Research Article

Received: 21 February 2013 Revised: 22 March 2013 Accepted: 24 March 2013 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/psc.2514

Nucleobase-caged peptide nucleic acids: PNA/PNA duplex destabilization and light-triggeredPNA/PNA recognitionSamit 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 inN-(2-aminoethyl)glycine peptide nucleic acids (aeg-PNA). The adeninyl and cytosinyl nucleo amino acid building blocksFmoc-aNPP-aeg-OH and Fmoc-cNPP-aeg-OH were synthesized and incorporated into PNA sequences by Fmoc solid phasesynthesis relying on high stability of the NPP nucleobase protecting group toward Fmoc-cleavage, coupling, capping, andresin cleavage conditions. Removal of the nucleobase caging group was achieved by UV-LED irradiation at 365 nm. Thenucleobase caging groups provided sterical crowding effecting the Watson–Crick base pairing, and thereby, the PNA doublestrand stabilities. Duplex formation can completely be suppressed for complementary PNA containing caging groups in bothstrands. 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

* Correspondence to: Ulf Diederichsen, Institut für Organische und BiomolekulareChemie, 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

Introduction

Caging with photolabile protecting groups involves the modificationof biologically active molecules to temporarily block and inactivatefunctionalities [1,2]. The activity of biomolecules is masked by intro-duction of caging groups at key positions allowing to restore thefunction externally triggered by irradiation [3]. Various caging groupswith high selectivity and orthogonality are addressed providingcleavage under mild conditions [4]. The caging group strategy canbe applied to various biomolecules such as ATP [1], amino acids [5],proteins [6], nucleic acids [7,8], sugars [9], lipids [10] and as atemporary photolabile N-terminal protecting group for solidphase 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 andRNA hybridization [12]. However, nucleobase caging groups arenot applied so far for the modulation of aeg-PNA double strandstabilities. Caging of aeg-PNA is of high relevance because aeg-PNAis well-established as DNA and RNA structural mimic nicelyresembling oligonucleotide pairing complexes with high duplexstabilities 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 alight-triggered modulation of PNA/PNA duplex formation wasderived from a recently reported application of aeg-PNA as recog-nition unit in artificial SNARE (soluble N-ethylmaleimide-sensitivefactor attachment protein receptor) protein-like peptide-PNAhybrids for membrane fusion [16]. The approximation of lipidvesicles is initiated by PNA/PNA duplex formation, thereby, startingthe membrane fusion process. In order to test the hypothesis forSNARE-mediated membrane fusion following a zipper-like recogni-tion process resulting in a tetrameric helical peptide bundle,aeg-PNA oligomers were envisioned introducing sterical hindrancefor nucleobase recognition at one end of the forming duplex.

J. Pept. Sci. 2013

The synthesis of adenine and cytosine-caged nucleo aminoacids Fmoc-aNPP-aeg-OH (1) and Fmoc-cNPP-aeg-OH (2) isreported using the 2-(o-nitrophenyl)-propyl (NPP) caging groupas introduced by Pfleiderer [17]. Incorporation of the cagednucleo amino acids in PNA oligomers by spss provided theopportunity to study the modulation of PNA/PNA double strandstability based on number and positioning of caging groups. Astepwise reduction of stability by ΔTm=5–3 �C for each caginggroup up to complete inhibition of duplex formation wasobtained. Complete recovery of PNA/PNA duplex formation wasinduced by photocleavage.

2-(o-Nitrophenyl)-propyl was chosen as caging group becauseit is compatible with the cleavage conditions in solid phase pep-tide synthesis and it can be cleaved by irradiation at 365 nmyielding a-methyl nitrostyrene as by-product that is less harmfulas, for example, nitrosoaldehyde resulting from ortho-nitrobenzylcaging groups and respective derivatives [17–20]. With respect topositioning, the NPP group at the nucleobase, Heckel reportedNPP-caged thymidine at the O4-position [21] and the O6-cagedguanosine [22]. Furthermore, he introduced the NPP caging groupin nucleotides at the N6-position of adenine and the N4-positionof cytosine [23]. In a first attempt, we decided to prepare theN6-adenine and N4-cytosine-caged nucleo amino acids Fmoc-aNPP-aeg-OH (1) and Fmoc-cNPP-aeg-OH (2). Caging groups at theexocyclic amino groups are expected to influence the nucleobasepairing for sterical reasons but still allow complete hydrogen bond-ing at theWatson–Crick site. Therefore, N6-adenine andN4-cytosine

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GUHA ET AL.

cagings offer the chance for fine-tuning the duplex formation andstabilities of PNA pairing complexes.

Materials and Methods

General

Startingmaterials 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 buildingblocks were purchased from ACM Research Chemicals (Malaysia)and Fmoc-Gly-Wang resin from Novabiochem (Merck, Darmstadt.Germany). Flash column chromatography was performed on Mercksilica gel 60 (40–60mm) at 0.3–1.1bar pressure. HPLC grade CH3CN andultra pure H2O (Millipore, Bedford, UK) were used for HPLC analysis.

Instruments

High-resolution mass spectra (HRMS-ESI) were obtained with theBruker Apex-Q IV FT-ICR-MS instrument (Bremen, Germany). HPLCwas performed on a Pharmacia Äkta Basic system (GE Healthcare,London, UK) with a pump type P-900, variable wavelength detectorUV-900 using Nucleodur RP C-18 (250� 4.6mm, 5mm) analyticalHPLC 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 ofamino acids and PNA oligomers was performed by irradiation withUV-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 werecollected at 260nm in buffer (20mM HEPES, 100mM NaCl, 1mMEDTA, pH=7.4, 4mm) applying the following temperature protocol:20 �C! 85 �C, 85 �C (equilibration for 5min), 85 �C! 0 �C (1 �C/min),0 �C (equilibration for 10min), 0 �C! 85 �C (1 �C/min), 85 �C! 20 �C.The hyperchromicity (H) was calculated according to the equation:H(%)= 100*[A(T)�A0]/A0, where A(T) is the absorbance given at anytemperature and A0 is the minimum absorbance.

Synthesis of Nucleo Amino Acids Fmoc-aNPP-aeg-OH (1) andFmoc-cNPP-aeg-OH (2)

tert-Butyl-2-(6-chloro-9H-purin-9-yl)acetate (3)

6-Chloropurine (1.20 g, 7.76mmol) and anhydrous K2CO3 (1.08 g,7.76mmol) were suspended in dry DMF (24ml). tert-Butylbromoacetate (1.40 g, 7.18mmol) was added dropwise and thereaction mixture was stirred for 20 h at 50 �C. The solid was fil-tered off and the organic solvent was evaporated. The residuewas treated with 0.5 N HCl (20ml) and stirred for 20min. After fil-tration, the residue was washed with H2O. The product 3 (1.3 g,5.2mmol, 67%) was obtained as a light yellow solid. m/z (HR ESIMS) calculated [M+H]+ 269.0800, found 269.0797. 1H NMR: dH(300MHz, 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 (75MHz, 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).

tert-Butyl 2-(6-((2-(2-nitrophenyl)propyl)amino)-9H-purin-9-yl)acetate (5)

Purinyl acetate 3 (0.66 g, 2.61mmol) was dissolved in anhydrousEtOH (7.30ml). 2-(2-Nitrophenyl)propylamine (4, prepared as de-scribed in [23], 0.94 g, 5.22mmol) and Et3N (0.70ml, 5.00mmol)were added and the reaction mixture was refluxed for 40 h. The

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resulting solution was evaporated under vacuum. The product5 (0.67 g, 1.70mmol, 65%) was obtained after purification by flashchromatography (pentane/acetone 1 : 1) as white solid. m/z (HRESI MS) calculated [M+H]+ 413.1932, found: 413.1932. 1H NMR:dH (300MHz, DMSO-d6) 8.16 (1H, s br, NH), 7.40–8.06 (6H, m, ArH),4.92 (2H, s, CH2-COO

tBu), 3.65�3.75 (3H, m, CH, CH2), 1.41 (9H, s,tBu), 1.30 (3H, d, J=6.0, CH3). 13C NMR: dC (75MHz, 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).

2-(6-((2-(2-Nitrophenyl)propyl)amino)-9H-purin-9-yl)acetic acid (6)

Adeninyl acetate 5 (0.51 g, 1.29mmol) was dissolved in 95% TFA(5ml) and stirred for 1 h at room temperature. The solvent wasremoved under vacuum, and the residue was treated with coldEt2O. The resulting white solid was washed with cold Et2O anddried in vacuum. The product 6 (0.41 g, 1.16mmol, 90%) wasobtained as a white solid. m/z (HR ESI MS) calculated [M+Na]+

379.1125, found 379.1117. 1H NMR: dH (300MHz, DMSO-d6)13.00 (1H, s br, COOH), 8.48 (1H, s br, NH), 8.31�7.41 (6H, m, ArH),5.01 (2H, s, CH2-COOH), 3.78�3.63 (3H, m, CH, CH2), 1.33 (3H, d,J=6.9, CH3).

13C NMR: dC (75MHz, 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).

Fmoc-aNPP-aeg-OtBu (7)

Adeninyl acetate 6 (0.40 g, 1.12mmol) was dissolved in dry DMF(3ml) and cooled to 0 �C under inert atmosphere. PyBOP(0.58 g, 1.12mmol) and Fmoc-aeg-OtBu HCl (0.40 g, 0.93mmol)were added and the solution was stirred for 10min at 0 �C. DIPEA(0.73ml, 4.20mmol) was added slowly at 0 �C and the solutionwas stirred for 4 h at room temperature. The solvent was evapo-rated and the residue was dissolved in ethyl acetate. The organiclayer was washed with 10% NH4Cl-solution (3� 20ml), 6%NaHCO3-solution (3� 20ml) and brine (3� 20ml), dried over an-hydrous Na2SO4, and the solvent was evaporated. Nucleo aminoacid 7 (0.50 g, 0.68mmol, 61%) was obtained after purification byflash 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 (300MHz, DMSO-d6) 8.13 (1H, s br, NH), 7.88�7.30(14H, m, ArH), 5.18 (1H, s br, NH), 4.40�4.20 (5H, m, CH2�COOtBu,OCH2, Fmoc-CH), 3.94 (2H, s, CH2), 3.78�3.65 (4H, m, 2�CH2),3.54�3.52 (2H, m, CH2), 3.45�3.44 (1H, m, CH), 1.40 (9H, s, C(CH3)

3), 1.29 (3H, d, J=6.0, CH3).13C NMR: dC (75MHz, 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.

Fmoc-aNPP-aeg-OH (1)

Nucleo amino acid 7 (0.48 g, 0.66mmol) was dissolved in TFA(5ml) and stirred for 1 h at room temperature. The solvent wasremoved under vacuum and the residue was treated with coldEt2O. The resulting solid was washed with cold Et2O and driedin vacuum. Nucleo amino acid 1 (0.39 g, 0.58mmol, 88%) wasobtained as a white solid. m/z (HR ESI MS) calculated [M-H]�

677.2478, found 677.2480. 1H NMR: dH (300MHz, DMSO-d6)12.50 (1H, s br, COOH), 8.66 (1H, s br, NH), 8.30�7.29 (14H, m,ArH), 5.27 (1H, s br, NH), 4.37�4.19 (5H, m, CH2�COOH, OCH2,Fmoc-CH), 4.01 (2H, s, CH2), 3.68�3.53 (4H, m, 2�CH2), 3.39�3.34

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CAGED PEPTIDE NUCLEIC ACIDS

(3H, m, CH, CH2), 1.32 (3H, d, J = 6.0, CH3).13C NMR: dC (75MHz,

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.

tert-Butyl-uracil-1-yl-acetate (8)

Uracil (2.20 g, 19.8mmol) and anhydrous K2CO3 (2.74 g,19.8mmol) were suspended in dry DMF (60ml). tert-Butylbromoacetate (3.51 g, 18.0mmol) was added dropwise, and thereaction mixture was stirred for 20 h at room temperature. Thesolid was filtered off, and the organic solvent was evaporated.The residue was treated with 0.5 N HCl (50ml) and stirred for20min. After filtration, the residue was washed with H2O. Theproduct 8 (3.37 g, 14.9mmol, 75%) was obtained as a white solid.m/z (HR ESI MS) calculated [M+Na]+ 249.0846, found 249.0847.1H NMR: dH (300MHz, 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 (75MHz, 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).

tert-Butyl-2-(4-((2-(2-nitrophenyl)propyl)amino)-2-oxopyrimidin-1(2H)-yl)-acetate (9)

DMAP (0.38 g, 3.09mmol), mesitylene-2-sulfonyl chloride (1.74 g,7.96mmol), and Et3N (3.07ml, 22.1mmol) were added to a solutionof 8 (1.00 g, 4.42mmol) in dry DCM (50ml) under inert atmosphere.Afterwards, the solution was stirred for 6 h at room temperature.The solvent was evaporated under vacuum and the residue wasdissolved in dry DCM (40ml) and 2-(2-nitrophenyl)propylamine(4) (1.59 g, 8.84mmol) was added. Afterwards, LiCl (1.05 g,24.8mmol) and Et3N (5.24ml, 37.6mmol) were added slowly at0 �C and the solution was stirred for 20 h at room temperature.The resulting solution was evaporated under vacuum. The product9 (1.28 g, 3.30mmol, 75%) was obtained after purification by flashchromatography (DCM/EtOAc 3:1! 2:1) as a white solid. m/z(HR ESI MS) calculated [M+H]+ 389.1819, found 389.1817. 1HNMR: dH (300MHz, DMSO-d6) 7.79 (1H, d, J=7.5, CH=CH),7.70�7.42 (4H, m, ArH), 5.62 (1H, d, J=7.5, CH=CH), 4.30 (2H, s,CH2), 3.67�3.55 (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(75MHz, 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).

2-(4-((2-(2-Nitrophenyl)propyl)amino)-2-oxopyrimidin-1(2H)-yl)-aceticacid (10)

Cytosinyl acetate 9 (0.50 g, 1.29mmol) was dissolved in 95% TFA(5ml) and stirred for 1 h at room temperature. The solvent wasremoved under vacuum, and the residue was treated with coldEt2O. The resulting white solid was washed with cold Et2O anddried in vacuum. The product 10 (0.41 g, 1.23mmol, 95%) wasobtained as a white solid. m/z (HR ESI MS) calculated [M+Na]+

355.1013, found 355.1015. 1H NMR: dH (300MHz, DMSO-d6) 12.4(1H, s br, COOH), 7.86�7.46 (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), 3.84�3.54(2H, m, CH2), 3.52�3.34 (1H, m, CH), 1.30 (3H, d, J= 6.7, CH3).

13CNMR: dC (75MHz, 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).

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Fmoc-cNPP-aeg-OtBu (11)

Cytosinyl acetate 10 (0.80 g, 2.41mmol) was dissolved in dry DMF(5ml) and cooled to 0 �C under inert atmosphere. PyBOP (1.25 g,2.41mmol) and N-(2-aminoethyl)glycine Fmoc-aeg-OtBu HCl (0.87g,2.01mmol) were added and the solution was stirred for 10min at0 �C. DIPEA (1.58ml, 9.05mmol) was added slowly at 0 �C and thesolution was stirred for 4 h at room temperature. The solvent wasevaporated, and the residue was dissolved in ethyl acetate. Theorganic layer was washed with 10% NH4Cl solution (3� 20ml), 6%NaHCO3 solution (3� 20ml) and brine (3� 20ml), dried over an-hydrous Na2SO4, and the solvent was evaporated. Nucleo aminoacid 11 (1.04 g, 1.46mmol, 73%) was obtained after purificationby flash chromatography (DCM/MeOH 30:1) as a light yellowsolid. m/z (HR ESI MS) calculated [M +H]+ 711.3137, found711.3136. 1H NMR: dH (300MHz, DMSO-d6) 7.87�7.25 (14H, m,12 ArH, Fmoc-NH, CHCH), 5.74 (1H, s br, NH), 5.62 (1H, d, J= 7.2,CHCH), 4.68�4.19 (5 H, m, CH2�COOtBu, OCH2, Fmoc-CH), 3.93(2H, s, CH2), 3.66�2.98 (7H, m, CH, CH2, CH2�CH2), 1.40 (9H, s, C(CH3)3), 1.27 (3H, d, J=6.5, CH3).

13C NMR: dC (75MHz, 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.

Fmoc-cNPP-aeg-OH (2)

Fmoc-cNPP-aeg-OtBu (11) (0.56 g, 0.79mmol) was dissolved in TFA(5ml) and stirred for 1 h at room temperature. The solvent was re-moved under vacuum, and the residue was treated with coldEt2O. The resulting white solid was washed with cold Et2O anddried in vacuum. Nucleo amino acid 2 (0.49 g, 0.75mmol, 96%)was obtained as white solid. m/z (HR ESI MS) calculated [M+H]+

655.2511, found 655.2510. 1H NMR: dH (300MHz, 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(75MHz, 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 phasesynthesis in a 15mm scale on an Fmoc-Gly-preloaded Wang resin(0.33mmol g�1). The resin was swollen in a BD (Becton Dickinson,Fraga, Spain) syringe for 2 h in NMP before starting the couplingcycle by Fmoc-deprotection applying 20% piperidine in NMP(2� 10min) followed by successive washing with NMP (3�), DCM(3�), and NMP (3�). Pre-activation and coupling: The respectivenucleo amino acid (4.0 equiv.) was dissolved in stock solutions ofO-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium (HATU)(0.24M, 3.9 equiv.) and 1-hydroxy-7-azabenzotriazole (HOAt)(0.25M, 4.0 equiv.) in NMP. Directly before transferring the couplingcocktail to the resin, DIEA (0.2M) and 2,6-lutidine (0.3M) wereadded to the pre-activated mixture. Double coupling for 1 h eachwas followed by washing with NMP, DCM, and NMP (3� each).Capping was carried out by treatment with a solution of Ac2Oand 2,6-lutidine in NMP (1 : 2 : 7 v/v/v, 2� 5min) followed bywashing with 5% DIEA in NMP (3�) and with DCM (3�), NMP (6�),DCM (3�), and NMP (3�). For N-terminal Fmoc-deprotection,20% piperidine in NMP (2� 5min) was employed followed bywashing with NMP, DCM, and NMP (3� each).

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GUHA ET AL.

After the final coupling cycle, the resin was washed with NMP,DCM, MTBT, and MeOH (5� each) and dried in vacuum. Cleavageof 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 ambienttemperature. The reaction mixture was concentrated under N2

stream followed by precipitation using cold Et2O (3�). Theresulting suspension was centrifuged at �5 �C. The supernatantwas discarded and the compound washed with Et2O severaltimes and dried. The following PNA oligomers were obtainedwith >98% purity as indicated by RP-HPLC and characterizedby ESI HR mass spectrometry as follows:

PNA1 (H-gtagatcact-Gly-OH): HPLC (Nucleodur RP-C18,250� 4.6mm, 5mm, gradient 0–25%): tR = 27.7min. m/z (HR ESIMS) calculated [M+ 3H]3+ 929.0399, found 929.0399.

PNA2 (H-catctagtga-Gly-OH): HPLC (Nucleodur RP-C18,250� 4.6mm, 5mm, gradient 0–25%): tR=27.9min. m/z (HR ESI MS)calculated [M+4H]4+ 697.0317, found 697.0312.

PNA3 (H-gtaNPPgatcact-Gly-OH): HPLC (Nucleodur RP-C18,250� 4.6mm, 5mm, gradient 0–25%): tR=35.4min. m/z (HR ESI MS)calculated [M+4H]4+ 737.7976, found 737.7970.

PNA4 (H-gtaNPPgaNPPtcact-Gly-OH): HPLC (Nucleodur RP-C18,250� 4.6mm, 5mm, gradient 0–25%): tR=36.7min. m/z (HR ESI MS)calculated [M+3H]3+ 1037.7488, found 1037.7486.

PNA5 (H-gtaNPPgaNPPtcaNPPct-Gly-OH): HPLC (Nucleodur RP-C18,250� 4.6mm, 5mm, gradient 0–25%): tR = 36.9min.m/z (HR ESI MS)calculated [M+5H]5+ 655.6649, found 655.6651.

PNA6 (H-cNPPatctagtga-Gly-OH): HPLC (Nucleodur, RP-C18,250� 4.6mm, 5mm, gradient 0–25%): tR=34.6min. m/z (HR ESI MS)calculated [M+4H]4+ 737.7976, found 737.7978.

PNA7 (H-catcNPPtagtga-Gly-OH): HPLC (Nucleodur, RP-C18,250� 4.6mm, 5mm, gradient 0–25%): tR=35.6min. m/z (HR ESI MS)calculated [M+3H]3+ 983.3944, found 983.3945.

PNA8 (H-cNPPatcNPPtagtga-Gly-OH): HPLC (Nucleodur, RP-C18,250� 4.6mm, 5mm, gradient 0–25%): tR=36.9min. m/z (HR ESI MS)calculated [M+4H]4+ 778.5634, found 778.5637.

NO2

H2NBrCH2COOtBu

K2CO3, DMF50 oC, 20 h

N

NNH

N

Cl

O

N

NN

N

Cl

OtBu

O

Fm

TFA, 1 h

FmocHNN

O

O

N

NN

N

HN

OH

NO2

EtOH, Et3Nreflux, 40 h

3

1

4

67% 65%

88%

Scheme 1. Synthesis of caged aeg-PNA building block Fmoc-aNPP-aegPyBOP= benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosph

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Results and Discussion

Synthesis of the nucleo amino acids Fmoc-aNPP-aeg-OH (1) andFmoc-cNPP-aeg-OH (2) (Schemes 1 and 2) was provided byNPP 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 nucleophilicsubstitution starting from 6-chloropurine, whereas uracil wasconverted to the cytosine derivative by O4 mesitylene sulfonateactivation followed by NPP protection. The NPP caging groupturned out to be stable under nucleo amino acid preparationand PNA oligomer synthesis conditions, thereby, serving aspermanent protection until photochemical cleavage. The NPPprotected adeninyl and cytosinyl acetic acid derivatives 6 and10 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-cagednucleo amino acids 1 and 2 as monomeric units for solid phasePNA synthesis.

Solid phase PNA oligomer synthesis following the Fmoc-protocol was applied to provide PNA oligomers PNA1and PNA2 and the NPP-caged nucleo amino acid containingPNA3–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 aminoacids were compatible with the Fmoc/tBu SPPS protocol usedfor PNA oligomer synthesis on a Wang resin. Peptide couplingwas provided with HATU/HOAt under optimized conditionsregarding a slight excess of nucleo amino acids (4 equiv.) usedwith respect to HATU (3.9 equiv.) and a pre-activation of theamino acid prior addition to the resin. After cleavage from theWang resin and purification, the PNAs 1–8 were obtained in apurity >98% as determined by HPLC. The oligomers were charac-terized by high-resolution mass spectrometry.

The influence of the NPP caging group on PNA/PNA doublestrand formation and stability was investigated for the PNA

TFA, 1 h

HN

FmocHNCOOtBu

DMF, PyBOP, DIEA, 4 h

N

NN

N

HNNO2

OtBu

O

N

NN

N

HNNO2

OH

ocHNN

O

O

N

NN

N

HN

OtBu

NO2

5 6

7

90%

61%

-OH (1); NPP = o-nitrophenylpropyl, DIEA =N,N-diisopropylethylamine;ate; Fmoc = 9-fluorenyl methoxycarbonyl; TFA = trifluoroacetic acid.

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N

NH

O

O

N

NH

H

O

O

NO2

H2N

N

N

O

NFmocHN

O

HNNO2

N

N

O

NFmocHN

O

HNNO2

N

N

O

HNNO2

N

N

O

HNNO2

O

OH

BrCH2COOtBu

K2CO3, DMF,20 h, 75%

1. DCM, DMAP, mesitylene sulfonyl chloride, Et3N, 6 h

LiCl, Et3N, 12 h

2.

TFA, 1h

TFA, 1h

HN

FmocHNCOOtBu

DMF, PyBOP, DIEA, 4 h

8 9 10

112

OtBu

O O

OtBu

75%

95%

96%

73%

4

O

OH OtBu

O

Scheme 2. Synthesis of caged aeg-PNA building block Fmoc-cNPP-aeg-OH (2).

Figure 1. PNA decamer sequenceswith the position of the NPP caging group indicated. PNAs 1, 3–5 have a sequence complementarity to PNAs 2, 6–8withrespect to double strand formation with parallel strand orientation.

CAGED PEPTIDE NUCLEIC ACIDS

decamers PNA1 and PNA2 with complementary sequence forWatson–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, 20mM HEPES, 100mM NaCl, 1mMEDTA, 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 nucleobasepairs indicating the transition from duplex to single strands.The double strand stability is expressed as melting temperatureat the inflection point. The stabilities of PNA3 + PNA2 andPNA1+ PNA6 with one NPP caging group in the PNA/PNA

J. Pept. Sci. 2013 Copyright © 2013 European Peptide Society and Joh

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 asindicated for the combinations PNA4+ PNA2 and PNA1 + PNA8.The influence of the caging groups was not dependent oncaging adenine or cytosine. Nevertheless, the additional caginggroup was placed within the same oligomer and adding a thirdcaging group to one of the oligomers provided an additionalloss in overall duplex stability of ΔTm= 3 �C (PNA5 + PNA2). Incase PNA5 carrying three NPP caging groups was combinedwith a complementary strand that had two additional caginggroups (PNA8), duplex formation was completely prevented

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0 20 40 60 800

5

10

15

20

H /

%PNA1/PNA2

Tm = 45 C

0 20 40 60 800

5

10

15

20

H /

%

T / C T / C

T / C T / C

T / C T / C

PNA3/PNA2

Tm = 40 C

0 20 40 60 800

5

10

15

20

H /

%

Tm = 36 C

PNA4/PNA2 PNA5/PNA2

Tm = 33 C

0 20 40 60 800

5

10

15

20

H /

%

0 20 40 60 800

5

10

15

20

H /

%

PNA1/PNA8

Tm = 36 C

(a) (b)

(c) (d)

(f)(e)

0 20 40 60 800

5

10

15

20

H /

%

Tm = 40 C

PNA1/PNA6

Figure 2. Temperature dependent UV absorption for double strand formation with parallel strand orientation of PNA1 and PNA2 (a) and PNAs withone (b, e), two (c, f ), and three caging groups (d) (4 mM, 20mM HEPES, 100mM NaCl, 1mM EDTA, pH= 7.4, 260 nm).

GUHA ET AL.

(Figure 3). Uncaging induced by irradiation at 365 nm usingUV-LED provided recovery of the duplex forming stability ofTm= 45 �C as it was determined for PNA1 + PNA2.The UV light-triggered release of the photolabile NPP

protecting group was further monitored by RP-HPLC and ESI-MStechniques (Figure 4). The time dependency of the NPPdeprotection of a 50pmol sample of caged nucleo amino acid orPNA oligomer was provided within 1h under irradiation with aUV-LED at 365 nm. These UV-LEDs have about 100 times the powerof mercury lamps and hence remarkably clean uncaging wasreached. Deprotection of the nucleo amino acids as shown inFigure 4 is comparable with respect to speed and cleanness

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to uncaging of PNA oligomers. Because the efficiency of photo-chemical uncaging depends on the molar extinction coefficientand photochemical quantum yield, NPP deprotection at 365 nmis relatively slow [26] and will be improved by modulating theUV light intensity or using alternative caging groups with higherextinction coefficient.

Conclusion

An efficient procedure for the synthesis of two new nucleobase-caged aeg-PNA nucleo amino acids Fmoc-aNPP-aeg-OH (1) and

ean Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013

0 20 40 60 800

5

10

15

20

H /

%

T / C T / C

PNA5/PNA8

0 20 40 60 800

5

10

15

20

H /

%

(a) (b)

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, 20mM HEPES, 100mM NaCl, 1mM EDTA, pH= 7.4, 260 nm); (b) UV-melting curve after irradiation at 365 nm using an UV-LED.

0 20 40 600

20

40

60

80

100

Per

cent

Time / min

Fmoc-aNPP-aeg-OH (caged) Fmoc-a-aeg-OH (uncaged)

0 10 20 30 40 50 600

20

40

60

80

100

Fmoc-cNPP-aeg-OH (caged) Fmoc-c-aeg-OH (uncaged)

Per

cent

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.

CAGED PEPTIDE NUCLEIC ACIDS

Fmoc-cNPP-aeg-OH (2) was developed. Incorporation into PNA se-quences by Fmoc-solid phase chemistry was provided with opti-mized coupling and cleavage conditions yielding respectivePNA oligomers with >98% purity prior to HPLC purification. TheNPP nucleobase caging group was highly stable under Fmoc spssconditions 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 dropof a ten-base pair duplex stability by ΔTm=5 �C, 9 �C, and 12 �C, re-spectively. Duplex formation was even suppressed by introductionof NPP caging groups in opposed PNA oligomers. This fine-tuningand fast recovery of the duplex stability by photochemicaldeprotection 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.

Acknowledgements

S. G. acknowledges the Alexander von Humboldt foundation,Germany, for financial assistance. Generous support from theDeutsche Forschungsgemeinschaft DFG (SFB 803) is gratefullyacknowledged.

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