"click and go": Simple and fast folic acid conjugation

Article abstract of DOI:10.1039/c4ob00150h

Folic acid targeting by functionalization of the terminal γ-carboxylic acid is one of the most important strategies to selectively deliver chemotherapeutics and dyes to cancer cells which overexpress folate receptors. However, conjugation of folic acid is

Full text of DOI:10.1039/c4ob00150h

Organic &
Biomolecular Chemistry
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PAPER
“Click and go”: simple and fast folic acid
conjugation†
Alexandre F. Trindade,*a,b Raquel F. M. Frade,b Ermelinda M. S. Maçôas,a
Cátia Graça,a Catarina A. B. Rodrigues,a José M. G. Martinhoa and
Carlos A. M. Afonso*b
Cite this: Org. Biomol. Chem., 2014,
12, 3181
Folic acid targeting by functionalization of the terminal γ-carboxylic acid is one of the most important
strategies to selectively deliver chemotherapeutics and dyes to cancer cells which overexpress folate
receptors. However, conjugation of folic acid is limited by its unique solubility and by selectivity issues
imposing the need for expensive preparative reverse-phase chromatographic purification to isolate
γ-folate conjugates. Herein is provided a novel synthetic tool for the synthesis of new folic acid conjugates
with excellent γ-purity based on strain-promoted alkyne–azide cycloadditions with a γ-folate–cyclo-
octyne conjugate 3. To demonstrate the potential of this methodology several new folate conjugates were
synthesized with high γ-purity and without using any type of chromatographic purification by reacting con-
jugate 3 with several fluorescent probes, polymers and siliceous materials bearing azide. In addition, the
cycloaddition reaction between conjugate 3 and an azido-derived fluorescent dye was successfully per-
formed in cellular media leading to an increase of fluorescence in the cells which overexpress folate recep-
tors (NCI-H460).
Received 20th January 2014,
Accepted 11th March 2014
DOI: 10.1039/c4ob00150h
www.rsc.org/obc
the folate.10 Folic acid docks with the pterin head buried in the
receptor pocket, being stabilized by numerous hydrogen bridges
and hydrophobic interactions. Even the glutamate residue is
stabilized by six hydrogen bonds, four of them with the α-carb-
oxylic acid. These observations clearly indicate that only γ-folate
conjugates can retain the affinity toward the receptor.
Introduction
In the last few decades, folic acid has been employed to
actively target cancer cells constituting a very active field of
research in medicinal chemistry. This small vitamin is crucial
for the normal cell functioning and its receptors were found to
be overexpressed in a considerable percentage of cancer cell
lines.1,2 Until now, a handful of small folic acid conjugates
with drugs or fluorescent probes have been described and are
being evaluated in clinical trials.3–5 Apart from these develop-
ments, the synthetic tools available for conjugation of folic acid
are relatively underdeveloped and remain highly challenging.6–8
This vitamin is structurally constituted of pteroic acid co-
valently bound to a glutamic acid residue. However, only the
γ-conjugates have the medicinal relevance, since they have
higher affinity toward the receptors compared with α-conjugates.9
Recently, the FRα protein was isolated and crystallized providing
important insights into the interaction between the receptor and
Fortunately, the γ-conjugates are intrinsically obtained as
the major product (from 55 to 90% selectivity7,9,11–15) using
carbodiimide chemistry. The separation of low molecular
weight folate conjugates by precipitation (from α-conjugates,
bi-conjugates or unreacted folic acid) becomes troublesome as
these molecules are only soluble in DMSO in their neutral
form. Therefore their separation requires in general tedious
and expensive preparative reverse phase chromato-
graphy.7,9,16–18 When the conjugation is performed with
macromolecules or heterogeneous materials the conjugate
acquires the properties of the material,18–27 allowing for the
non-conjugated folic acid to be easily removed, while α-conju-
gates and bis-conjugates will remain inseparable. Svenson
raised the issue of introducing targeting ligands into supramo-
lecular structures that also display passive targeting. This type
of active targeting can introduce concerns to the regulatory
evaluation and approval because it adds one synthetic
step that can have a negative impact on the reproducibility of
the synthesis and on the polydispersity of the macromolecule.28
In order to circumvent the limitations of direct conjugation
of alcohols and amines to folic acid, Low and Fuchs have
aCQFM, Centro de Química-Física Molecular, IN-Institute of Nanosciences and
Nanotechnology, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco
Pais, 1049-001 Lisboa, Portugal. E-mail: alexandretrindade@ff.ul.pt;
Fax: (+351) 218 464 455/7
bInstituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia,
Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
E-mail: carlosafonso@ff.ul.pt; Fax: (+351) 217 946 470
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob00150h
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Scheme 1 General main synthetic methodologies available for folic acid conjugation and this work proposal.
shown that it is possible to synthesize regioselectively γ-conju- ethylenediamine spacer in order to ensure high affinity to
gates using a folate reconstructive strategy (Scheme 1, route A). folate receptors.9,44
The concept is based on the conversion of folic acid into
Hence, herein is described a new folic acid conjugation
pteroic acid, by the cleavage of the glutamic acid residue,6–29 route based on strain-promoted alkyne–azide cycloadditions
followed by the reattachment of a previously γ-functionalized (spAAC)45,46 for folate-based cancer cell targeting. The cyclo-
glutamic moiety. The methodology was applied to the syn- octyne BCN (BCN stands for bicyclo[6.1.0]non-4-yn-9-ylmethanol)
thesis of small folic acid conjugates with drugs and dyes was selected as the most appropriate clickable probe as it is
bearing
spacer,5,30–35 and to the preparation of a highly soluble s−1 in spAAC) and has low lipophilicity.47 The γ-conjugate
2-(trimethylsilyl)ethoxy-bisprotected folic acid conjugate folate-diethylenediamine-BCN 3 was synthesized in high purity
(Scheme 1, route A).8 Unfortunately, this methodology also employing a direct amide coupling strategy (Scheme 1, route
required purification by reverse phase chromatography. B), without using chromatographic purification. The procedure
a
hydro-soluble and bioresponsive polypeptide commercially available, symmetrical, highly reactive (0.29 M−1
1
Amines featuring both azides and terminal alkynes have was demonstrated for the conjugation of folic acid to PEG,
also been conjugated with folic acid in order to exploit copper- common fluorescent probes and silica-based materials.
catalysed “click” chemistry.23,36–38 Even though this strategy
is more appealing than the direct amide or alcohol coupling
since it is bio-orthogonal, it employs a transition metal which
Results and discussion
forms stable complexes with pteridines,39–41 and is responsible
for undesirable toxicity in cells.42 More recently, Burke’s group
explored cyclooctyne-tetrazine cycloaddition chemistry to link
drugs to a folate-cyclooctyne conjugate 2 having a PEG-SU-Lys
spacer (Scheme 1, route A). Unfortunately, the preparation of
the reactive conjugate relied on chromatographic separation
and the cycloaddition conjugation reaction gave a mixture of
regioisomers in low to good yields (28–79% isolated yields).43
Therefore, we envisioned the development of a small folate
conjugate that incorporates a non-metal based “clickable”
functional group42 allowing fast, selective and quantitative
conjugation to folic acid. This conjugate should also have an
The proposed strategy to prepare “clickable” conjugate 3 relies
on a simple reaction between folate ethylenediamine and
BCN–NHS mixed carbonate. The synthesis of the ethylenedi-
amine folate precursor is known;9,17,48–50 however, no consist-
ent information regarding the conjugation selectivity to folic
acid is available. Hence, we began to study the influence of
reagent stoichiometry, temperature and basic additives (added
in the initial DCC activation period) on the conjugation yield
and γ-selectivity (Table 1), using ethanol amine as a surrogate
for N-Boc-ethylenediamine. After an extensive screening, con-
jugate 4 was obtained with optimal yield and selectivity when
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conjugation was performed with N-Boc-ethylenediamine, the
conjugate 5 was isolated in 73% yield (Scheme 2) with excel-
lent purity (over 95% based on RP-HPLC). Treatment with TFA
for 2 hours delivered the folate N-ethylenediamine 6 in 83%
isolated yield (Scheme 2).
Table 1 Selected examples for the optimization of folic acid γ-conju-
gation to ethanol amine
Subsequently, the synthesis of the desired folate ethylene-
diamine BCN conjugate 3 was attempted (Scheme 3), by react-
ing BCN–NHS mixed carbonate in the presence of DIPEA for
16 hours as reported.52 We observed the formation of a folate-
containing impurity along with the desired carbamate 3. Such
an impurity was formed due to the extended reaction time,
since 1H NMR experiments indicated that the mixed carbonate
aminolysis occurs in less than five minutes yielding carbamate
3 as the single folate-derived product in quantitative yield
(>95%) (see ESI Fig. 1† for more details). This was further con-
firmed when the addition of two equivalents of benzyl azide
and MeO-PEG350-N3 resulted in the complete conversion
(>95%) toward the respective triazoles in 1 and 2.5 hours,
respectively (ESI Fig. 2†). Based on these data, two method-
ologies were developed for the preparation of new folate conju-
gates via copper-free click chemistry (Scheme 3). In method A,
folate conjugate 6 was reacted for 30 minutes with 1.4 eq. of
BCN/NHS mixed carbonate in the presence of 4 eq. of DIPEA
yielding the desirable folate ethylenediamine BCN conjugate 3
in 73% isolated yield.
Folic
DCC Y NHS Z Base
Conv.a Select.b
Entry acid X eq. eq.
eq.
(2 eq.)
(%)
(%)
1c
2c
3c
4c
5c
6d
7d
8d
9d
1
1
2
2
1
1
1
1
1
1
2
2
1
2
2
2
2
2
1
2
1
2
1
1
1
1
1
—
—
—
—
67
96
88
92
48
33
88
84
—
98
0–99
87
Organic bases
73–94
56–87
94
Inorganic bases 2–85
DIPEAe
21
99
Pyridine
90
a Determined by RP-HPLC (C18 Luna Phenomenex) and represents the
percentage of new compounds formed relatively to the initial folic acid
amount. b Percentage of the γ-carboxylic amine obtained relatively to
other products formed. c 35 °C (temperature close to physiological for
protein functionalization). d 25 °C. e Diisopropylethyl amine.
folic acid was activated in the presence of two equivalents of
DCC and NHS at 25 °C for 17 hours (for more details see
Table 1 in ESI†). Under these conditions the γ-conjugate was
obtained with 92% selectivity (Table 1, entry 2). Kinetic studies
revealed that suboptimal conversion and selectivity were
obtained for shorter reaction times (Table 2 in ESI†). The
excess of DCC is mandatory to achieve quantitative conversion
of folic acid (Table 1, entries 1–5), while the addition of
basic additives did not impact positively the selectivity or
the conversion (Table 1, entries 6–7).51 Gratifyingly, when the
Then, the respective triazoles 7a and 7b were isolated in
95–96% yield with high purity (by 1H NMR) after being reacted
for 2 hours with 3 eq. of benzyl azide and MeO-PEG350-N3,
respectively. This constitutes an overall yield of up to 70%
starting from folate conjugate 6 (or 42% from folic acid after
4 steps). These results confirm the effectiveness of copper-free
strain-promoted click chemistry for the synthesis of new folate
Scheme 2 Synthetic sequence for folic acid selective activation.
Scheme 3 Synthesis of conjugate 3 and its efficient conjugation to azides via strain-promoted click chemistry.
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Scheme 4 Examples of folic acid conjugates prepared from 3.
conjugates, easily purified from the excess azide (Scheme 3, isolated in 75% yield (Scheme 4). In order to confirm the
method A). chemical immobilization of folic acid onto the silica particles,
The two-step triazole yields can be further improved up to the isolated powder was packed in a column and eluted with
88% from folate conjugate 6 (or 53% from folic acid after 4 DMSO without any observable leaching of folates (for more
steps), while maintaining the same purity level, by employing details please see ESI†).
method B; conjugate 3 was prepared using only 1.05 eq. of
As presented herein, the copper-free cycloaddition between
BCN/NHS carbonate and 3 eq. of DIPEA and then reacted conjugate 3 and azides constitutes a powerful synthetic tool to
in situ with only 2 eq. azides (Scheme 3, method B). It is worth introduce folic acid-based targeting into polymers (exemplified
mentioning that folate triazoles were synthesized with excel- by PEG), heterogeneous supports (like silica) and low-
lent γ-selectivity and high purity without using any chromato- molecular weight fluorescent dyes, due to the simplicity of syn-
graphic purification, and that conjugate 3 was stable for at thesis and isolation. This synthetic tool may also contribute
least 24 hours in DMSO solutions. Subsequently these to the low polydispersity of macromolecules subject to
methods were evaluated towards the synthesis of new folic functionalization.
acid-derived fluorescent probes (Scheme 3). Dansyl (c), fluor-
Then, we decided to study how the photophysical properties
escein (d), rhodamine 6G (R6G, e) and 7-nitro-4-aminobenzo- of the dyes were impacted with conjugation to folic acid.
furazan (f) featuring azide functionalities were synthesized Scheme 5 compares the absorption and emission spectra of
and conjugated with folic acid, to prepare a family of fluo- the azido free dyes and the respective folate conjugates
rescent conjugates that covers a broad range of the UV-Vis (obtained using µM concentrations in 1% DMSO–water). The
spectrum. Method A was employed in the conjugation of fluo- folate conjugation leads to very little effects on the shape of
rescein, R6G and benzofurazan furnishing the respective tri- the absorption and emission bands associated with transitions
azoles in 64–68% isolated yield, while with method B the dansyl localized in the fluorophores. This is mostly evident when
conjugate was obtained in 70% isolated yield (Scheme 4), with comparing 7d and 7e with the respective azido free dyes
excellent purity.
(Scheme 5b and c). In the aqueous mixture the emission
Method B was also successfully applied in folic acid grafting quantum yields of 7d and 7e (7–9%) are about one order of
onto heterogeneous supports, like silica gel. For this purpose, magnitude lower compared with the azido free dyes.
flash chromatography silica gel grade was first treated with
The absorption spectra of 7c are dominated by the contri-
3-((6-azidohexyl)thio)propyl triethoxysilane and then reacted bution from the folate residue (red line in Scheme 5a), which
with conjugate 3. After 24 hours a pale yellow powder 7g was has its lowest energy transition at about 360 nm, overlapping
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Scheme 6 Laser scanning confocal images of the HEK 293 (a) and
NCI-H460 (b) cells incubated with 5 µM of 7e for 1 h. The images are an
overlay of the emission of 7e collected in the 530–580 nm region upon
excitation at 476 nm (green color), and the emission of the membrane
marker collected in the 670–730 nm region upon excitation at 633 nm
(red color). The same incubation and excitation conditions were used in
the two images. The corresponding normalized intensity distribution per
pixel in the cytoplasmatic compartment is displayed in the lower panel
(c).
Scheme 5 Comparison between the normalized absorption and emis-
sion spectra of the modified free dyes (blue dashed line) and the corres-
ponding folate conjugates (red straight line) in
a 1% DMSO–H2O
mixture. (a) 7c and N-(2-azidoethyl)dansyl, (b) 7d and fluorescein, (c) 7e
and rhodamine 6G and (d) 7f and furazan. The double band structure in
the emission of 7c shown in panel (a) is due to the contribution from
emission localized in the folate (λem = 455 nm) in addition to the emis-
sion band localized in the dansyl dye (λem = 552 nm). The excitation
spectra of 7c and 7f are shown in panels (a) and (d) as straight lines with
circles in order to aid the discussion.
immediately clear by comparing the images of the HEK 293
cells (Scheme 6a) with the NCI-H460 cancer cells (Scheme 6b).
The distribution of pixel counts of a given intensity within
the cytoplasmatic compartment of a single cell, shown in the
lower panel of Scheme 6, allows for a more quantitative analy-
sis of the increased uptake by the cancer cells. Integration of
these distribution curves shows a 5-fold increase in fluo-
rescence inside NCI-H460 cancer cells relative to HEK 293 non-
cancer cells, showing that the conjugate 7e can mark with
good selectivity cancer cells overexpressing folate receptors
relative to non-cancer cells. The origin of the two peak struc-
ture of the pixel count distribution is not clear but it could be
related to the formation of dimers and higher order aggregates
in the cytoplasm due to the high local concentration of the
conjugate.
The main advantage of the spAAC strategy is the attractive
chance of undergoing in situ conjugation in living cells, tissue
or organisms. To address the possibility of performing in situ
folate conjugation in living cells, we began by evaluating the
efficiency of the click reaction between conjugate 3 and
MeO-PEG350-N3 under physiological conditions. Due to the
low solubility of conjugate 3 in water, the reaction was per-
formed in 1% DMSO–water, at 37 °C (112 µM conjugate 3 +
224 µM MeO-PEG350-N3). After 24 hours, 1H NMR of the iso-
lated folates showed that about 50% of the triazole product
was formed. Finally, the synthesis of a dye labelled conjugate
was attempted in NCI-H460 cells. The comparison between
treating the cells with the free dye in the presence and absence
of conjugate 3 will determine the efficiency of the click reac-
tion in vitro, given that the free dye should have the lowest
with the low absorption cross-section transition localized in
the dansyl unit (at 340 nm). This overlap makes the 7c conju-
gate a poor candidate for cell staining. The absorption spec-
trum of 7f in aqueous media is also dominated by the folate
residue, but the weak absorption of the benzofurazan unit is
detected at 470 nm (Scheme 5d, red line). This band assign-
ment is supported by the observation of two bands at 340 and
475 nm (typical of this dye53) in the corresponding excitation
spectrum collected at 560 nm (straight line with circles),
which overlaps almost perfectly with the absorption of the
free dye (blue dashed line). A strong overlap is also observed
between the emission of 7f and the free dye. The quantum
yield of 7f in the aqueous mixture is quite low (∼1–2%) but, as
shown by the emission spectra in Scheme 5d, it is possible to
excite selectively the dye and separate its emission from emis-
sion of the folate residue (observed at 455 nm in Scheme 5a).
The decrease in the absorption of 7f observed in aqueous solu-
tion relative to DMSO suggests the formation of aggregates.54
In order to illustrate the potential use of these conjugates
as markers for cancer cells, the internalization of conjugate 7e
was studied in vitro using cancer (human non-small cell lung
cancer, NCI-H460) and non-cancer cell lines (human kidney
embryonic cells, HEK 293). Internalization of 7e is demon-
strated in Scheme 6 where an overlaid image shows the mem-
brane labelled with WGA-Alexa594 (a typical membrane
selective probe) in red and the cytoplasm labelled with 7e in
green. The enhanced uptake of 7e by the cancer cell lines was
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The main features presented above allow us to anticipate
the potential use of this methodology to easily prepare novel
folate conjugates for different specific applications. The fact
that the conjugated dyes synthesized herein have decreased
quantum yield of fluorescence prompts us to undertake optim-
ization of the spacer unit, in particular by altering the distance
between the triazole ring and the chromophores.
Experimental
N-Boc-ethylenediamine-folate 5
Scheme 7 Laser scanning confocal images of treated NCI-H460 cells
for 24 hours: (a) cells incubated with 25 µM of 8 and (b) cells incubated
with 25 µM of 8 together with 12.5 µM of 3. Images are an overlay of the
emission collected in the 549–591 nm region upon excitation at 488 nm
(green color), and the emission of the membrane marker collected in
the 700–753 nm region upon excitation at 514 nm (red color). The same
incubation and excitation conditions were used in the two images.
In a round-bottomed flask was dissolved 640 mg folic acid
(1.34 mmol, 1 eq. dehydrated powder) in 25 ml of DMSO. After
the dissolution was complete (about 30 minutes with mild
heating), 308 mg (2 eq.) of NHS and 552 mg (2 eq.) of DCC
were added successively. The reaction mixture was stirred for
16 h at room temperature, after which the urea precipitate was
filtered off. Then, 0.376 ml (2 eq.) of triethylamine was added
followed by 429 mg (2 eq.) of N-Boc-ethylenediamine dissolved
in 5 ml of DMSO. The mixture was again stirred overnight
before a mixture of 20% acetone in diethylether was added.
The thin yellow precipitate was carefully centrifuged and
washed four times with acetone and two times with diethyl
ether and dried under vacuum (658 mg, 73% yield). The folic
acid was conjugated with N-Boc-ethylenediamine almost exclu-
sively in the terminal carboxylic acid as confirmed by
possible bioavailability. For this reason the benzofurazan dye 8
was chosen instead of R6G. NCI-H460 cells were separately
incubated for 24 hours with dye 8 (Scheme 7a) and with a
2 : 1 mixture of dye 8 and conjugate 3 (Scheme 7b). In the
absence of conjugate 3, no dye 8 is detected inside the cells
nor within the cellular membrane. In the presence of conju-
gate 3 a considerable emission intensity from the 7f conjugate
is observed in the cell membrane (in yellow in Scheme 7b),
localized with the membrane specific dye (in red in
Scheme 7b). The trafficking of the dye labelled conjugate in
the cell interior exists but is limited. Nevertheless, these
results clearly show that the click reaction took place in situ.
RP-HPLC (injection of sample dissolved in DMSO, 1 ml min−1
0 to 5.5 min 100% water, linear gradient from 5.5 min at 100%
water to 15.5 min at 100% acetonitrile, Gemini 5 μm C18
110 Å, 4.6 × 250 mm, Phenomenex column) and H NMR. H
NMR (400 MHz, DMSO) δ 8.64 (s, 1H), 8.04–7.86 (m, 2H),
7.68–7.62 (m, 2H), 7.10–6.82 (m, 3H), 6.65–6.63 (d, 2H), 4.48
(bs, 2H), 4.27 (m, 1H), 3.43 (bs, water) 3.06–2.88 (m, 4H),
2.28–1.85 (m, 4H), 1.35 (s, 9H). 13C NMR (100 MHz, DMSO)
δ 172.38, 166.60, 161.64, 156.07, 154.46, 151.23, 151.19,
149.00, 129.53, 129.32, 128.44, 122.01, 111.72, 78.07, 52.84,
46.43, 46.12, 40.94, 39.27, 32.50, 31.51, 28.68, 10.14.
,
1
1
Conclusions
Herein is described a novel synthetic tool for the functionali-
zation of folic acid and its conjugation to small molecules
(including fluorescent dyes), polymers and heterogeneous
materials using efficient non-metal based click chemistry. This
methodology presents the following main features:
Ethylenediamine-folate 6
(a) the synthesis of a small folate-cyclooctyne conjugate 3
via an efficient synthetic route displaying over 95% γ-purity The ethylenediamine-folate conjugate was prepared according
and without employing any chromatographic purification; to a literature protocol. N-Boc-ethylenediamine folate (300 mg)
(b) the cycloaddition reaction between conjugate 3 and was dissolved in 2 ml of trifluoroacetic acid and stirred for two
several fluorescent dyes, polymers and heterogeneous silica hours. The solvent was removed under pressure with the aid of
bearing azide enables a fast and quantitative synthesis of new dichloromethane and the red-dark residue was dissolved in a
folic acid conjugates with high γ-purity;
minimal amount of dry DMF. The addition of triethylamine
(c) the new folate-rhodamine 6G conjugate 7e has shown resulted in the precipitation of a yellow powder which was
higher uptake inside cancer cell lines overexpressing folate washed and centrifuged four times with acetone and two
receptors (NCI-H460) compared with non-tumoral cell lines;
times with diethyl ether (207 mg, 83% yield). 1H NMR
(d) the cycloaddition reaction was performed in cellular (400 MHz, DMSO) δ 8.64 (s, 1H), 8.19–7.96 (m, 1H), 7.66–7.58
media leading to an increase of fluorescence inside cancer cell (m, 2H), 7.18–6.91 (m, 3H), 6.66–6.63 (m, 2H), 4.47 (bs, 2H),
lines overexpressing folate receptors (NCI-H460) due to 4.31–4.07 (m, 1H), 3.52 (water), 3.32–3.19 (m), 2.86–2.80 (m,
accumulation of conjugate 7f.
2H), 2.24–1.93 (m, 4H).
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BCN-ethylenediamine-folate 3
diethyl ether to yield the respective triazole product in 86%
yield (14 mg).
To a solution of ethylenediamine-folate (13 mg, 0.027 mmol)
was added diisopropylethyl amine (17 μl, 0.1 mmol, 4 eq.) and
(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl succinimidyl car-
bonate (12 mg, 0.038 mmol, 1.4 eq.) and stirred until a clear
solution is obtained (about 20 minutes). The reaction mixture
was poured into a mixture of 20% acetone in diethylether. The
thin yellow precipitate was carefully centrifuged and washed
two times with acetone and two times with diethyl ether and
dried under vacuum (13 mg, 73% yield). 1H NMR (400 MHz,
DMSO) δ 8.64 (s, 1H, pterin), 8.14–7.88 (m, 2H), 7.68–7.63 (m,
2H, aromatic), 7.11–6.95 (m, 4H), 6.66–6.64 (m, 2H, aromatic),
4.50 (bs, 2H, benzylic), 4.29 (m, 1H, αH), 4.03–4.01 (m, 2H,
–HNCO2CH2), 3.36 (water), 3.10–3.02 (m, 4H, OvC–
NHCH2CH2), 2.22–1.92 (m, 10H, CH2CH2CO2+ CHH–CH2–
alkyne–CH2–CHH+ –CH2–alkyne–CH2–), 1.52 (m, 2H,CHH–
CH2–alkyne–CH2–CHH), 1.24–1.16 (m, 3H, –HNCO2CH2CH),
0.84 (m, 2H, HNCO2CH2CH(CH)2). APT NMR (100 MHz,
DMSO) δ 172.41, 172.37, 172.26, 166.67, 166.60, 156.91,
154.34, 151.21, 149.07, 148.97, 129.56, 129.40, 128.41, 121.86,
111.65, 99.46, 61.85, 52.60, 46.37, 40.70, 39.13, 32.44, 31.60,
29.03, 21.31, 19.99, 18.07. MS (ESI+) = 660.26 (M + H)+. HRMS
(ESI-FIA-TOF) calc. = 660.2894, found = 660.2875.
Conjugate 7b
Method A: BCN-ethylenediamine-folate (3.5 mg, 0.0051 mmol,
1 eq.) was dissolved in 1 ml of DMSO and added to a flask con-
taining 6 mg of MeO-PEG350-N3 (0.015 mmol, 3 eq.). The
reaction mixture was stirred for 5 hours before it was poured
into a solution of 20% acetone–diethyl ether. The yellow pre-
cipitate was washed two times with acetone and diethyl ether
56
1
to yield the respective triazole product in 96% yield (5 mg). H
NMR (400 MHz, DMSO) δ 8.65 (1H, s), 8.16–8.05 (1H, bm),
7.89 (1H, bm), 7.68–7.63 (2H, m), 7.13–6.94 (m, 4H), 6.66–6.64
(2H, d), 4.50–4.48 (2H, d), 4.40 (2H, m), 4.29 (1H, bm), 4.03
(2H, bm), 3.74 (2H, bm), 3.50–3.47 (24H), 3.37 (water), 3.24
(3H, s), 3.10–2.95 (6H, bm), 2.77–2.71 (2H, bm), 2.55–1.92 (6H,
bm), 1.55–1.52 (2H, bd), 1.10–1.05 (1H, bm,), 0.92 (2H, bm).
13C NMR (75 MHz, DMSO) δ 172.24, 166.68, 156.89, 154.24,
151.20, 149.09, 149.02, 148.96, 143.64, 134.27, 129.57, 129.41,
128.38, 121.84, 111.63, 71.72, 70.22, 70.0, 69.82, 58.49, 55.37,
47.59, 46.33, 25.81, 22.68, 22.42, 21.72, 19.63, 19.08, 17.72. MS
(ESI+) = 1025.5, a mixture of oligomers with and without Na.
Conjugate 7c
Method B: Ethylenediamine folate conjugate (10 mg,
0.021 mmol, 1 eq.) was dissolved in 2 ml of DMSO, followed by
the addition of diisopropylethyl amine (11 μl, 0.063 mmol,
Conjugate 7a
Method A: BCN-ethylenediamine-folate (3.5 mg, 0.0051 mmol, 3 eq.) and (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl succini-
1 eq.) was dissolved in 1 ml of DMSO and added to a flask con- midyl carbonate (6.5 mg, 0.022 mmol, 1.05 eq.). The reaction
taining 2 mg of benzyl azide55 (0.015 mmol, 3 eq.). The reac- mixture was stirred until a yellow solution is obtained (about
tion mixture was stirred for 2 hours before it was poured into a 30 minutes), followed by the addition of N-(2-azidoethyl)
solution of 20% acetone–diethyl ether. The yellow precipitate dansyl57 (23 mg, 0.072 mmol, 3.4 eq.). The reaction mixture
was washed two times with acetone and diethyl ether to yield was stirred for 5 hours before it was poured into a solution of
the respective triazole product in 95% yield (4 mg). 1H NMR 20% acetone–diethyl ether. The yellow precipitate was washed
(400 MHz, DMSO) δ 11.58, 8.65 (s, 1H), 8.15–8.02 (m, 1H), 7.88 two times with acetone and diethyl ether to yield the respective
(m, 1H), 7.68–7.66 (m, 2H), 7.37–7.27 (m, 3H), 7.14–6.94 (m, triazole product in 70% yield (14 mg). 1H NMR (400 MHz,
2H + m, 4H), 6.66–6.64 (m, 2H), 5.54 (s, 2H), 4.50 (bs, 2H), DMSO) δ 8.65 (s, 1H), 8.47–8.45 (d, 1H), 8.22–8.20 (d, 1H),
4.29 4.29 (m, 1H, αH), 4.01–3.97 (m, 2H), 3.36 (water), 8.08–8.03 (d, 1H), 7.90–7.88 (bs, 1H), 7.68–7.55 (m, 4H),
3.09–3.00 (m, 6H), 2.86–2.75 (m, 2H), 2.31–1.90 (m, 6H), 7.26–7.24 (d, 1H), 7.12–6.92 (m, 4H), 6.66–6.64 (d, 2H),
1.57–1.46 (m, 2H), 1.08–1.05 (m, 1H), 0.88–0.76 (m, 2H). 13C 4.50–4.49 (d, 2H), 4.31–4.24 (m, 3H), 4.01 (m, 2H), 3.36 (H2O),
NMR (100 MHz, DMSO) δ 172.40, 172.38, 172.25, 166.73, 3.19–3.03 (m, 6H), 2.83–2.68 (m, 8H), 2.34–1.91 (m, 6H), 1.48
166.66, 156.93, 156.84, 154.37, 151.21, 148.97, 144.37, 133.50, (m, 2H), 1.22–1.20 (m, 1H), 0.88 (bs, 2H). 13C NMR (100 MHz,
129.55, 129.40, 128.41, 128.23, 127.42, 121.87, 111.65, 61.87, DMSO) δ 174.54, 174.44, 172.40, 172.24, 166.69, 156.90,
51.11, 46.38, 39.13, 32.45, 25.93, 22.58, 22.09, 21.31, 19.16, 154.33, 151.83, 151.21, 149.02, 148.98, 143.70, 135.96, 133.83,
19.04, 17.60. MS (ESI+) = 793.33 (M + H)+. HRMS (ESI-FIA-TOF) 130.03, 129.53, 129.41, 129.36, 128.70, 128.40, 128.37, 124.01,
calc. = 793.3534, found = 793.3522.
121.88, 119.42, 115.61, 111.66, 65.37, 52.41, 47.57, 46.39,
Method B: Ethylenediamine folate conjugate (10 mg, 45.52, 42.82, 40.62, 40.41, 40.20, 39.99, 39.78, 39.57, 39.36,
0.021 mmol, 1 eq.) was dissolved in 2 ml of DMSO, followed by 32.46, 31.12, 25.86, 22.57, 22.37, 21.66, 19.62, 19.18, 17.77. MS
the addition of diisopropylethyl amine (11 μl, 0.063 mmol, (ESI+) = 979.24. HRMS (ESI-FIA-TOF) calc. = 979.3997, found =
3 eq.) and (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl succini- 979.3967.
midyl carbonate (6.5 mg, 0.022 mmol, 1.05 eq.). The reaction
mixture was stirred until a yellow solution is obtained (about
Conjugate 7d
30 minutes), followed by the addition of benzyl azide (5.6 mg, Method A: BCN-ethylenediamine-folate (6.9 mg, 0.01 mmol,
0.042 mmol, 2 eq.). The reaction was stirred for 2 hours before 1 eq.) was dissolved in 2 ml of DMSO and added to a flask con-
it was poured into a solution of 20% acetone–diethyl ether. taining 10 mg of 3-(1-(2-azidoethyl)thiourea) fluorescein58
The yellow precipitate was washed two times with acetone and (0.021 mmol, 2 eq.). The reaction was stirred for 4 hours
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before it was poured into a solution of 20% acetone–diethyl 25.91, 22.67, 22.12, 21.39, 19.22, 19.15, 19.06, 17.84, 17.84,
ether. The light orange precipitate was washed two times with 17.61, 14.07. MS (ESI+) = 1219.5; HRMS (ESI-FIA-TOF) calc. =
acetone and diethyl ether to yield the respective triazole 1219.5477, found = 1219.5432.
product in 88% yield (10.5 mg). 1H NMR (300 MHz, DMSO)
Conjugate 7f
δ 11.46 (bs, 1H), 10.16 (bs, 3H), 8.64 (s, 1H), 8.16–7.88 (m, 3H),
7.67 (m, 3H), 7.20–7.17 (d, 1H), 7.10–6.94 (m, 3H), 6.67–6.55 Method A: BCN-ethylenediamine-folate (7 mg, 0.01 mmol,
(m, 8H), 4.49 (m, 4H), 4.28 (m, 1H), 4.00–3.91 (m, 4H) 3.39 1 eq.) was dissolved in 2 ml of DMSO and added to a flask con-
(H2O), 3.00 (m, 6H) 2.73 (m, 2H), 2.31–1.91 (m, 6H), 1.55 (m, taining
5
mg of N-(2-azidoethyl)-7-nitrobenzofurazan 844
2H), 1.23 (s, 1H), 0.93 (bs, 2H). 13C NMR (101 MHz, DMSO) (0.02 mmol, 2 eq.). The reaction mixture was stirred for
δ 181.32, 174.43, 174.27, 172.42, 172.25, 168.91, 166.79, 4 hours before it was poured into a solution of 20% acetone–
159.94, 157.03, 156.92, 154.20, 152.34, 151.23, 149.17, 148.98, diethyl ether. The brown precipitate was washed four times
148.06, 144.00, 141.32, 134.07, 129.49, 128.39, 127.07, 124.64, with acetone and two times with diethyl ether to yield the
121.79, 113.05, 111.66, 110.12, 102.72, 83.50, 65.38, 52.54, respective triazole product in 68% yield (8 mg).
46.36, 43.99, 40.58, 40.37, 40.16, 39.95, 39.74, 39.53, 39.33,
1H NMR (300 MHz, DMSO) δ 8.64 (bs, 1H), 8.46–8.46 (d,
32.47, 30.91, 25.90, 22.61, 22.50, 21.86, 19.76, 19.34, 17.88. MS 1H, furazan), 8.01–7.89 (m, 2H), 7.66 (m, 2H), 7.09–6.92 (m,
(ESI+) = 1135.02. HRMS (ESI-FIA-TOF) calc. = 1135.3844, found 4H), 6.65–6.62 (m, 2H), 6.30 (d, 1H, furazan) 4.56–4.48 (m,
= 1135.3819.
4H), 4.29 (bs, 1H), 3.96 (m, 3H), 3.02–2.90 (m, 6H), 2.64 (m,
2H), 2.09–1.99 (m, 6H), 1.48 (m, 2H), 1.11–1.07 (m, 3H), 0.8
(m, 2H).
Conjugate 7e
Rhodamine 6G (4-(azidomethyl)phenyl)methyl ether. The
13C NMR (75 MHz, DMSO) δ 172.37, 166.67, 156.94, 156.86,
rhodamine 6G 4-chloromethyl-1-phenylmethyl ester chloride59 154.30, 151.20, 149.06, 148.98, 143.93, 138.09, 134.07, 129.54,
derivative (30.4 mg, 1 eq.), NaN3 (6.7 mg, 2 eq.) and NaI (cat.) 129.40, 128.40, 121.86, 121.60, 111.60, 99.83, 65.37, 61.88,
were dissolved in a mixture of acetone (1 ml) and water 53.47, 46.37, 32.50, 31.15, 25.77, 22.74, 22.44, 21.64, 19.76,
(0.5 ml); the resulting mixture was stirred at room temperature 17.74, 15.63.
for 19.5 h. The mixture was extracted with CH2Cl2. The organic
MS (ESI+, C40H44N16NaO10) = 931.33; HRMS (ESI-FIA-TOF,
fraction was dried with Na2SO4, filtered and the organics were M + H) calc. = 909.3504, found = 909.3488.
removed under vacuum. The remaining solid was recrystallized
Conjugate 7g
from AcOEt, EtOH and Et2O to give a greenish pink solid
(8.7 mg, yield = 28%). 1H NMR (300 MHz, DMSO) δ (ppm) 8.24 1st step – In an Aldrich glass reactor, 3-mercaptopropyl tri-
(dd, J = 7.6, 1.2 Hz, 1H), 7.83 (dtd, J = 19.3, 7.5, 1.4 Hz, 2H), methoxysilane (0.36 g, 1.8 mmol), 6-bromo-1-hexene (0.244 g,
7.36 (dd, J = 7.4, 1.1 Hz, 1H), 7.12 (d, J = 8.1 Hz, 2H), 6.86 (d, 1.5 mmol) and 2,2′-azo-bis-isobutyronitrile (0.12 g, 0.75 mmol)
J = 8.0 Hz, 2H), 6.74–6.66 (m, 4H), 4.89 (s, J = 10.5 Hz, 2H), were dissolved in 10 ml of chloroform. The reaction mixture
4.77 (s, 4H), 4.42 (s, J = 14.5 Hz, 2H), 2.04 (s, 6H), 1.25 (t, J = was heated for 24 h at 80 °C, before the solvent was removed
1
7.1 Hz, 6H). 13C NMR (75 MHz, DMSO) δ (ppm) 164.80, under reduced pressure. The crude was analyzed by H NMR,
156.57, 156.42, 155.65, 135.57, 134.51, 133.20, 131.01, 130.34, confirming the complete conversion of the alkene, and used in
129.43, 128.26, 128.17, 125.41, 112.78, 93.56, 66.42, 66.10, the next step without further purification.60
54.97, 53.39, 38.01, 17.74, 13.62. MS (ESI+) = 560.27. HRMS
calc. for C34H34ClN5O3: 560.2750, found: 560.2726.
2nd step – The crude thioether was refluxed in 15 ml of dry
toluene in the presence of flash chromatography grade silica-
Method A: BCN-ethylenediamine-folate (7 mg, 0.01 mmol, gel for 24 h under an argon atmosphere. After filtration and
1 eq.) was dissolved in 2 ml of DMSO and added to a flask con- washing with dichloromethane, 1.9 g of functionalized silica-
taining 14 mg of rhodamine 6G (4-(azidomethyl)phenyl) gel was isolated accounting for 95% yield over the two steps.60
methyl ether (0.024 mmol, 2.4 eq.). The reaction mixture was
3rd step – The bromide functionalized silica-gel (maximum
stirred for 4 hours before it was poured into a solution of 20% theoretical of 1.5 mmol) was reacted with 650 mg of sodium
acetone–diethyl ether. The purple precipitate was washed four azide (10 mmol) in DMF for 72 h at 80 °C. The crude solid was
times with acetone and two times with diethyl ether to yield filtered and washed thoroughly with water to remove the in-
1
the respective triazole product in 93% yield (13 mg). H NMR organic by-product salts, yielding 1.4 g of a white solid (72%
(400 MHz, DMSO) δ 8.57 (bs, 1H), 8.26–8.23 (d, 1H), 7.88–7.45 isolated yield).
(m, 11H), 7.10–6.60 (m, 12H), 5.53 (s, 2H), 4.90 (s, 2H),
4th step – Method B: Ethylenediamine folate conjugate
4.42–4.22 (m, 3H), 3.98 (m, 2H), 3.46–3.37 (water + CH2 rhoda- (20 mg, 0.041 mmol, 1 eq.) was dissolved in 4 ml of DMSO, fol-
mine), 3.01 (m, 6H), 2.80 (m, 2H), 2.05–1.92 (m, 12H), 1.51 (m, lowed by the addition of diisopropylethyl amine (22 µl,
2H), 1.29–1.25 (m, 7H), 0.85 (m, 2H). 13C NMR (75 MHz, 0.12 mmol, 3 eq.) and (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-
DMSO) δ 172.37, 165.12, 156.93, 156.93, 156.09, 151.25, ylmethyl succinimidyl carbonate (13 mg, 0.044 mmol,
150.97, 148.66, 148.22, 144.35, 136.56, 134.72, 133.71, 133.47, 1.05 eq.). The reaction mixture was stirred until a yellow solu-
131.42, 130.84, 130.76, 129.74, 128.88, 128.69, 128.49, 128.43, tion is obtained (about 30 minutes), followed by the addition
127.27, 125.79, 113.21, 113.21, 111.77, 111.74, 111.53, 93.94, of azide-functionalized silica-gel (260 mg, the maximum
66.79, 61.84, 50.80, 46.48, 46.42, 38.65, 38.47, 38.47, 25.99, theoretical 0.2 mmol of azides). The reaction was stirred for
3188 | Org. Biomol. Chem., 2014, 12, 3181–3190
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16 h, centrifuged and the light yellow solid (275 mg) was
washed thoroughly with acetone and ether. The yellow color
was absent from the supernatant indicating full immobiliz-
ation. This yellow powder was further eluted with DMSO, and
since no leaching of color to the mobile phase was detected it
further confirmed the chemical immobilization. Folic acid
physically immobilized onto silica-gel is eluted with DMSO. To
immobilize physically folic acid onto silica-gel, it was dissolved
in DMSO, and stirred for 30 minutes with silica. Then, the
DMSO was removed by washing with a mixture of 20%
acetone–diethyl ether.
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The linear absorption spectra were recorded on a Jasco V-660
UV-vis spectrophotometer, and the fluorescence measure-
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Spectrofluorimeter. The spectra were recorded in µmol solu-
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Cell staining procedure
Cell lines were cultured in RPMI-1640 medium supplemented
with 10% FBS and antibiotic antimycotic solution (100 units
ml−1 penicillin, 0.1 mg ml−1 streptomycin and 0.25 mg ml−1
amphotericin B) and grown in an incubator at 37 °C under a
5% CO2 atmosphere.
µ-Slide 8 well plates were coated with poly-L-lysine for at
least 30 min and washed before seeding with the cells. After
an incubation period of approximately 2 days, cells were
treated for 1 hour with 5 µM of the probe to be tested. For con-
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12.5 µM of 3) were added to the cell medium and kept for
incubation for a 24 hour period. After treatment, cells were
washed with phenol-red free medium and treated with 5 µg
ml−1 of the membrane staining WGA-Alexa594 that was kept
for 20–30 minutes in contact with the cells. After this staining
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medium was added to the wells before imaging.
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