B. Campbell et al.
Bioorganic Chemistry 84 (2019) 394–398
chosen sequence the same experiments were performed on A/DS-PNA/
T-Comp. As expected, the two fully intact PNA oligomers are observed
in the presence or absence of TCEP.
equipped with an X-Bridge column (BEH130PREP C18 5 μM,
10 × 250 mm) and eluted with water (solvent A) 0.1% TFA and an
increasing amount of 9:1 ACN:water (solvent B). Typical gradient is 5%
solvent B to 40% solvent B over 40 min. Samples collected from the
HPLC were analyzed by Matrix Assisted Laser Dissociation Ionization-
Time of Flight (MALDI-TOF) on a Bruker Daltonics Microflex LT.
MALDI data analysis was performed with a Polytools 1.0 software
package. UV–vis spectra (for PNA concentrations) and thermal dena-
turation studies were recorded on a Cary 100 UV–vis. PNA concentra-
tions were determined at 260 nm at 90 °C using extinction coefficients
obtained from Applied Biosystems extinction coefficients for PNA
Collectively, these observations indicate that the disulfide bond is
likely cleaved by TCEP, which results in two smaller strands originating
from DS-PNA that do not appear to form a stable duplex, even at low
temperatures (the experiment was started at 15 °C). However, these
results may not accurately reflect the availability of the antisense PNA
to interact with a transient mRNA target that is typically present at a
low concentration. Since PNA lack electrostatic repulsion, even short 5-
or 6-mer oligomers are still capable of forming homoduplexes.
Accordingly, additional measures may be necessary to ensure that the
fragments of the DS-PNA are ushered clear of the antisense strand. One
strategy would be to introduce additional disulfide linkages in the
backbone of the DS-PNA, albeit maintaining duplex stability, to render
small DS-PNA cleavage products once the disulfide bonds are cleaved.
Alternatively, since the incorporation of the disulfide can have rather
dramatic effects on the stability of the temporary but requisite duplex,
the DS-PNA could be truncated on either termini to yield 4-mer or
smaller cleavage produces. Future studies will be aimed at optimizing
disulfide position, determining appropriate cleavage products lengths,
and understanding the thermodynamic and kinetic parameters asso-
ciated with these design elements as they pertain to binding an RNA
target.
monomers
were
obtained
from
Applied
Biosystems
−1 −1
−
1
−1
−1
T = 8600 M cm−1; A = 13,700 M cm
−1
(C = 6600 M cm
;
;
G = 11,700 M−1 cm ;).
4.2. Thermal denaturation studies
Samples were heated to 90 °C, held there for 5 min, and then cooled
to 15 °C at 1 °C/min. Samples were held at this temperature for 5 min
and then heated to 90 °C at 1 °C/min. Data points were collected at
260 nm every 0.5 °C. All strand concentrations were analyzed at 1 μM in
1× PBS (pH 7.4).
4.3. HPLC cleavage studies
3
. Conclusion
Complementary PNA strands were combined at a concentration of
1
0 μM, heated to 90 °C, and then allowed to cool to room temperature
We have successfully synthesized a new Fmoc-disulfide monomer
over 2 h. The samples were then transferred to a 37 °C oven and TCEP
was added (final concentration 0.5 mM). After incubating for an hour
the samples were analyzed by HPLC using a Shimadzu LC-2-AD UFLC
equipped with a Phenomenex Kinetex colum (C 18 5 μ, 100 A,
250 × 4.6 mm) eluting with water with (solvent A) 0.1% TFA and an
increasing amount of (solvent B) 9:1 ACN:water (5% solvent B to 40%
solvent B over 40 min). Control samples were treated the same way
except TCEP was not added before the 1 hr incubation period.
that can be incorporated into a PNA oligomer using standard Fmoc-
based solid phase synthesis protocols. Incorporation of the monomer in
the middle of a 12-mer PNA reduces the stability of the duplexes by
roughly10 °C compared to a fully complementary strand, and interest-
ingly, duplex stability is most effected when the disulfide is paired with
T. Cleavage studies show that the disulfide bond is indeed susceptible to
chemical reduction when hybridized with a complementary PNA
strand. These findings provide the initial evidence that this approach
may have the potential to release an antigene or antisense oligomer.
Further investigations with different disulfide monomer derivatives and
spacing of the cleavage sites will yield additional insights and provide
direction towards designing second and third generation DS-PNA.
4.4. 2-Mercapto-2-methylpropanoic acid (1)
A flask equipped with a reflux condenser and a stir bar was charged
with of 2-bromo-2-methylpropionic acid (8.0 g, 47.9 mmol, 1 eqv). The
solid was melted gently with a heat gun (∼44–47 °C) and then a so-
lution of thiourea (4.56 g, 60 mmol, 1.25 eqv) in 8.00 mL of water was
added while stirring vigorously. The reaction was heated to 80 °C, at
which point the solution cleared (∼25 min), and then a solution of
NaOH (6 g, 150 mmol, 3.13 eqv) dissolved in 36 mL of water was
added. The reaction was heated to 95 °C for two hours. The solvent was
evaporated to leave a thick residue which was taken up in water
(20 mL) and then the pH was adjusted to ∼10 with HCl. The aqueous
solution was extracted with DCM (50 mL). The aqueous layer was then
acidified to pH 2–3 and extracted with DCM (3 × 50 mL). The organic
4
. Materials and methods
Synthetic reagents were purchase from Fisher Scientific or Sigma
Aldrich and used without further purification. PNA monomers were
purchased from PolyOrg, Inc. Reactions were performed under a
blanket of nitrogen with analytical grade solvents. Silica gel was ob-
1
13
tained from Sorbent Technologies. H and C spectra were obtained on
a JEOL 300 NMR spectrometer at 300 and 75 MHz, respectively, in
CDCl . Mass spectrometry on compounds 1–4 was performed on an
3
Agilent Infinity LC/MSD (G6125C) single quadrupole in the positive
washes were collected, dried with MgSO
4
, and then the solvent was
) δ 1.65
H) ppm. ESI-MS: m/z
1
mode from 60 to 500 m/z.
stripped to leave an oil. (Yield: 23%) H NMR (300 MHz, CDCl
3
(
s, 6H, 2xCH
3
H
), 2.52 (s, 1H, SH), 9.88 (s, 1H, CO
2
+
4.1. PNA synthesis, purification, and characterization
cacld. For C
4
8
O
2
S, [M] : 120.17, found 121.00.
PNA oligomers were synthesized using Fmoc conditions on PAL-
4.5. Di-tert-butyl (disulfanediylbis(ethane-2,1-diyl))dicarbamate (2)
mPeg resin. Fmoc protecting groups were removed with 20% piperidine
in DMF followed and checked using a Kaiser test. Monomers (1 eqv)
including compound 4, were dissolved in 135 μLNMP and then acti-
vated using HBTU (0.92 eqv) in DMF in the presence of DIPEA (1 eqv)
and lutidine (1 eqv) for 2.5 min before being added to the deprotected
resin where they were allowed to react for 1 h. A mini-peg unit was
added to the N-terminus of each PNA to increase water solubility [22].
Oligomers were cleaved from the resin using 5% m-cresol in TFA for 1 h
Di-tert-butyl-dicarbonate (1.75 g, 8 mmol, 2 eqv) and cystamine di-
hydrochloride (1 g, 4 mmol, 1 eqv) were charged to a 50 mL flask with a
stir bar and then the flask was sealed with a rubber septa and placed
under a blanket of nitrogen. Dioxane (3.6 mL) was charged to the flask
and the reactants were allowed to dissolve before adding NaOH (4.4 mL
of 2 M) dropwise over 5 min. The reaction was allowed to stir for 90 min
at room temperature. Dioxane and water were removed in vacuo and
the residue was taken up in ethyl acetate (200 mL). The organic layer
was washed with 1% HCl (3 × 50 mL) followed by brine, and then
(
×2).
PNA oligomers were purified using a Shimadzu LC-2-AD UFLC
397
Campbell, Brandon
