Nucleobase-caged peptide nucleic acids: PNA/PNA duplex destabilization and light-triggered PNA/PNA recognition

Article abstract of DOI:10.1002/psc.2514

The 2-(o-nitrophenyl)-propyl (NPP) group is used as caging group to mask the nucleobases adenine and cytosine in N-(2-aminoethyl)glycine peptide nucleic acids (aeg-PNA). The adeninyl and cytosinyl nucleo amino acid building blocks Fmoc-a^NPP-aeg

Full text of DOI:10.1002/psc.2514

Research Article
Received: 21 February 2013
Revised: 22 March 2013
Accepted: 24 March 2013
Published online in Wiley Online Library: 3 May 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2514
Nucleobase-caged peptide nucleic acids: PNA/
PNA duplex destabilization and light-triggered
PNA/PNA recognition
Samit Guha, Julia Graf, Björn Göricke and Ulf Diederichsen*
The 2-(o-nitrophenyl)-propyl (NPP) group is used as caging group to mask the nucleobases adenine and cytosine in
N-(2-aminoethyl)glycine peptide nucleic acids (aeg-PNA). The adeninyl and cytosinyl nucleo amino acid building blocks
Fmoc-aNPP-aeg-OH and Fmoc-cNPP-aeg-OH were synthesized and incorporated into PNA sequences by Fmoc solid phase
synthesis relying on high stability of the NPP nucleobase protecting group toward Fmoc-cleavage, coupling, capping, and
resin cleavage conditions. Removal of the nucleobase caging group was achieved by UV-LED irradiation at 365 nm. The
nucleobase caging groups provided sterical crowding effecting the Watson–Crick base pairing, and thereby, the PNA double
strand stabilities. Duplex formation can completely be suppressed for complementary PNA containing caging groups in both
strands. PNA/PNA recognition can be completely restored by UV light-triggered release of the photolabile protecting group.
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: caging group; double strand recognition; nucleobase pairing; peptide nucleic acid (PNA); photolabile protecting group
The synthesis of adenine and cytosine-caged nucleo amino
Introduction
acids Fmoc-aNPP-aeg-OH (1) and Fmoc-cNPP-aeg-OH (2) is
Caging with photolabile protecting groups involves the modification
of biologically active molecules to temporarily block and inactivate
functionalities [1,2]. The activity of biomolecules is masked by intro-
duction of caging groups at key positions allowing to restore the
function externally triggered by irradiation [3]. Various caging groups
with high selectivity and orthogonality are addressed providing
cleavage under mild conditions [4]. The caging group strategy can
be applied to various biomolecules such as ATP [1], amino acids [5],
proteins [6], nucleic acids [7,8], sugars [9], lipids [10] and as a
temporary photolabile N-terminal protecting group for solid
phase synthesis for N-(2-aminoethyl)glycine peptide nucleic acid
(aeg-PNA) [11]. An azobenzene photoswitch was further incorpo-
rated in aeg-PNA in order to gain photocontrol over DNA and
RNA hybridization [12]. However, nucleobase caging groups are
not applied so far for the modulation of aeg-PNA double strand
stabilities. Caging of aeg-PNA is of high relevance because aeg-PNA
is well-established as DNA and RNA structural mimic nicely
resembling oligonucleotide pairing complexes with high duplex
stabilities and resistance toward enzymatic degradation [13,14].
Despite numerous applications known for the use of aeg-PNA [15]
that will benefit from the availability of caged PNA, our need for a
light-triggered modulation of PNA/PNA duplex formation was
derived from a recently reported application of aeg-PNA as recog-
nition unit in artificial SNARE (soluble N-ethylmaleimide-sensitive
factor attachment protein receptor) protein-like peptide-PNA
hybrids for membrane fusion [16]. The approximation of lipid
vesicles is initiated by PNA/PNA duplex formation, thereby, starting
the membrane fusion process. In order to test the hypothesis for
SNARE-mediated membrane fusion following a zipper-like recogni-
tion process resulting in a tetrameric helical peptide bundle,
aeg-PNA oligomers were envisioned introducing sterical hindrance
for nucleobase recognition at one end of the forming duplex.
reported using the 2-(o-nitrophenyl)-propyl (NPP) caging group
as introduced by Pfleiderer [17]. Incorporation of the caged
nucleo amino acids in PNA oligomers by spss provided the
opportunity to study the modulation of PNA/PNA double strand
stability based on number and positioning of caging groups. A
stepwise reduction of stability by ΔTm = 5–3 ꢀC for each caging
group up to complete inhibition of duplex formation was
obtained. Complete recovery of PNA/PNA duplex formation was
induced by photocleavage.
2-(o-Nitrophenyl)-propyl was chosen as caging group because
it is compatible with the cleavage conditions in solid phase pep-
tide synthesis and it can be cleaved by irradiation at 365 nm
yielding a-methyl nitrostyrene as by-product that is less harmful
as, for example, nitrosoaldehyde resulting from ortho-nitrobenzyl
caging groups and respective derivatives [17–20]. With respect to
positioning, the NPP group at the nucleobase, Heckel reported
NPP-caged thymidine at the O4-position [21] and the O6-caged
guanosine [22]. Furthermore, he introduced the NPP caging group
in nucleotides at the N6-position of adenine and the N4-position
of cytosine [23]. In a first attempt, we decided to prepare the
N6-adenine and N4-cytosine-caged nucleo amino acids Fmoc-
aNPP-aeg-OH (1) and Fmoc-cNPP-aeg-OH (2). Caging groups at the
exocyclic amino groups are expected to influence the nucleobase
pairing for sterical reasons but still allow complete hydrogen bond-
ing at the Watson–Crick site. Therefore, N6-adenine and N4-cytosine
*
Correspondence to: Ulf Diederichsen, Institut für Organische und Biomolekulare
Chemie, Universität Göttingen, Tammannstraße 2, 37077 Göttingen, Germany.
E-mail: udieder@gwdg.de
Institut für Organische und Biomolekulare Chemie, Universität Göttingen,
Tammannstraße 2, 37077 Göttingen, Germany
J. Pept. Sci. 2013; 19: 415–422
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

GUHA ET AL.
cagings offer the chance for fine-tuning the duplex formation and
stabilities of PNA pairing complexes.
resulting solution was evaporated under vacuum. The product
5 (0.67 g, 1.70 mmol, 65%) was obtained after purification by flash
chromatography (pentane/acetone 1 : 1) as white solid. m/z (HR
1
ESI MS) calculated [M + H]+ 413.1932, found: 413.1932. H NMR:
Materials and Methods
dH (300 MHz, DMSO-d6) 8.16 (1H, s br, NH), 7.40–8.06 (6H, m, ArH),
4.92 (2H, s, CH2-COOtBu), 3.65ꢂ3.75 (3H, m, CH, CH2), 1.41 (9H, s,
tBu), 1.30 (3H, d, J = 6.0, CH3). 13C NMR: dC (75 MHz, DMSO-d6)
166.8 (CO), 154.4 (ArC), 152.3 (ArC), 150.3 (ArC), 150.3 (ArC), 150.3
(ArC), 138.2 (ArC), 132.6 (ArC), 128.4 (ArC), 127.2 (ArC), 123.4 (ArC),
119.1 (ArC), 82.0 (C(CH3), 45.1 (CH2), 44.4 (CH2), 33.2 (CH), 27.6 (C
(CH3)3), 19.5 (CH3).
General
Starting materials and reagents were purchased from Sigma Aldrich,
TCI, Fluka (Taufkirchen, Germany); N-methyl-2-pyrrolidone (NMP)
was obtained from Carl Roth GmbH. Fmoc-protected PNA building
blocks were purchased from ACM Research Chemicals (Malaysia)
and Fmoc-Gly-Wang resin from Novabiochem (Merck, Darmstadt.
Germany). Flash column chromatography was performed on Merck
silica gel 60 (40–60 mm) at 0.3–1.1 bar pressure. HPLC grade CH3CN and
ultra pure H2O (Millipore, Bedford, UK) were used for HPLC analysis.
2-(6-((2-(2-Nitrophenyl)propyl)amino)-9H-purin-9-yl)acetic acid (6)
Adeninyl acetate 5 (0.51 g, 1.29 mmol) was dissolved in 95% TFA
(5 ml) and stirred for 1 h at room temperature. The solvent was
removed under vacuum, and the residue was treated with cold
Et2O. The resulting white solid was washed with cold Et2O and
dried in vacuum. The product 6 (0.41 g, 1.16 mmol, 90%) was
obtained as a white solid. m/z (HR ESI MS) calculated [M + Na]+
379.1125, found 379.1117. 1H NMR: dH (300 MHz, DMSO-d6)
13.00 (1H, s br, COOH), 8.48 (1H, s br, NH), 8.31ꢂ7.41 (6H, m, ArH),
5.01 (2H, s, CH2-COOH), 3.78ꢂ3.63 (3H, m, CH, CH2), 1.33 (3H, d,
J = 6.9, CH3). 13C NMR: dC (75 MHz, DMSO-d6) 168.9 (COOH), 158.7
(ArC), 158.4 (ArC), 150.2 (ArC), 148.2 (ArC), 142.1 (ArC), 137.8 (ArC),
132.8 (ArC), 128.4 (ArC), 127.5 (ArC), 123.6 (ArC), 118.2 (ArC), 64.8
(CH2), 44.3 (CH2), 33.1 (CH), 19.4 (CH3).
Instruments
High-resolution mass spectra (HRMS-ESI) were obtained with the
Bruker Apex-Q IV FT-ICR-MS instrument (Bremen, Germany). HPLC
was performed on a Pharmacia Äkta Basic system (GE Healthcare,
London, UK) with a pump type P-900, variable wavelength detector
UV-900 using Nucleodur RP C-18 (250 ꢁ 4.6 mm, 5 mm) analytical
HPLC column. All HPLC runs were performed by using a linear gra-
dient of 0.1% aq. TFA and 80% aq. CH3CN/0.1% TFA. Uncaging of
amino acids and PNA oligomers was performed by irradiation with
UV-LEDs (M365L2, driver LEDD1B, Thorlabs) operated at 0.5 A.
UV-melting curves were recorded with a Jasco V-550 UV spec-
trometer (Gross-Umstadt, Germany) by using a Jasco ETC.-
505S/ETC.-505 T peltier temperature controller. Data were
collected at 260 nm in buffer (20 mM HEPES, 100 mM NaCl, 1 mM
EDTA, pH = 7.4, 4 mm) applying the following temperature protocol:
20 ꢀC ! 85 ꢀC, 85 ꢀC (equilibration for 5 min), 85 ꢀC ! 0ꢀC (1ꢀC/min),
0ꢀC (equilibration for 10 min), 0 ꢀC ! 85 ꢀC (1ꢀC/min), 85 ꢀC! 20 ꢀC.
The hyperchromicity (H) was calculated according to the equation:
H(%)= 100*[A(T)ꢂA0]/A0, where A(T) is the absorbance given at any
temperature and A0 is the minimum absorbance.
Fmoc-aNPP-aeg-OtBu (7)
Adeninyl acetate 6 (0.40 g, 1.12 mmol) was dissolved in dry DMF
(3 ml) and cooled to 0 ꢀC under inert atmosphere. PyBOP
(0.58 g, 1.12 mmol) and Fmoc-aeg-OtBu HCl (0.40 g, 0.93 mmol)
were added and the solution was stirred for 10 min at 0 ꢀC. DIPEA
(0.73 ml, 4.20 mmol) was added slowly at 0 ꢀC and the solution
was stirred for 4 h at room temperature. The solvent was evapo-
rated and the residue was dissolved in ethyl acetate. The organic
layer was washed with 10% NH4Cl-solution (3ꢁ 20 ml), 6%
NaHCO3-solution (3ꢁ 20ml) and brine (3 ꢁ 20 ml), dried over an-
hydrous Na2SO4, and the solvent was evaporated. Nucleo amino
acid 7 (0.50 g, 0.68 mmol, 61%) was obtained after purification by
flash chromatography (DCM/MeOH 30:1) as a light yellow solid.
m/z (HR ESI MS) calculated [M+ Na]+ 757.3069, found 757.3065.
1H NMR: dH (300 MHz, DMSO-d6) 8.13 (1H, s br, NH), 7.88ꢂ7.30
(14H, m, ArH), 5.18 (1H, s br, NH), 4.40ꢂ4.20 (5H, m, CH2ꢂCOOtBu,
OCH2, Fmoc-CH), 3.94 (2H, s, CH2), 3.78ꢂ3.65 (4H, m, 2 ꢁ CH2),
3.54ꢂ3.52 (2H, m, CH2), 3.45ꢂ3.44 (1H, m, CH), 1.40 (9H, s, C(CH3)
3), 1.29 (3H, d, J = 6.0, CH3). 13C NMR: dC (75 MHz, DMSO-d6) 168.4,
167.9, 166.8, 156.3, 156.1, 152.1, 150.3, 143.8, 141.4, 140.6, 138.2,
132.6, 128.4, 127.5, 127.2, 126.9, 125.0, 123.4, 120.0, 82.0, 80.9,
65.4, 50.1, 50.0, 48.7, 47.0, 46.7, 43.6, 43.3, 37.9, 33.3, 27.6, 19.5.
Synthesis of Nucleo Amino Acids Fmoc-aNPP-aeg-OH (1) and
Fmoc-cNPP-aeg-OH (2)
tert-Butyl-2-(6-chloro-9H-purin-9-yl)acetate (3)
6-Chloropurine (1.20 g, 7.76 mmol) and anhydrous K2CO3 (1.08 g,
7.76 mmol) were suspended in dry DMF (24 ml). tert-Butyl
bromoacetate (1.40 g, 7.18 mmol) was added dropwise and the
reaction mixture was stirred for 20 h at 50 ꢀC. The solid was fil-
tered off and the organic solvent was evaporated. The residue
was treated with 0.5 N HCl (20 ml) and stirred for 20 min. After fil-
tration, the residue was washed with H2O. The product 3 (1.3 g,
5.2 mmol, 67%) was obtained as a light yellow solid. m/z (HR ESI
MS) calculated [M + H]+ 269.0800, found 269.0797. 1H NMR: dH
(300 MHz, DMSO-d6) 8.78 (1H, s, ArH), 8.66 (1H, s, ArH), 5.14 (2H,
s, CH2), 1.41 (9H, s, C(CH3)3). 13C NMR: dC (75 MHz, DMSO-d6) 166.1
(CO), 152.0 (ArC), 151.7 (ArC), 149.0 (ArC), 147.8 (ArC), 130.4 (ArC),
82.5 (C(CH3)3), 45.2 (CH2), 27.5 (C(CH3)3).
Fmoc-aNPP-aeg-OH (1)
Nucleo amino acid 7 (0.48 g, 0.66 mmol) was dissolved in TFA
(5 ml) and stirred for 1 h at room temperature. The solvent was
removed under vacuum and the residue was treated with cold
Et2O. The resulting solid was washed with cold Et2O and dried
in vacuum. Nucleo amino acid 1 (0.39 g, 0.58 mmol, 88%) was
obtained as a white solid. m/z (HR ESI MS) calculated [M-H]ꢂ
677.2478, found 677.2480. 1H NMR: dH (300 MHz, DMSO-d6)
12.50 (1H, s br, COOH), 8.66 (1H, s br, NH), 8.30ꢂ7.29 (14H, m,
ArH), 5.27 (1H, s br, NH), 4.37ꢂ4.19 (5H, m, CH2ꢂCOOH, OCH2,
Fmoc-CH), 4.01 (2H, s, CH2), 3.68ꢂ3.53 (4H, m, 2 ꢁ CH2), 3.39ꢂ3.34
tert-Butyl 2-(6-((2-(2-nitrophenyl)propyl)amino)-9H-purin-9-yl)
acetate (5)
Purinyl acetate 3 (0.66 g, 2.61 mmol) was dissolved in anhydrous
EtOH (7.30 ml). 2-(2-Nitrophenyl)propylamine (4, prepared as de-
scribed in [23], 0.94 g, 5.22 mmol) and Et3N (0.70 ml, 5.00 mmol)
were added and the reaction mixture was refluxed for 40 h. The
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 415–422

CAGED PEPTIDE NUCLEIC ACIDS
(3H, m, CH, CH2), 1.32 (3H, d, J = 6.0, CH3). 13C NMR: dC (75 MHz,
DMSO-d6) 170.7, 168.0, 158.5, 158.2, 157.9, 156.3, 156.1, 150.1,
143.8, 140.7, 137.7, 132.8, 128.4, 127.5, 127.0, 125.0, 123.6, 120.1,
116.8, 114.5, 65.5, 49.1, 47.7, 46.9, 46.6, 44.2, 37.9, 33.1, 19.4.
Fmoc-cNPP-aeg-OtBu (11)
Cytosinyl acetate 10 (0.80 g, 2.41 mmol) was dissolved in dry DMF
(5 ml) and cooled to 0 ꢀC under inert atmosphere. PyBOP (1.25 g,
2.41 mmol) and N-(2-aminoethyl)glycine Fmoc-aeg-OtBu HCl (0.87 g,
2.01 mmol) were added and the solution was stirred for 10 min at
0 ꢀC. DIPEA (1.58 ml, 9.05 mmol) was added slowly at 0 ꢀC and the
solution was stirred for 4 h at room temperature. The solvent was
evaporated, and the residue was dissolved in ethyl acetate. The
organic layer was washed with 10% NH4Cl solution (3ꢁ 20 ml), 6%
NaHCO3 solution (3 ꢁ 20 ml) and brine (3 ꢁ 20 ml), dried over an-
hydrous Na2SO4, and the solvent was evaporated. Nucleo amino
acid 11 (1.04 g, 1.46 mmol, 73%) was obtained after purification
by flash chromatography (DCM/MeOH 30:1) as a light yellow
solid. m/z (HR ESI MS) calculated [M + H]+ 711.3137, found
tert-Butyl-uracil-1-yl-acetate (8)
Uracil (2.20 g, 19.8 mmol) and anhydrous K2CO3 (2.74 g,
19.8 mmol) were suspended in dry DMF (60 ml). tert-Butyl
bromoacetate (3.51 g, 18.0 mmol) was added dropwise, and the
reaction mixture was stirred for 20 h at room temperature. The
solid was filtered off, and the organic solvent was evaporated.
The residue was treated with 0.5 N HCl (50 ml) and stirred for
20 min. After filtration, the residue was washed with H2O. The
product 8 (3.37 g, 14.9 mmol, 75%) was obtained as a white solid.
m/z (HR ESI MS) calculated [M + Na]+ 249.0846, found 249.0847.
1H NMR: dH (300 MHz, CDCl3) 9.60 (1H, s br, NH), 7.10 (1H, d,
J = 7.9, CHCH), 5.72 (1H, d, J = 7.9, CHCH), 4.35 (2H, s, CH2), 1.45
(9H, s, C(CH3)3). 13C NMR: dC (75 MHz, CDCl3) 166.3 (CO), 163.7
(CO), 150.8 (CO), 144.5 (CC), 102.4 (CC), 83.5 (C(CH3)3), 49.3
(CH2), 27.9 (C(CH3)3).
1
711.3136. H NMR: dH (300 MHz, DMSO-d6) 7.87ꢂ7.25 (14H, m,
12 ArH, Fmoc-NH, CHCH), 5.74 (1H, s br, NH), 5.62 (1H, d, J = 7.2,
CHCH), 4.68ꢂ4.19 (5 H, m, CH2ꢂCOOtBu, OCH2, Fmoc-CH), 3.93
(2H, s, CH2), 3.66ꢂ2.98 (7H, m, CH, CH2, CH2ꢂCH2), 1.40 (9H, s, C
(CH3)3), 1.27 (3H, d, J = 6.5, CH3). 13C NMR: dC (75 MHz, DMSO-d6)
168.3, 167.9, 167.8, 163.9, 156.0, 155.4, 149.8, 145.5, 143.6, 140.5,
137.8, 132.7, 128.1, 127.3, 126.8, 124.9, 123.4, 119.8, 93.5, 80.7,
65.4, 65.3, 61.8, 54.8, 46.7, 45.3, 44.3, 33.3, 27.6, 19.5.
tert-Butyl-2-(4-((2-(2-nitrophenyl)propyl)amino)-2-oxopyrimidin-1
(2H)-yl)-acetate (9)
Fmoc-cNPP-aeg-OH (2)
DMAP (0.38 g, 3.09 mmol), mesitylene-2-sulfonyl chloride (1.74 g,
7.96 mmol), and Et3N (3.07 ml, 22.1 mmol) were added to a solution
of 8 (1.00 g, 4.42 mmol) in dry DCM (50 ml) under inert atmosphere.
Afterwards, the solution was stirred for 6 h at room temperature.
The solvent was evaporated under vacuum and the residue was
dissolved in dry DCM (40 ml) and 2-(2-nitrophenyl)propylamine
(4) (1.59 g, 8.84 mmol) was added. Afterwards, LiCl (1.05 g,
24.8 mmol) and Et3N (5.24 ml, 37.6 mmol) were added slowly at
0 ꢀC and the solution was stirred for 20 h at room temperature.
The resulting solution was evaporated under vacuum. The product
9 (1.28 g, 3.30 mmol, 75%) was obtained after purification by flash
chromatography (DCM/EtOAc 3:1 ! 2:1) as a white solid. m/z
(HR ESI MS) calculated [M+ H]+ 389.1819, found 389.1817. 1H
NMR: dH (300 MHz, DMSO-d6) 7.79 (1H, d, J = 7.5, CH = CH),
7.70ꢂ7.42 (4H, m, ArH), 5.62 (1H, d, J = 7.5, CH= CH), 4.30 (2H, s,
CH2), 3.67ꢂ3.55 (1H, m, CH), 3.52–3.36 (2H, m, CH2), 3.29 (1H, s br,
NH), 1.40 (9H, s, C(CH3)3), 1.27 (3H, d, J = 6.5, CH3). 13C NMR: dC
(75 MHz, DMSO-d6) 167.5 (CO), 164.0 (CO), 155.5 (CN), 149.9 (ArC),
145.0 (ArC), 137.8 (ArC), 132.7 (ArC), 128.1 (ArC), 127.3 (ArC), 123.4
(CC), 93.7 (CC), 80.9 (C(CH3)3), 50.4 (CH2), 45.4 (CH2), 33.2 (CH), 27.6
(C(CH3)3), 19.5 (CH3).
Fmoc-cNPP-aeg-OtBu (11) (0.56 g, 0.79 mmol) was dissolved in TFA
(5 ml) and stirred for 1 h at room temperature. The solvent was re-
moved under vacuum, and the residue was treated with cold
Et2O. The resulting white solid was washed with cold Et2O and
dried in vacuum. Nucleo amino acid 2 (0.49 g, 0.75 mmol, 96%)
was obtained as white solid. m/z (HR ESI MS) calculated [M + H]+
655.2511, found 655.2510. 1H NMR: dH (300 MHz, DMSO-d6)
12.30 (1H, s br, COOH), 7.96–7.27 (14H, m, 12 ArH, Fmoc-NH, CHCH),
5.92 (1H, d, J = 7.6, CHCH), 4.83–4.62 (2H, m, CH2), 4.35–4.23 (4H, m,
Fmoc-CH, OCH2, NH), 4.00 (2H, s, CH2), 3.81–3.57 (3H, m, CH2, CH),
3.49–3.10 (4H, m, 2 ꢁ CH2), 1.30 (3H, d, J = 6.6, CH3). 13C NMR: dC
(75 MHz, DMSO-d6) 170.4, 166.9, 166.5, 159.5, 158.2, 157.9, 156.1,
149.6, 147.4, 143.6, 140.5, 137.1, 132.9, 128.1, 127.6, 127.4, 126.8,
124.9, 123.7, 119.9, 93.8, 65.3, 64.7, 54.4, 48.2, 47.1, 46.7, 32.2, 19.3.
Solid Phase Synthesis of Caged PNA Oligomers
Synthesis of the PNA oligomers was performed as solid phase
synthesis in a 15 mm scale on an Fmoc-Gly-preloaded Wang resin
(0.33 mmol gꢂ1). The resin was swollen in a BD (Becton Dickinson,
Fraga, Spain) syringe for 2 h in NMP before starting the coupling
cycle by Fmoc-deprotection applying 20% piperidine in NMP
(2 ꢁ 10 min) followed by successive washing with NMP (3ꢁ), DCM
(3ꢁ), and NMP (3ꢁ). Pre-activation and coupling: The respective
nucleo amino acid (4.0 equiv.) was dissolved in stock solutions of
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium (HATU)
(0.24 M, 3.9 equiv.) and 1-hydroxy-7-azabenzotriazole (HOAt)
(0.25 M, 4.0 equiv.) in NMP. Directly before transferring the coupling
cocktail to the resin, DIEA (0.2 M) and 2,6-lutidine (0.3M) were
added to the pre-activated mixture. Double coupling for 1 h each
was followed by washing with NMP, DCM, and NMP (3ꢁ each).
Capping was carried out by treatment with a solution of Ac2O
and 2,6-lutidine in NMP (1 : 2 : 7 v/v/v, 2 ꢁ 5 min) followed by
washing with 5% DIEA in NMP (3ꢁ) and with DCM (3ꢁ), NMP (6ꢁ),
DCM (3ꢁ), and NMP (3ꢁ). For N-terminal Fmoc-deprotection,
20% piperidine in NMP (2 ꢁ 5 min) was employed followed by
washing with NMP, DCM, and NMP (3ꢁ each).
2-(4-((2-(2-Nitrophenyl)propyl)amino)-2-oxopyrimidin-1(2H)-yl)-acetic
acid (10)
Cytosinyl acetate 9 (0.50 g, 1.29 mmol) was dissolved in 95% TFA
(5 ml) and stirred for 1 h at room temperature. The solvent was
removed under vacuum, and the residue was treated with cold
Et2O. The resulting white solid was washed with cold Et2O and
dried in vacuum. The product 10 (0.41 g, 1.23 mmol, 95%) was
obtained as a white solid. m/z (HR ESI MS) calculated [M + Na]+
1
355.1013, found 355.1015. H NMR: dH (300 MHz, DMSO-d6) 12.4
(1H, s br, COOH), 7.86ꢂ7.46 (5H, m, 4 ArH and CHCH), 5.93 (1H,
d, J = 7.6, CHCH), 4.5 (1H, s br, NH), 4.49 (2H, s, CH2), 3.84ꢂ3.54
(2H, m, CH2), 3.52ꢂ3.34 (1H, m, CH), 1.30 (3H, d, J = 6.7, CH3). 13C
NMR: dC (75 MHz, DMSO-d6) 168.9 (COOH), 159.6 (CO), 150.5
(CN), 149.7 (ArC), 146.9 (ArC), 137.2 (ArC), 132.9 (ArC), 127.9
(ArC), 127.6 (ArC), 123.5 (CC), 94.0 (CC), 49.8 (CH2), 46.6 (CH2),
32.9 (CH), 19.3 (CH3).
J. Pept. Sci. 2013; 19: 415–422 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

GUHA ET AL.
After the final coupling cycle, the resin was washed with NMP,
DCM, MTBT, and MeOH (5ꢁ each) and dried in vacuum. Cleavage
of PNA oligomers from Wang resin and simultaneous benzhydryl-
oxycarbonyl (Bhoc)-deprotection were obtained using a TFA/m-
cresol/TIS cleavage-cocktail (92.5/5.0/2.5 v/v/v) for 2 h at ambient
temperature. The reaction mixture was concentrated under N2
stream followed by precipitation using cold Et2O (3ꢁ). The
resulting suspension was centrifuged at ꢂ5 ꢀC. The supernatant
was discarded and the compound washed with Et2O several
times and dried. The following PNA oligomers were obtained
with >98% purity as indicated by RP-HPLC and characterized
by ESI HR mass spectrometry as follows:
Results and Discussion
Synthesis of the nucleo amino acids Fmoc-aNPP-aeg-OH (1) and
Fmoc-cNPP-aeg-OH (2) (Schemes 1 and 2) was provided by
NPP protection of the respective nucleobase derivatives 6-
chloropurine-9-tert-butylacetate (3) and tert-butyl-uracil-1-yl-
acetate (8). The NPP group was introduced by nucleophilic
substitution starting from 6-chloropurine, whereas uracil was
converted to the cytosine derivative by O4 mesitylene sulfonate
activation followed by NPP protection. The NPP caging group
turned out to be stable under nucleo amino acid preparation
and PNA oligomer synthesis conditions, thereby, serving as
permanent protection until photochemical cleavage. The NPP
protected adeninyl and cytosinyl acetic acid derivatives 6 and
10 were coupled to the secondary amine of the Fmoc-
protected aminoethylglycine tert-butylester using PyBOP activa-
tion. Finally, ester hydrolysis with TFA provided the NPP-caged
nucleo amino acids 1 and 2 as monomeric units for solid phase
PNA synthesis.
Solid phase PNA oligomer synthesis following the Fmoc-
protocol was applied to provide PNA oligomers PNA1
and PNA2 and the NPP-caged nucleo amino acid containing
PNA3–PNA8 (Figure 1). For the exocyclic amino groups of cyto-
sine, adenine and guanine of the regular aeg-nucleo amino acids,
the Bhoc (benzhydryloxycarbonyl) protecting group was applied
[24]. The Fmoc/NPP and Fmoc/Bhoc protected nucleo amino
acids were compatible with the Fmoc/tBu SPPS protocol used
for PNA oligomer synthesis on a Wang resin. Peptide coupling
was provided with HATU/HOAt under optimized conditions
regarding a slight excess of nucleo amino acids (4 equiv.) used
with respect to HATU (3.9 equiv.) and a pre-activation of the
amino acid prior addition to the resin. After cleavage from the
Wang resin and purification, the PNAs 1–8 were obtained in a
purity >98% as determined by HPLC. The oligomers were charac-
terized by high-resolution mass spectrometry.
PNA1 (H-gtagatcact-Gly-OH): HPLC (Nucleodur RP-C18,
250 ꢁ 4.6 mm, 5 mm, gradient 0–25%): tR = 27.7 min. m/z (HR ESI
MS) calculated [M + 3H]3+ 929.0399, found 929.0399.
PNA2 (H-catctagtga-Gly-OH): HPLC (Nucleodur RP-C18,
250 ꢁ 4.6 mm, 5mm, gradient 0–25%): tR = 27.9 min. m/z (HR ESI MS)
calculated [M + 4H]4+ 697.0317, found 697.0312.
PNA3 (H-gtaNPPgatcact-Gly-OH): HPLC (Nucleodur RP-C18,
250 ꢁ 4.6 mm, 5mm, gradient 0–25%): tR = 35.4 min. m/z (HR ESI MS)
calculated [M + 4H]4+ 737.7976, found 737.7970.
PNA4 (H-gtaNPPgaNPPtcact-Gly-OH): HPLC (Nucleodur RP-C18,
250 ꢁ 4.6 mm, 5mm, gradient 0–25%): tR = 36.7 min. m/z (HR ESI MS)
calculated [M + 3H]3+ 1037.7488, found 1037.7486.
PNA5 (H-gtaNPPgaNPPtcaNPPct-Gly-OH): HPLC (Nucleodur RP-C18,
250 ꢁ 4.6 mm, 5 mm, gradient 0–25%): tR = 36.9 min. m/z (HR ESI MS)
calculated [M + 5H]5+ 655.6649, found 655.6651.
PNA6 (H-cNPPatctagtga-Gly-OH): HPLC (Nucleodur, RP-C18,
250 ꢁ 4.6 mm, 5mm, gradient 0–25%): tR = 34.6 min. m/z (HR ESI MS)
calculated [M + 4H]4+ 737.7976, found 737.7978.
PNA7 (H-catcNPPtagtga-Gly-OH): HPLC (Nucleodur, RP-C18,
250 ꢁ 4.6 mm, 5mm, gradient 0–25%): tR = 35.6 min. m/z (HR ESI MS)
calculated [M + 3H]3+ 983.3944, found 983.3945.
PNA8 (H-cNPPatcNPPtagtga-Gly-OH): HPLC (Nucleodur, RP-C18,
250 ꢁ 4.6 mm, 5mm, gradient 0–25%): tR = 36.9 min. m/z (HR ESI MS)
calculated [M + 4H]4+ 778.5634, found 778.5637.
The influence of the NPP caging group on PNA/PNA double
strand formation and stability was investigated for the PNA
HN
N
Cl
H2N
HN
N
Cl
N
BrCH2COOtBu
K2CO3, DMF
NO2
NO2
NO2
N
N
TFA, 1 h
90%
4
N
N
N
N
N
N
N
N
N
EtOH, Et3N
reflux, 40 h
N
H
N
50 oC, 20 h
O
O
O
67%
65%
OH
OtBu
OtBu
3
6
5
H
N
COOtBu
FmocHN
DMF, PyBOP, DIEA, 4 h
61%
HN
HN
NO2
NO2
N
N
N
N
N
N
N
N
TFA, 1 h
88%
O
O
O
O
N
N
7
FmocHN
OH
FmocHN
OtBu
1
Scheme 1. Synthesis of caged aeg-PNA building block Fmoc-aNPP-aeg-OH (1); NPP = o-nitrophenylpropyl, DIEA = N,N-diisopropylethylamine;
PyBOP = benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; Fmoc = 9-fluorenyl methoxycarbonyl; TFA = trifluoroacetic acid.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 415–422

CAGED PEPTIDE NUCLEIC ACIDS
1. DCM, DMAP,
mesitylene sulfonyl
chloride, Et3N, 6 h
O
N
HN
HN
N
O
BrCH2COOtBu
NO2
TFA, 1h
95%
NO2
NH
N
N
O
NH
O
2.
N
O
N
H
O
O
OH
K2CO3, DMF,
20 h, 75%
O
O
H2N
OtBu
OtBu
NO2
10
4
9
8
LiCl, Et3N, 12 h
75%
H
N
COOtBu
FmocHN
DMF, PyBOP, DIEA, 4 h
73%
HN
HN
N
NO2
NO2
N
TFA, 1h
N
O
N
O
96%
O
O
O
O
N
N
OtBu
11
FmocHN
FmocHN
OH
2
Scheme 2. Synthesis of caged aeg-PNA building block Fmoc-cNPP-aeg-OH (2).
Figure 1. PNA decamer sequences with the position of the NPP caging group indicated. PNAs 1, 3–5 have a sequence complementarity to PNAs 2, 6–8 with
respect to double strand formation with parallel strand orientation.
decamers PNA1 and PNA2 with complementary sequence for
Watson–Crick base pair formation in parallel strand orientation
(Figure 2). As indicated by temperature dependent UV spectros-
copy, the duplex PNA1 + PNA2 is formed with a melting temper-
ature of Tm = 45 ꢀC (4 mM, 20 mM HEPES, 100 mM NaCl, 1 mM
EDTA, pH = 7.4, 260 nm) [25]. The increase of absorption
(hyperchromicity) with increasing temperature provides a sig-
moidal curve due to the cooperative destacking of nucleobase
pairs indicating the transition from duplex to single strands.
The double strand stability is expressed as melting temperature
at the inflection point. The stabilities of PNA3 + PNA2 and
PNA1 + PNA6 with one NPP caging group in the PNA/PNA
pairing complexes provided a drop in stability of ΔTm = 5 ꢀC. In-
troduction of a second NPP caging group in the PNA/PNA du-
plex resulted in an additional loss of stability of ΔTm = 4 ꢀC as
indicated for the combinations PNA4 + PNA2 and PNA1 + PNA8.
The influence of the caging groups was not dependent on
caging adenine or cytosine. Nevertheless, the additional caging
group was placed within the same oligomer and adding a third
caging group to one of the oligomers provided an additional
loss in overall duplex stability of ΔTm = 3 ꢀC (PNA5 + PNA2). In
case PNA5 carrying three NPP caging groups was combined
with a complementary strand that had two additional caging
groups (PNA8), duplex formation was completely prevented
J. Pept. Sci. 2013; 19: 415–422 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

GUHA ET AL.
(a)
(b)
20
20
15
10
5
15
10
5
PNA1/PNA2
Tm = 45 C
PNA3/PNA2
Tm = 40 C
0
0
0
0
0
20
40
40
40
60
60
60
80
80
80
0
20
40
60
60
60
80
80
80
T / C
T / C
T / C
T / C
(c)
(d)
20
15
10
5
20
15
10
5
PNA5/PNA2
Tm = 33 C
PNA4/PNA2
Tm = 36 C
0
0
0
20
40
20
T / C
(e)
(f)
20
15
10
5
20
15
10
5
PNA1/PNA8
Tm = 36 C
PNA1/PNA6
Tm = 40 C
0
0
0
20
40
20
T / C
Figure 2. Temperature dependent UV absorption for double strand formation with parallel strand orientation of PNA1 and PNA2 (a) and PNAs with
one (b, e), two (c, f), and three caging groups (d) (4 mM, 20 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH = 7.4, 260 nm).
(Figure 3). Uncaging induced by irradiation at 365 nm using
UV-LED provided recovery of the duplex forming stability of
Tm = 45 ꢀC as it was determined for PNA1 + PNA2.
to uncaging of PNA oligomers. Because the efficiency of photo-
chemical uncaging depends on the molar extinction coefficient
and photochemical quantum yield, NPP deprotection at 365 nm
is relatively slow [26] and will be improved by modulating the
UV light intensity or using alternative caging groups with higher
extinction coefficient.
The UV light-triggered release of the photolabile NPP
protecting group was further monitored by RP-HPLC and ESI-MS
techniques (Figure 4). The time dependency of the NPP
deprotection of a 50 pmol sample of caged nucleo amino acid or
PNA oligomer was provided within 1 h under irradiation with a
UV-LED at 365 nm. These UV-LEDs have about 100 times the power
of mercury lamps and hence remarkably clean uncaging was
reached. Deprotection of the nucleo amino acids as shown in
Figure 4 is comparable with respect to speed and cleanness
Conclusion
An efficient procedure for the synthesis of two new nucleobase-
caged aeg-PNA nucleo amino acids Fmoc-aNPP-aeg-OH (1) and
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CAGED PEPTIDE NUCLEIC ACIDS
(a)
(b)
20
20
15
10
5
15
10
5
PNA5/PNA8
0
0
0
20
40
T / C
60
80
0
20
40
T / C
60
80
Figure 3. (a) Overall five NPP caging groups in the combination of PNA5 and PNA8 prevent double strand formation as indicated by temperature de-
pendent UV profile (4 mM, 20 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH = 7.4, 260 nm); (b) UV-melting curve after irradiation at 365 nm using an UV-LED.
100
80
60
40
20
0
100
80
60
40
20
0
Fmoc-cNPP-aeg-OH (caged)
Fmoc-c-aeg-OH (uncaged)
Fmoc-aNPP-aeg-OH (caged)
Fmoc-a-aeg-OH (uncaged)
0
20
40
60
0
10
20
30
40
50
60
Time / min
Time / min
Figure 4. NPP deprotection of nucleo amino acids Fmoc-aNPP-aeg-OH (1) and Fmoc-cNPP-aeg-OH (2) initiated by irradiation at 365 nm using an UV-LED.
Fmoc-cNPP-aeg-OH (2) was developed. Incorporation into PNA se-
and biology: reaction mechanisms and efficacy. Chem. Rev. 2013;
113: 119–191.
quences by Fmoc-solid phase chemistry was provided with opti-
2 Deiters A. Light activation as a method of regulating and studying
mized coupling and cleavage conditions yielding respective
gene expression. Curr. Opin. Chem. Biol. 2009; 13: 678–686.
PNA oligomers with >98% purity prior to HPLC purification. The
NPP nucleobase caging group was highly stable under Fmoc spss
conditions and thus can be readily incorporated into caged PNA/
peptide hybrids for biological applications. Incorporation of one,
two, or three caging groups in one PNA strand resulted in drop
of a ten-base pair duplex stability by ΔTm = 5 ꢀC, 9 ꢀC, and 12 ꢀC, re-
spectively. Duplex formation was even suppressed by introduction
of NPP caging groups in opposed PNA oligomers. This fine-tuning
and fast recovery of the duplex stability by photochemical
deprotection provides a perfect tool for the manipulation of recog-
nition complexes that imitate a zipper-like or a two-stage recogni-
tion process as we are currently investigating in context of SNARE-
mediated membrane fusion.
3 Mayer G, Heckel A. Biologically active molecules with a “light switch”.
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8 Jain PK, Shah S, Friedman SH. Patterning of gene expression using
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S. G. acknowledges the Alexander von Humboldt foundation,
Germany, for financial assistance. Generous support from the
Deutsche Forschungsgemeinschaft DFG (SFB 803) is gratefully
acknowledged.
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