-O), 70.62
(O-CH2-CH2-O), 70.30 (O-CH2-CH2-O), 70.29 (O-CH2-CH2-O), 69.63 (O-CH2-CH2-O), 69.59
(CH2-O), 69.49 (CH-O-), 64.93 (CH2-O), 64.86 (CH2-O), 55.63 (N-CH-CH2), 55.62 (CH2-N), 51.03
(CH2-N), 32.44 (CH2-CO), 32.16 (CH2-CO), 29.93 (CH2-CH2), 28.98 (CH2-CH2), 28.62 (CH3-C),
20.57 (CH3-CH) ppm. HRMS (ESI+): m/z calcd for C23H46N3O9 ([M + H]+) 508.3229 found 508.3231;
m/z calcd for C23H45N3NaO9 ([M + Na]+) 530.3048 found 530.3050.

Molecules 2014, 19
10510
3.2.2. N-[N'-(tert-Butoxycarbonyl)glycylglycylglycyl]-L-threoninol (7b)
1
Yield: 612 mg (78%). H-NMR (400 MHz, DMSO-d6): δH = 8.09–8.03 (m, 2H, NH), 7.27 (d,
J = 8.8 Hz, 1H, NH), 6.97 (t, J = 5.6 Hz, 1H, NH), 4.61–4.54 (br, 2H, OH), 3.85–3.81 (m, 1H,
OH-CH-CH3), 3.72–3.70 (m, 4H, NH-CH2-CO), 3.63–3.58 (m, 2H, NH-CH-CH2), 3.56 (d, J=6.0 Hz, 2H,
NH-CH2-CO), 3.44–3.40 (m, 1H, OH-CH(A)H(B)-CH)), 3.33–3.28 (m, 1H, OH-CH(A)H(B)-CH)),1.36 (s,
9H, (CH3)3-C), 0.97 (d, J = 6.4 Hz, 3H, CH3-CH-CH) ppm.13C-NMR (125 MHz, DMSO-d6):
δC = 170.58 (NH-CO), 169.67 (NH-CO), 169.37 (NH-CO), 156.50 (O-CO-NH), 78.83 (CH3-C), 64.88
(CH3-CH), 61.12 (OH-CH2-CH), 56.43 (NH-CH-CH), 43.99 (NH-CH2-CO), 42.77 (NH-CH2-CO), 42.70
(NH-CH2-CO), 28.87 (CH3-C), 20.73 (CH3-CH). HRMS (ESI+): m/z: calcd. for C15H29N4O7([M + H]+)
377.2036 found 377.2031; m/z: calcd. for C15H28N4NaO7 ([M + Na]+) 399.1856 found 399.1856.
3.2.3. tert-Butyl-N-[8-[[(1R,2R)-2-hydroxy-1-(hydroxymethyl)propyl]amino]-8-oxo-octyl]carbamate (7c)
Yield: 701 mg (96%). IR (film): 3323, 2974, 2930, 2858, 1689, 1652, 1525, 1366, 1251, 1171 cm−1.
1H-NMR (400 MHz, CDCl3): δH = 6.30 (br, 1H, NH), 4.56 (br, 1H, OH), 4.19 (qd, J = 6.4 Hz, 1.9 Hz,
1H, OH-CH-CH3), 3.83 (m, 3H, CH-CH2-OH), 3.07 (m, 2H, CH2-NH), 2.26 (t, J = 7.3 Hz, 2H, CH2-CH2),
1.66 (m, 2H, alkyl chain), 1.44 (s, 9H, (CH3)3-C), 1.33 (m, 6H, alkyl chain), 1.21 (d, J = 6.3 Hz, 3H,
CH3-CH) ppm. 13C-NMR (125 MHz, CDCl3): δC = 174.31 (CO-NH), 77.43 (CH3-C-O), 69.43 (CH-O),
65.62 (CH2-O), 54.61 (N-CH-CH2), 40.63 (CH2-CO), 36.86, 29.94, 29.04, 28.62, 26.54, 25.57
(alkyl chain), 20.82 (CH3-CH-) ppm. HRMS (ESI+): m/z calcd for C17H35N2O5 ([M + H]+) 347.2540
found 347.2543; m/z calcd for C34H68N4NaO10 ([2M + Na]+) 715.4828 found 715.4824.
3.3. General Procedure for the Introduction of Lipoic Acid Residue: Synthesis of Compounds 8a–8c
Using the same experimental procedure as described above for the activation of carboxylic acids, a
solution of (±)-α-lipoic acid (322 mg, 1.56 mmol) and p-nitrophenol (239 mg, 1.72 mmol) in
anhydrous pyridine (15 mL) was prepared under argon. Then, the solution was cooled to 0 °C and
DCC (354 mg, 1.72 mmol) was added under argon. The reaction was stirred at 0 °C for 30 min and at
room temperature overnight. The precipitate was filtered out and the solvent was removed under
reduced pressure. The residue was dissolved in toluene and concentrated to dryness under reduced
pressure (×3). Then the crude was dissolved in DCM (75 mL) and the organic phase was washed with
5% aqueous NaHCO3 (2 × 30 mL) and saturated aqueous NaCl (30 mL). The organic phase was dried
(MgSO4) and concentrated to dryness under reduced pressure and then dried in a desiccator.
The p-nitrophenyl ester 6 (yellow solid) was used in the next step without further purification. Then,
the corresponding protected amines 7a–7c (1.30 mmol) were subjected to acidic conditions
(DCM/TFA 10%) in order to obtain the respective free amines as trifluoroacetate salts. Each protected
amine was dissolved in DCM (20mL) and TFA (2 mL) was added slowly at room temperature. Stirring
was continued until TLC showed the consumption of all starting material (usually 1–1.5 h), then the
mixture was concentrated to dryness under reduced pressure. The residue was dissolved in toluene and
concentrated to dryness under reduced pressure. This operation was repeated three times to remove
traces of TFA to yield the TFA salt of the deprotected amino compound (colorless oil). The residue
was dried in vacuo and used in the next step without further purification. Then, each deprotected amino

Molecules 2014, 19
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compound was dissolved in anhydrous DMF (10 mL) and anhydrous DIEA (0.94 mL, 5.20 mmol) was
added under argon. The solution was added dropwise under argon to a prepared solution (anhydrous
DMF, 10 mL) of the activated lipoic acid derivative. The mixture was allowed to react at room
temperature and under argon overnight. The solvent was removed under reduced pressure and the residue
was dissolved in toluene (3 × 10 mL) and concentrated to dryness. Crudes of compounds 8a and 8c were
dissolved into 75 mL of dichloromethane and were washed with 5% NaHCO3 (3 × 30 mL) and with a
saturated aqueous solution NaCl (30 mL). The organic layers were dried with anhydrous MgSO4 and
the corresponding crudes were purified by flash chromatography on silica gel using as solvent pure
DCM to DCM with 4% MeOH. Crude of 8b once dried with toluene was directly purified by flash
chromatography on silica gel (crude adsorbed onto the silica, DCM/MeOH 100:5→100:15).
3.3.1. N'-[3-[2-[2-[3-[4-(Dithiolan-3-yl)butanoylamino]propoxy]ethoxy]ethoxy]propyl]-N-[(1R,2R)-2-
hydroxy-1-(hydroxymethyl)propyl]butanediamide (8a)
The compound was obtained as a mixture of two diastereoisomers. Yield: 565 mg (73%). IR (film):
3320, 2928, 1652, 1539, 1456, 1216, 1130 cm−1. 1H-NMR (400 MHz, CDCl3): δH = 6.73 (br, 1H, NH),
6.65 (br, 1H, OH), 6.43 (br, 1H), 4.16 (dq, J = 6.3 Hz, 2.6 Hz, 1H, OH-CH-CH3), 3.88 (dd, J = 11.3 Hz,
3.4 Hz, 1H, CH-CH-OH), 3.75 (m, 2H, CH-CH-OH), 3.65 (m, 4H, O-CH2-CH2-O), 3.61 (m, 4H,
O-CH2-CH2-O), 3.55 (m, 4H, CH2-CH2-O and O-CH2-CH2), 3.35 (m, 4H, 2 NH-CH2-), 3.18 (m, 2H,
S-CH2-CH2), 3.14 (m, 2H, CH2-NHCO), 2.64 (m, 1H, S-CH-CH2), 2.49 (m, 4H, NH-CH2-CH2-CH2-O),
2.17 (t, J = 7.6 Hz, 2H, CH2-CH2-), 1.74 (m, 8H, CO-CH2-CH2-CO and alkyl residue), 1.44 (d, J = 6.9 Hz,
3H, CH3-CH), 1.38 (d, J = 6.7 Hz, 3H, CH3-CH) ppm.13C-NMR (125 MHz, CDCl3): δC = 173.72
(CO), 173.35 (CO), 172.51 (CO), 70.73 (O-CH2-CH2-O), 70.62 (O-CH2-CH2-O), 70.20 (O-CH2-CH2-O),
70.19 (O-CH2-CH2-O), 70.18 (CH2-O), 70.08 (CH2-O), 68.89 (CH-O), 64.54 (CH2-O), 56.75 (CH2-CO),
55.71 (CH2-CO), 41.86 (CH2-CH-N), 40.43 (CH2-S), 38.62 (CH2-N), 38.16 (CH2-N), 37.82 (CH2-CH-S),
36.67 (CH2-CO), 34.88 (S-CH2-CH2-CH-S) 37.85 (S-CH-CH2), 36.63 (-CH2-CH2-), 34.85 (-CH2-CH2-),
32.31 (NH-CH2-CH2-), 32.21 (-CH2-CH2-CH2-O), 32.09, 29.34, 29.05 (CH3-CH) ppm. HRMS (ESI+):
m/z calcd for C26H50N3O8S2 ([M + H]+) 596.3034, found 596.3038.
3.3.2. N-[N'-{5-(1,2-Dithiolan-3-yl)pentanoyl}glycylglycylglycyl]-L-threoninol (8b)
The compound was obtained as a mixture of two diastereoisomers. Yield: 459 mg (76%). 1H-NMR
(400 MHz, CD3OD): δH = 3.91–3.86 (m, 1H, OH-CH-CH3), 3.82–3.81 (m, 2H, NH-CH2-CO), 3.79 (d,
J = 2.8 Hz, 4H, NH-CH2-CO), 3.74–3.70 (m, 1H, NH-CH-CH2), 3.58–3.53 (m, 1H, OH-CH(A)H(B)-CH),
3.51–3.46 (m, 1H, OH-CH(A)H(B)-CH), 3.13–2.97 (m, 3H, S-CH2-CH2 and S-CH-CH2), 2.41–2.33 (m,
1H, S-CH-CH(A)H(B)-CH2), 2.20 (t, J = 7.4 Hz, 2H, CH2-CH2-CO-NH), 1.84–1.76 (m, 1H,
S-CH-CH(A)H(B)-CH2), 1.68–1.50 (m, 4H, alkyl chain), 1.42–1.35 (m, 2H, alkyl chain), 1.05 and 1.04
13
(2d, J = 6.4 Hz and J = 6.4 Hz, 3H, CH3-CH) ppm. C-NMR (125 MHz, DMSO-d6): δC = 173.30
(NH-CO), 170.36 (NH-CO), 169.70 (NH-CO), 169.41 (NH-CO), 64.91 (CH3-CH), 61.13 (OH-CH2-CH),
56.79 (NH-CH-CH2-), 56.45 (S-CH-CH2), 46.40 (S-CH2-CH2), 43.96 (NH-CH2-CO), 42.74 (NH-CH2-
CO), 42.72 (NH-CH2-CO), 38.77 (S-CH-CH2), 35.63 (CH2-CH2-CO-NH), 34.81 (CH2 alkyl chain),
29.01 (CH2alkyl chain), 25.5 (CH2 alkyl chain), 20.72 (CH3-CH) ppm. HRMS (ESI+): m/z calcd for

Molecules 2014, 19
10512
C18H33N4O6S2 ([M + H]+) 465.1842, found 465.1848; m/z calcd for C18H32N4NaO6S2 ([M + Na]+)
487.1661 found 487.1658.
3.3.3. 8-[4-(Dithiolan-3-yl)butanoylamino]-N-[(1R,2R)-2-hydroxy-1-(hydroxymethyl)propyl]octan-
amide (8c)
The compound was obtained as a mixture of two diastereoisomers. Yield: 231 mg (41%). IR (film):
1
3304, 2927, 2855, 1646, 1557, 1540, 1457, 1201 cm−1. H-NMR (400 MHz, CDCl3): δH = 6.37 (br,
1H, NH), 5.59 (br, 1H; OH), 4.18 (qd, J = 6.2 Hz, 1.5 Hz, 1H, OH-CH-CH3) 3.84 (m, 2H, CH-CH2-OH),
3.57 (m, 1H,CH-CH2-OH), 3.23 (m, 2H, S-CH2-CH2-), 3.13 (m, 2H, CH2-NHCO), 2.47 (m, 1H,
S-CH-CH2-), 2.27 (d, J = 7.3 Hz, 2H, -CH2-CH2, lipoic residue), 2.17 (t, J = 7.0 Hz, 2H, -CH2-CH2-),
1.91 (m, 2H, -CH2-CH2-), 1.67 (m, 6H, alkyl chain), 1.48 (m, 2H, alkyl chain), 1.33 (m, 8H, alkyl chain),
1.21 (d, J = 6.4 Hz, 3H, CH3-CH) ppm.13C-NMR (125 MHz, CDCl3): δC = 174.45 (CONH), 173.44
(CONH), 69.69 (CH-O-), 65.72 (CH2-O), 56.62 (NH-CH-CH2-), 54.65 (NH-CH2), 45.85 (CH2-CO),
40.47 (CH2-CO), 39.55 (CH2-S), 38.72 (CH2-CH-S), 36.81, 34.72, 29.42, 29.07, 28.76, 28.40, 28.39,
26.37, 25.72 (alkyl chain), 20.72 (CH3-CH) ppm. HRMS (ESI+): m/z calcd for C20H39N2O4S2 ([M + H]+)
435.2346, found 435.2351; m/z calcd for C40H76N4NaO8S4 ([2M + Na]+) 869.4619 found 869.4615.
3.4. General Procedure for the Preparation of the DMT Protected Compounds 2 and 9a–9c
Each diol (0.80 mmol) was dried by evaporation of anhydrous acetonitrile (ACN) under reduced
pressure (×2). Then to a solution of the corresponding diol in anhydrous pyridine (15 mL) at 0 °C,
DIEA (217 µL, 1.20 mmol), 4,4'-dimethoxyltritylchloride (DMT-Cl) (297 mg, 0.88 mmol) and
dimethylaminopyridine (DMAP) (catalytic amount) were added. After 15 min, the mixture was
allowed to reach room temperature. Then the reaction was stirred for an additional 24 h and finally was
quenched with methanol except for diol 7b. In this case DMT-Cl (135 mg, 0.40 mmol), DIEA (109 µL
0.60 mmol) and DMAP (catalytic amount) were dissolved in dry pyridine (5 mL) and were added to
the mixture. The solution was stirred at room temperature for 6 h and quenched with methanol.
The solvent was evaporated under reduced pressure. The residue was dissolved in toluene (3 × 10 mL)
and concentrated to dryness. Then, the resulting crude was dissolved in dichloromethane (100 mL) and
the organic phase was washed with 5% aqueous NaHCO3 (50 mL) and with saturated aqueous NaCl
(50 mL). The solvent was evaporated and the residue was purified by flash chromatography on silica gel.
The column was packed using a 1% triethylamine (TEA) solution in DCM. Compounds 2, 9a and 9c
were eluted with a gradient of MeOH from 0% to 4% in DCM and compound 9b was eluted with a
gradient of MeOH from 0% to 10% in DCM.
3.4.1. N-[(1R,2R)-1-[[bis(4-Methoxyphenyl)phenylmethoxy]methyl]-2-hydroxypropyl]-5-(dithiolan-3-
yl)pentanamide (2)
The compound was obtained as a mixture of two diastereoisomers. Yield: 295 mg (62%). IR (film):
3176, 2927, 1646, 1508, 1249, 1175, 1033 cm−1. 1H-NMR (400 MHz, CDCl3): δH = 7.67 (tt, J = 7.56 Hz,
7.7 Hz; 1H,CHarom), 7.36 (m, 2H, (CHarom), 7.27 (m, 8H,CHarom), 6.84 (d, J = 8.8 Hz, 2H,CHarom), 6.7
(br, 1H, NH), 4.07 (m, 1H, OH-CH-CH3), 3.92 (m, 1H, CH-CH-OH), 3.79 (s, 6H, 2 OCH3), 3.72 (m,

Molecules 2014, 19
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1H, CH-CH-OH), 3.56 (m, 1H, N-CH-CH2), 3.41 (dd, J = 9.6 Hz, 4.2 Hz, 1H, S-CH-CH2-), 3.30 (dd,
J = 9.4 Hz, 4.2 Hz, 1H, S-CH-CH2-), 3.17 (m, 2H, S-CH2-CH2-), 3.00 (br, 1H, OH), 2.45 (m, 1H,
S-CH-CH2-), 2.23 (t, J = 7.4 Hz; 2H, CO-CH2), 1.90 (m,2H, -CH2-CH2-), 1.69 (m, 4H, alkyl residue),
1.49 (m, 2H, alkyl residue), 1.13 (d, J = 6.4 Hz, 3H, CH-CH3) ppm.13C-NMR (125 MHz, CDCl3):
δC = 173.25 (CO), 158.84 (Carom), 150.06 (Carom), 144.51 (Carom), 136.14 (Carom), 135.73 (Carom),
135.66 (Carom), 135.53 (CHarom), 130.01 (CHarom), 128.20 (CHarom), 128.18 (CHarom), 127.23 (CHarom),
123.92 (CHarom), 113.57 (CHarom), 87.03 (C-Carom), 68.94 (CH-O-), 65.55 (CH2-O), 56.57 (N-CH-CH2-),
55.63 (OCH3), 55.49 (OCH3), 53.54 (COCH2), 40.43 (CH2-S), 38.64 (CH2-CH-S), 36.73, 34.93, 29.16,
25.76 (alkyl chain), 20.18 (CH3-CH) ppm. HRMS (ESI+): m/z calcd for C66H82N2NaO10S4 ([2M + Na]+)
1213.4745, found 1213.4745.
3.4.2. N-[(1R,2R)-1-[[bis(4-Methoxyphenyl)phenylmethoxy]methyl]-2-hydroxypropyl]-N'-[3-[2-[2-[3-
[5-(dithiolan-3-yl)pentanoylamino]propoxy]ethoxy]ethoxy]propyl]butanediamide (9a)
The compound was obtained as a mixture of two diastereoisomers. Yield: 467 mg (65%). IR (film):
1
2929, 2662, 1653, 1608, 1545, 1508, 1463, 1393, 1249, 1178, 1131, 1095, 1033, 753 cm−1. H-NMR
(400 MHz, CDCl3): δH = 7.39 (d, J = 7.3 Hz, 2H, CHarom), 7.29 (m, 6H, CHarom), 7.21 (d, J = 7.3 Hz,
1H, CHarom), 6.84 (d, J = 8.9 Hz, 4H,CHarom), 6.69 (br, 1H, NH), 6.64 (br, 1H, NH), 6.49 (br, 1H, NH),
4.07 (m, 1H, CH3-CH-OH), 3.91 (m, 3H, N-CH-CH2-OH), 3.79 (s, 6H, 2 OCH3), 3.64 (m, 8H,
2 O-CH2-CH2-O), 3.59–3.54(m, 4H, O-CH2-CH2-O), 3.34 (m, 4H, 2 NH-CH2-), 3.09 (m, 4H,
S-CH2-CH2 and CH2-NHCO), 2.52 (m, 5H, S-CH-CH2- and NH-CH2-CH2-CH2-O), 2.14 (t, J = 7.2 Hz,
2H, CO-CH2), 1.85–1.49 (m, 8H, alkyl residues), 1.12 (2d, J = 6.4 Hz, 6H, 2 CH3-CH) ppm. 13C-NMR
(125 MHz, CDCl3): δC = 172.83 (CO), 172.74 (CO), 172.06 (CO), 158.55 (Carom), 145.36 (Carom),
135.87 (Carom), 135.79 (Carom), 130.06 (CHarom), 129.89 (Carom), 127.82 (CHarom), 127.79 (CHarom),
126.43 (CHarom), 113.44 (CHarom), 86.54 (C-O), 70.57 (CH-O-), 70.49 (CH-O-), 70.05 (CH-O-), 69.98
(CH-O-), 69.88 (CH-O-), 68.26 (CH-O-), 64.8 (CH-O-), 56.44 (OCH3), 55.23 (OCH3), 54.06 (CH-N),
53.64 (CH-N), 41.93 (CH2-S), 38.46 (CH2-S), 37.83 (CH2-CH-S), 36.37(COCH2), 34.68 (N-CH2),
31.95 (N-CH2), 31.72, 29.62, 29.08, 28.96, 28.86, 25.43, 19.82, 17.33 (alkyl residue), 17.31 (CH3CH)
ppm. HRMS (ESI+): m/z calcd for C26H50N3O8S2 ([M + H-DMT]+) 596.3033, found 596.3032.
3.4.3. O1-(4, 4'-Dimetoxytriphenylmethyl)-N-[N'-{5-(1,2-dithiolan-3-yl)pentanoyl}glycylglycylglycyl]-
L-threoninol (9b)
The compound was obtained as a mixture of two diastereoisomers. Yield: 325 mg (53%). 1H-NMR
(400 MHz, DMSO-d6): δH = 8.13 (t, J = 5.6 Hz, 1H, CH2-NH-CO), 8.07–8.04 (m, 2H, CH2-NH-CO),
7.50 (d, J = 7.6 Hz, CH-NH-CO), 7.37–7.17 (m, 9H, CHarom), 6.86 (d, J = 8.8 Hz, 4H, CHarom), 4.54
(d, J = 5.2 Hz, 1H, OH-CH), 3.95–3.90 (m, 1H, OH-CH-CH3), 3.89–3.82 (m, 1H, NH-CH-CH2),
3.78–3.69 (m, 12H, 2 OCH3 and 3 NH-CH2-CO), 3.61–3.53 (m, 1H, S-CH-CH2), 3.18–3.02 (m, 3H,
DMTO-CH(A)H(B)-CH and S-CH2-CH2), 2.86–2.82 (m, 1H, DMTO-CH(A)H(B)-CH), 2.41–2.34 (m, 1H,
S-CH-CH(A)H(B)-CH2), 2.12 (t, J = 7.2 Hz, 2H, CH2-CH2-CO-NH), 1.88–1.79 (m, 1H, S-CH-CH(A)H(B)-CH2),
1.68–1.59 (m, 2H, alkyl chain), 1.54–1.46 (m, 2H, alkyl chain), 1.36–1.31 (m, 2H, alkyl chain), 0.94
13
(d, J = 6.0 Hz, 3H, CH3-CH) ppm. C-NMR (125 MHz, DMSO-d6): δC = 173.25 and 173.16 (NH-
CO)(*), 170.31, 170.29, 170.23 and 170.21 (NH-CO)(*), 169.64, 169.62, 169.57 and 169.55 (NH-

Molecules 2014, 19
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CO)(*), 169.29, 169.23 and 169.21 (NH-CO)(*), 158.65 (Carom), 145.76 (Carom), 136.49 (Carom), 136.45
(Carom), 130.37 (CHarom), 128.45 (CHarom), 128.39 (CHarom), 127.22 (CHarom), 113.80 (CHarom), 85.82
(C-O), 65.44 and 65.33 (CH3-CH)(*), 63.39 (DMTO-CH2-CH), 56.79 (S-CH-CH2), 55.69 (O-CH3),
54.74, 54.72, 54.66 and 54.63 (NH-CH-CH)(*), 46.24 (S-CH2-CH2), 42.77 and 42.75 (NH-CH2-CO)(*),
42.66 and 42.63 (NH-CH2-CO)(*), 42.47 and 42.45 (NH-CH2-CO)(*), 38.77 (S-CH-CH2), 35.64 and 35.59
(CH2-CH2-CO-NH)(*), 34.81 (CH2 alkyl chain), 29 01 (CH2 alkyl chain), 25.51 (CH2 alkyl chain),
20.87 and 20.81 (CH3-CH)(*) ppm. HRMS (ESI+): m/z calcd for C39H50N4NaO8S2 ([M + Na]+)
789.2968, found 789.2960; m/z calcd for C18H33N4O6S2 ([M-DMT+H]+) 465.1842, found 465.1845;
m/z calcd for C18H32N4NaO6S2 ([M-DMT + Na]+) 487.1661 found 487.1661. (*)Multiple peaks
observed due to rotamers of the amide bond.
3.4.4. N-[(1R,2R)-1-[[bis(4-Methoxyphenyl)phenylmethoxy]methyl]-2-hydroxypropyl]-8-[5-(dithiolan-
3-yl)pentanoylamino]octanamide (9c)
The compound was obtained as a mixture of two diastereoisomers. Yield: 277 mg (47%). IR (film):
1
3019, 2925, 1652, 1058, 1215 cm−1. H-NMR (400 MHz, CDCl3): δH = 7.67 (tt, J = 7.6 Hz, 1.8 Hz,
1H, CHarom), 7.39 (m, 2H, CHarom), 7.27 (m, 8H, CHarom), 6.83 (d, J = 8.2 Hz, 2H, CHarom), 6.1 (br, 1H,
NH), 4.08 (m, 1H, OH-CH-CH3), 3.94 (m, 1H, CH-CH-OH), 3.78 (s, 6H, 2 OCH3), 3.56 (m, 1H,
N-CH-CH2), 3.39 (dd, J = 9.5 Hz, 4.5 Hz, 1H, CH-CH-OH), 3.23 (m, 2H, S-CH2-CH2-), 3.10 (m, 2H,
CH2-N), 2.42 (m, 1H, S-CH-CH2-), 2.21 (t, J = 7.5 Hz, 2H, CO-CH2), 2.14 (t, J = 7.5 Hz, 2H,
COCH2), 1.89 (m, 2H, -CH2-CH2-), 1.64 (m, 6H, alkyl residue), 1.45 (m, 4H, alkyl residue), 1.32 (m,
6H, alkyl residues), 1.12 (d, J = 6.3 Hz, 3H, CH3-CH) ppm. 13C-NMR (125 MHz, CDCl3): δC = 173.63
(CO), 172.91 (CO), 158.82 (Carom), 149.93 (Carom), 144.66 (Carom), 136.24 (Carom), 135.84 (Carom),
135.66 (Carom), 130.21 (CHarom), 130.19 (CHarom), 128.20 (CHarom), 128.18 (CHarom), 127.21 (CHarom),
124.05 (CHarom), 113.57 (CHarom), 86.86 (C-Carom), 68.55 (CH-O-), 65.37 (CH2-O), 56.73 (N-CH-CH2-),
55.46 (OCH3), 53.63 (COCH2), 40.47 (CH2-S), 39.65 (CH2-S), 38.63 (CH2-CH-S), 36.94 (COCH2),
36.69 (NCH2), 34.83, 29.76, 29.20, 29.19, 29.08, 26.83, 25.88, 25.68 (alkyl residues), 20.25 (CH3CH)
ppm. HRMS (ESI+): m/z calcd for C41H56N2NaO6S2 ([M + Na]+) 759.3472, found 759.3475; m/z calcd
for C82H112N4NaO12S4 ([2M + Na]+) 1495.7052, found 1495.7032.
3.5. Functionalization of CPG Solid Supports 4 and 11a–11c
The DMT derivatives (2, and 9a–9c) were incorporated on a long-chain alkylamine-controlled pore
glass support (LCAA-CPG) as described [56] using their corresponding hemisuccinate derivatives as
follows. The DMT derivative (0.05 mmol) was dried by evaporation with anhydrous ACN under
reduced pressure. The residue was dissolved in anhydrous pyridine (5 mL) under argon. Succinic
anhydride (26 mg, 0.26 mmol) and DMAP (2.5 mg, 0.02 mmol) were dissolved in 1 mL of pyridine
and added to the solution. The solution was stirred overnight at room temperature. The solvent was
removed under reduced pressure and the residue was dissolved in toluene (3 × 10 mL) and
concentrated to dryness. The resulting material was dissolved in dichloromethane (20 mL). The
solution was washed with 0.1 M sodium monophosphate (15 mL) and saturated aqueous NaCl (15 mL).
The organic layer was dried over anhydrous MgSO4, filtered and evaporated to dryness. Yellowish oils

Molecules 2014, 19
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were obtained in each case. The resulting hemisuccinates 3 and derivatives10a–10c were used in the
next step without further purification.
Amino-LCAA-CPG (CPG New Jersey, 73 μmol amino/g, 200 mg) was placed into a polypropylene
syringe fitted with a polypropylene disc and washed sequentially with DMF, MeOH, THF, DCM and
ACN (2 × 5 mL). Then, a solution of the appropriate hemisuccinate (25 µmol) and triethylamine
(25 µL, 175 µmol) in anhydrous ACN (500 µL) was prepared. The solution was added to the support
and then a solution of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)
(56 mg, 175 µmol) in anhydrous ACN (300 µL) was added. The mixture was left to react for 1 h.
The support was washed with DMF, MeOH, DCM and ACN (2 × 5 mL). The coupling procedure was
repeated once more if needed and the functionality of the resin was determined by DMT
quantification. The functionalization obtained in each case were f = 19.0 μmol/g for solid support 4,
18.5 μmol/g for 11a, 25.3 μmol/g for 11b and 15.1 μmol/g for 11c. Finally, each solid support was
treated with a mixture of Ac2O/DMF (1:1, 500 μL) to cap free amino groups.
3.6. N-[(1R,2R)-1-{[bis(4-Methoxyphenyl)phenylmethoxy]methyl}-2-[2-cyanoethoxydiisopro-
pylamino)phosphanyl]oxypropyl]-4-(dithiolan-3-yl)butanamide (12)
Compound 2 (100 mg, 0.13 mmol) was dried by evaporation of anhydrous ACN (3 × 10 mL) under
reduced pressure and dried in a desiccator for one hour. Then, compound 2 was dissolved in anhydrous
DCM (2 mL) and DIEA (69 µL, 0.39 mmol) was added dropwise at room temperature. The solution
was cooled at 0 °C and 2-cyanoethoxy-N,N-diisopropylaminochlorophosphine (46 µL, 0.19 mmol)
was added dropwise with a syringe. Reaction was stirred 5 min at 0 °C and 1 h at room temperature.
After this time the reaction was complete as judged by TLC. Then, DCM was added
(10 mL) and organic layer was washed with a 5% NaHCO3 aqueous solution (1 × 5 mL) and brine
(1 × 5 mL). Finally, solvent was evaporated and the crude was purified by flash chromatography
(Hex/AcOEt 20% to 50%) to give compound 12 (80 mg, 77% yield). The compound was obtained as a
mixture of diastereoisomers. IR (film): 2966, 2929, 2360, 2342, 1673, 1607, 1508, 1249, 1177, 1034 cm−1.
1H-NMR (400 MHz, CDCl3): δH = 7.38 (m, 2H), 7.26 (m, 7H), 6.29 (m, 4H), 5.80 (broad s, NH), 4.30
(m, 1H), 4.23 (m, 5H), 3.75 (s, 6H, 2 OCH3), 3.47 (m, 4H), 3.14 (m, 2H), 2.70 (m, 2H), 2.36 (m, 1H),
13
2.17 (m, 2H), 1.90 (m, 2H), 1.63 (m, 4H), 1.30–1.15 (m, 15H) ppm; C-NMR (125 MHz, CDCl3)
δC = 173.3 (CO), 158.4 (Carom), 144.8 (Carom), 135.9 (Carom), 130.1 (Carom), 130.0 (Carom), 129.9 (Carom),
128.2 (CHarom), 128.0 (CHarom), 127.7 (CHarom), 126.7 (CHarom), 113.0 (CN), 86.0 (C-Carom), 68.7 (CH-O),
65.7 (CH2-O), 65.3 (P-O-CH2), 58.3 (N-CH-CH2), 58.1 (N-CH-CH2), 55.2 (OCH3), 53.3 (COCH2),
43.0 (CH2-S), 40.2(CH-N), 38.4 (CH2-CH-S), 36.5, 34.6, 29.6, 24.7 (alkyl chain), 24.2 (CH3-CH), 22.6
31
(CH3-CH), 19.6 (CH2-CN) ppm; P-NMR (163 MHz, CDCl3) δP = 148.0 ppm; HRMS (ESI+): m/z
calcd for C42H59N3O6PS2 ([M+H]+) 796.3583, found 796.3577.
3.7. Synthesis of the Model Oligonucleotides (T12)TA_I, and (T12)TA_III(a)–(c)
The TA terminated dodecathymidine oligonucleotides (T12)TA_I, and (T12)TA_III(a)-(c) (5'-TTT
TTT TTT TTT-X-3', X being a TA modification) were synthesized on a DNA/RNA synthesizer
(Applied Biosystems) on a 0.2 µmol scale using the corresponding modified CPG solid supports. The
solid support 4 was used to synthesize the oligonucleotide (T12)TA_I and the corresponding solid

Molecules 2014, 19
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supports 11(a)–(c) for oligonucleotides (T12)TA_III(a)–(c). The last DMT group was removed at the
end of the synthesis. The resulting oligonucleotides were deprotected and cleaved from the solid
supports with aqueous concentrated ammonia using three different conditions: (i) at room temperature
for 4 h; (ii) at 55 °C for 1 h and (iii) at 55 °C overnight. The mixtures were filtered and ammonia
solutions were concentrated to dryness. The resulting products were analyzed by HPLC. Solvent A:
5% ACN in 100 mM triethylammonium acetate (TEAA) (pH = 7) and solvent B: 70% ACN in 100 mM
TEAA (pH = 7). Column: XBridgeTM OST C18 column; (4.6 × 50 mm, 2.5 µm). Flow rate: 1 mL/min.
Conditions: 10 min of linear gradient from 5% to 35% of B. The isolated products were analyzed by
mass spectrometry (MALDI-TOF) (Supplementary Information, Table S1).
3.8. Synthesis of 3'-TA-Modified Oligonucleotides and the 5'-Fluorescently Labeled 3'-TA-Modified
Oligonucleotide Probes
The thioctic acid terminated 21mer oligonucleotides TA_I, TA_II and TA_III(a)-(c) (5'-CGG
AGG TAC ATT CGA CTT GA-X-3', where X was a TA residue) were synthesized a 1 µmol scale
using the corresponding modified CPG solid supports. Oligonucleotide TA_II was synthesized using
the solid support 4 once the commercially available spacer 18-O-dimethoxytritylhexaethyleneglycol,
1-[(2-cyanoethyl)-(N,N-diisopropyl)] phosphoramidite was incorporated. Once the synthesis of TA_I,
TA_II and TA_III(a)–(c) was completed, each solid support was divided in two. The last DMT group
was removed at the end of the synthesis. One half was deprotected and the other half was used for the
synthesis of the corresponding fluorescently labeled oligonucleotides (F)TA_I, (F)TA_II and
(F)TA_III(a)–(c). Coupling yields were >97%. TA_I, TA_II and their corresponding fluorescently
labeled versions were treated with aqueous concentrated ammonia at 55 °C for 12 h to cleave the
i
products from the supports and remove the Bz and Bu groups. The TA_III series were treated with
aqueous concentrated ammonia at room temperature for 4 h to cleave the products from the supports
and remove the Bz and dmf groups. The mixtures were filtered and ammonia solutions were
concentrated to dryness. The resulting oligonucleotides were desalted and purified by HPLC using the
same solvents described above. Column: XBridgeTM OST C18 semipreparative column (10 × 50 mm,
2.5 µm). Flow rate: 3 mL/min. Conditions: 10 min of linear gradient from 0% to 30% of B for non
fluorescently labeled oligonucleotides and 10 min. of linear gradient from 6% to 40% for the
corresponding fluorescently labeled oligonucleotides. The pure fractions were combined and
evaporated to dryness. The resulting oligomers were analyzed by mass spectrometry, UV/Vis
spectroscopy and HPLC as well using the conditions and gradients described above but using an
analytic column (XBridgeTM OST C18 (4.6 × 50 mm, 2.5 μm). Flow rate: 1 mL/min), (Table S3, SI).
The yields obtained ranged from 33% to 49%.
3.9. Synthesis of a 5'-TA-Modified Oligonucleotide
The thioctic acid terminated 21mer oligonucleotide 5'TA_I, (5'-X-CGG AGG TAC ATT CGA
CTT GA-3', where X was a TA residue) was synthesized on a 0.2 µmol scale employing LV200®
polystyrene supports. Phosphoramidite 12 was used to incorporate the TA derivative. The DMT
determination showed that the efficiency of coupling of the phosphoramidite was >90%. The last DMT
group was removed at the end of the synthesis. The solid support was treated with aqueous

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concentrated ammonia at 55 °C for 12 h. and oligonucleotide 5'TA_I purification by HPLC was using
the same protocol as described above. (Table S3, SI). The purified oligonucleotide was obtained in
51% yield.
3.10. Synthesis of 3'-Alkylthiolated-Modified Oligonucleotides and the 5'-Fluorescently Labeled 3'-
Alkylthiolated Modified Oligonucleotide Probes
The 3'-disulfide-modified oligonucleotide ALK_DS (5'-CGG AGG TAC ATT CGA CTT GA-Y-3')
(Y stands for 3'- linear alkyldisulfide or alkylthiol modifications) was synthesized on a 1 µmol scale
using the 3'-Thiol modifier C3 S-S CPG following the procedure described previously. The last DMT
group was removed at the end of the synthesis. As well, once the synthesis was completed, half of the
solid support was used to incorporate the fluorescent dye at the 5'-termini to obtain the fluorescently
labeled (F)ALK_DS analogue. Each solid support was treated with aqueous concentrated ammonia at
55 °C for 12 h. Work-up was similar as described above. The oligonucleotides were analyzed by
HPLC using the conditions described above and characterized by mass spectrometry (MALDI-TOF)
and UV/Vis spectroscopy (Table S3, SI) and used without further purification. The yields obtained
were 74% for ALK_DS and 72% for (F)ALK_DS. The 3'-thiol-modified oligonucleotide ALK_SH and
its fluorescently labeled version (F)ALK_SH were obtained from the corresponding disulfide
oligonucleotides ALK_DS and (F)ALK_DS respectively by thiol deprotection [44,45]. The experimental
procedure is as follows: disulfide oligonucleotides (5 OD260) were dissolved in 300 µL of an aqueous
solution 0.1 M TEAA (pH = 7). Then, 34 L of an aqueous solution 0.1 M of tris(2-carboxyethyl)phosphine
hydrochloride (TCEP·HCl) were added to the solution and allowed to react at 55 °C overnight.
Under these conditions, the disulfide group was completely removed and the deprotected thiol
oligonucleotides (ALK_SH and (F)ALK_SH, respectively) were purified with Sephadex G-25
(NAP-5 column) and used in the next step without further purification.
3.11. Functionalization of Gold Nanoparticles
Colloidal gold nanoparticles (9.7 nm diameter) were functionalized with the TA-modified
oligonucleotides (TA_I, TA_II, TA_III(a)–(c)), with 3'-alkylthiolated-modified oligonucleotides
(ALK_DS and ALK_SH) and with their respectively fluorescently labeled versions as well.
To prepare the conjugates, 5 nmol of desalted oligonucleotide were dissolved in 1 mL of MilliQ water
and were added to a 1 mL of the gold nanoparticles solution. After 24 h, the solution was brought to a
final concentration of 10 mM sodium phosphate (pH = 7.2). The mixture was then allowed to
equilibrate for 30 min before bringing the concentration to 0.15 M NaCl over a 7 h 30 min period in a
stepwise manner (0.05 M NaCl increments each 2 h 30 min). The solutions were sonicated for 10 s.
before each addition to keep the particles dispersed during the salting procedure. The salting process
was followed by an incubation period of 48 h at room temperature. Finally, to remove all unbound
oligonucleotides, the solution was centrifuged at 13,200 rpm (16,100 ×g) for 30 min. This procedure
was repeated three times. As we wanted to compare stabilities of the different DNA-AuNP conjugates,
we dispersed the reddish residue in 500 µL of 0.15 M NaCl, 10 mM sodium phosphate (pH = 7.2),
0.01% NaN3 solution. Then, each solution was analyzed by UV-visible absorption spectroscopy and
the concentration of gold nanoparticle conjugates was adjusted to 6.9–7.1 nM by adding the required

Molecules 2014, 19
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volume of buffer. The conjugates obtained with the fluorescent oligonucleotides were directly
resuspended in 1 mL of a 0.15 M NaCl, 10 mM sodium phosphate (pH = 7.2), 0.01% NaN3 solution.
The extinction coefficient used was the same as that used for unmodified nanoparticles
(ε520 = 7.6 × 107 M−1·cm−1, data provided by the manufacturer). Each solution was then stored in the
fridge (4 °C) prior to use.
3.12. Stability Tests of Oligonucleotide-Gold Nanoparticle Conjugates
The stability of the different TA-terminated oligonucleotides TA_I, TA_II, TA_III(a)–(c) gold
nanoparticle conjugates was assessed by treating the AuNP-conjugates with a DTT solution based on
protocols described in the literature [22,37]. The colloidal stability of AuNP conjugates obtained with
disulfide and thiol modified oligonucleotides (ALK_DS and ALK_SH respectively) were studied as
well for comparison purposes. Then, to a 500 µL of each solution were added 12.5 µL of a 4 M DTT
solution (the final concentration of DTT was 100 mM). Prior to the addition of DTT, the solutions
were allowed to equilibrate at 40 °C and were sonicated for 20 s to keep the particles dispersed.
To give an indication of the durability of the oligonucleotide conjugates, absorbance at 675 nm was
monitored against time at 40 °C. For AuNPs conjugated with TA-terminated oligonucleotides absorbance
was recorded at 1 min intervals and for AuNPs conjugated with alkylthiolated oligonucleotides absorbance
was recorded at 1 s intervals. The measures were performed with 0.5 cm quartz cuvettes. The cells
used had a ground hole at the top to adapt a PTFE stopper to provide a more suitable seal to reduce
evaporation of the sample. Then, the half-life (time to reach half the value for complete aggregation)
was calculated for each conjugate by fitting the data obtained to a Boltzmann equation using the Origin
8.0 software.
3.13. Oligonucleotide Loading Quantification on Gold Nanoparticles
The number of oligonucleotides loaded onto gold nanoparticles was analyzed following procedures
previously described [37,47]. First, the concentration of nanoparticles and the concentration of
fluorescent DNA in each sample were measured followed by the displacement of fluorescent
oligonucleotides from the nanoparticle surface using a DTT solution. To this end, 200 µL of the gold
nanoparticle conjugate solution were treated with 200 µL of a 1 M DTT solution in 0.18 M sodium
phosphate buffer, (pH = 8) at room temperature for 2 h. The sample was centrifuged to remove the
sediment from the supernatant and then the fluorescence spectra were recorded (conditions: λEx = 495 nm
and λEm = 510 − 550 nm). For each oligonucleotide two different oligonucleotide-AuNP conjugates
were prepared and two different samples were prepared for each conjugate. Fluorescent measurements
were done in duplicate. Standard curves were prepared with known concentrations of the fluorescently
labeled TA-terminated oligonucleotide (F)TA_I using the same buffer, salt and DTT concentrations.
The average number of oligonucleotides per particle was obtained by dividing the measured
oligonucleotide molar concentration by the original oligonucleotide-AuNP conjugate concentration.
The average area that each oligonucleotide occupies on the nanoparticle surface (footprint) was calculated
by dividing the calculated surface area (nm2) by the average number of oligonucleotides per particle.

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3.14. Stability of Thioctic Derivatives to Cathepsin
Cathepsin B was dissolved in this buffer: 50 mM NaCl, 10 mM sodium phosphate pH = 6, 1 mM
EDTA and 30% glycerol and stored at −20 °C until needed. In order to find the optimal conditions for
the enzymatic reaction, different assays were carried out at different pH, reaction times and also
changing the proportions of the agents in the reaction. The optimal conditions were as follow: 13.8 µL
(0.02 unt/µL) of Cathepsin B were added to 200 µL of a solution containing 0.25 OD’s of different
modified oligonucleotides (TA_I, TA_II, TA_III(a)–(c)) and 5 mM of reduced glutathione.
The reaction buffer was 100 mM NaCl, 40 mM sodium acetate, 1 mM EDTA and 1% triton-100x,
pH = 5. The reaction mixture was left at 37 °C, with soft stirring during 12, 24, 48, 72 and 168 h
respectively. The reaction was quenched by heating the samples at 70 °C during 20 min. The resulting
products were analyzed by HPLC. Solvent A: 5% ACN in 100 mM TEAA (pH = 7) and solvent B:
70% ACN in 100 mM TEAA (pH = 7). Column: Nucleosil 120‒10 C18; (250 × 4 mm). Flow rate:
1 mL/min. Conditions: 20 min of linear gradient from 10% to 40% of B. The isolated products were
analyzed by mass spectrometry.
4. Conclusions
L-Threoninol has been used as a building block to introduce in a covalent way to a DNA sequence
four different ligands in length containing a TA moiety. All TA-appended oligonucleotide conjugates
were efficiently loaded on gold nanoparticles. Stability experiments confirmed that all DNA-gold
nanoparticle conjugates with TA modified at 3'-termini showed better stabilities through thiol
exchange in the presence of DTT than those conjugates containing acyclic disulfides such as the
commercially available 3'-thiol modifiers. On the other hand, we observed that oligonucleotides loaded
onto gold nanoparticles containing a TA-alkyl ligand (AuNP-TA_III(c)) or a TA-oligo(ethylene
glycol) ligand (AuNP-TA_III(a)) showed a remarkable increase in probe stability probably due to the
disposal of the spacers, covering and protecting the gold nanoparticle surface. This increase in the stability
of the system may be also due to the position of the amide group, which may induce favorable interactions
between the neighboring alkyl or oligo(ethylene glycol) chains. Finally, the thioctic derivative carrying the
triglycine linker is shown to be the most sensitive to cathepsin B. These preliminary results may open the
door to an interesting application for these oligonucleotide derivatives in cellular delivery applications.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/7/10495/s1.
Acknowledgments
This work is supported by the European Commission (Grants FP7-NMP-213382-2, FUNMOL and
NMP4-LA-2011-262943, MULTIFUN), by the Spanish Ministry of Education (Grant CTQ2010-20541),
the Generalitat de Catalunya (2009/SGR/208) and the Instituto de Salud Carlos III (CB06_01_0019).
CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio

Molecules 2014, 19
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2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with
assistance from the European Regional Development Fund.
Author Contributions
Sónia Pérez-Rentero and Santiago Grijalvo have performed most of the synthetic work, the analysis
of the stability of the oligonucleotide-NP conjugates to DTT. Sónia Pérez-Rentero, Guillem Peñuelas
and Carme Fàbrega performed the analysis of the stability of thioctic-oligonucleotides to cathepsin.
All authors have participated in the preparation of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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