Solid-phase synthesis of acid-sensitive N-(2-aminoethyl)glycine-PNA oligomers by the Fmoc/Bhoc strategy

Article abstract of DOI:10.1002/ejoc.200600747

In the context of investigation of nucleic acid-mediated excess electron transfer, a bis-functionalized (2-aminoethyl)glycine-PNA modified with a flavin and an oxetane moiety was synthesized. The solid-phase synthesis of the required PNA oligomer was espe

Full text of DOI:10.1002/ejoc.200600747

FULL PAPER
DOI: 10.1002/ejoc.200600747
Solid-Phase Synthesis of Acid-Sensitive N-(2-Aminoethyl)glycine-PNA
Oligomers by the Fmoc/Bhoc Strategy
Thorsten Stafforst[a] and Ulf Diederichsen*[a]
Keywords: Peptide nucleic acids / Solid-phase synthesis / Flavin / Oxetane / Electron transfer
In the context of investigation of nucleic acid-mediated ex-
cess electron transfer, a bis-functionalized (2-aminoethyl)gly-
cine-PNA modified with a flavin and an oxetane moiety was
synthesized. The solid-phase synthesis of the required PNA
oligomer was especially intriguing because of the high acid
sensitivity of the oxetane moiety, so the Fmoc/Bhoc strategy
was adapted to the mild cleavage conditions of the Siebera-
mide resin. Along with smooth oligomerization conditions,
the syntheses of oxetane- and flavin-functionalized Fmoc-
protected (2-aminoethyl)glycine building blocks are re-
ported.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
Introduction
model thanks to the extraordinary binding properties of
N-(2-Aminoethyl)glycine-PNA was introduced in 1991, PNA to single- and double-stranded nucleic acids and the
originally as a triple-strand binder to DNA.[1] The main potential for triple strand formation, strand displacement,
intention was to generate an oligonucleotide surrogate that and strong mismatch discrimination.[2] Furthermore, (2-ami-
would function in the antisense or antigene strategy to con- noethyl)glycine-PNA has been extensively used to address
trol gene regulation, but this artificial oligomer has since oligonucleotides with labels or functional units.[3] As part
become an important diagnostic tool and nucleic acid of our investigation of long-distance excess electron trans-
Figure 1. Bis-functionalized (2-aminoethyl)glycine-PNA oligomers for the investigation of excess electron transfer. A flavin chromophore
serves for light-induced electron injection and a thymine oxetane for monitoring of anion migration through cycloreversion of the oxetane
moiety[4] (n = 1–8).
fer, (2-aminoethyl)glycine-PNA needed to be functionalized
with both an electron donor and an acceptor group (Fig-
ure 1).[4a] The PNA strand was used to address the PNA/
DNA double or triple strands with the required modifica-
[a] Institut für Organische und Biomolekulare Chemie, Georg-
August-Universität Göttingen,
Tammannstrasse 2, 37077 Göttingen, Germany
Fax: +49-551-392944
E-mail: udieder@gwdg.de
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tions, since PNA/DNA complexes are known to be struc- mild basic conditions (20% piperidine) for the removal of
tural mimics for the corresponding oligonucleotide pairing the Fmoc group. The Bhoc group used for side chain pro-
complexes in the B-DNA conformation.[5] Our electron tection is usually removed with 95% TFA during the final
transfer studies required the synthesis of bis-functionalized cleavage from PEG-PS or Wang resin.[8]
PNA oligomers containing a flavin moiety for “charge in-
For the synthesis of even more acid-sensitive oligomers
jection” and a highly acid-sensitive thymine oxetane for such as the PNA/DNA chimera, the Fmoc/acyl and the
“charge trapping”,[4] and so a technique for the preparation Mmt/acyl strategies have been described for application in
of bis-functionalized acid-sensitive (2-aminoethyl)glycine- DNA synthesis,[9] although the relevant nucleo amino acids
PNA oligomers based on the Fmoc/Bhoc solid-phase pep- are not commercially available and require elaborate syn-
tide synthesis is presented, together with syntheses of the thesis. For the synthesis of flavin-/oxetane-functionalized
functionalized amino acid building blocks. In general, the PNA neither the Fmoc/acyl nor the Mmt/acyl strategy ap-
optimization of the solid-phase methodology for PNA peared applicable, since the removal of the acyl protecting
oligomers broadens the application of the Fmoc/Bhoc strat- groups requires treatment with ammonia at elevated tem-
egy, particularly with regard to the incorporation of highly peratures, which would be likely to cause degradation of the
acid-sensitive functionalities into (2-aminoethyl)glycine- flavin and oxetane moieties, and so we decided to adapt the
PNA. It might also be applicable to the preparation of pep- Fmoc/Bhoc strategy for acid-sensitive oligomers through
tides modified with comparable acid-sensitive functionali- the use of Sieberamide[10] or trityl resin.[11] An orientational
ties.
experiment with the Fmoc/Bhoc monomers indicated that
the Bhoc group could readily be removed within 20 min
under the usual conditions for Sieberamide and trityl resin
(1% TFA in DCM). The thymine oxetane moiety, repre-
senting the most acid-sensitive functionality in the oligo-
mer, showed 10% hydrolysis within 20 min under the same
conditions.
Results and Discussion
For the synthesis of (2-aminoethyl)glycine-PNA, two
strategies based on commercially available nucleo amino ac-
ids are known in the literature.[6] The Boc/Z strategy, with
N-terminal Boc protection and use of the Z group for side
chain protection, requires the removal of the Boc group
with 95% TFA after each elongation cycle and TFMSA
treatment during the final cleavage from solid support.[7]
Synthesis of Functionalized (2-Aminoethyl)glycine-PNA
Building Blocks
The flavin-containing PNA building block 1 was synthe-
The Fmoc strategy, on the other hand, was developed for sized from commercially available Fmoc-aeg-tBu (2, Fig-
the synthesis of acid-sensitive oligomers, with the use of ure 2). The flavin moiety was generated by the alloxane ap-
Figure 2. Synthesis of flavin building block 1 by the alloxane approach. (Dimethylnitrophenyl)glycine 3 was synthesized as described in
the literature.[15]
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Acid-Sensitive N-(2-Aminoethyl)glycine-PNA Oligomers
FULL PAPER
Figure 3. Synthesis of thymine oxetane building block rac,exo-6. Lithium iodide-mediated dealkylation of methyl ester 9 as reported by
Fisher.[16]
proach after condensation with (dimethylnitrophenyl)gly- firmed by 2D-NOESY NMR experiments.[18,19] The alter-
native regioisomer was not observed.[20] The thymine
oxetane acetic acid was purified by precipitation and ob-
tained in 28% yield.[21]
cine 3 by in situ HBTU/HOBt activation.[12] The nitro
group in intermediate 4 was reduced with H2 over Pd/C
and then condensed with alloxane under boric acid catalysis
conditions in an overall yield of 43%.[13] The corresponding
building block 1 was obtained nearly quantitatively by aci-
dolysis of tert-butyl ester 5 in 4  HCl/1,4-dioxane.[14]
Synthesis of the oxetane-functionalized amino acid 6 by
direct acylation of (2-aminoethyl)glycine was unsuccessful.
Since it was necessary to avoid hydrolysis of the oxetane-
functionalized amino acid under acidic conditions, the syn-
thesis of the oxetane building block 6 was started from the
Fmoc-protected (2-aminoethyl)glycine methyl ester 7, which
was in turn synthesized quantitatively from the tert-butyl
ester 2 by transesterification (Figure 3). After condensation
of thymine oxetane acetic acid rac,exo-8 with Fmoc-aeg-
OMe (7) by in situ HBTU/HOBt activation, the obtained
methyl ester 9 was hydrolyzed in the same step by lithium
iodide-mediated dealkylation with conservation of the
Fmoc group in an 86% overall yield.[16] The mild hydrolysis
of a methyl ester through dealkylation requires the presence
of a γ-carbonyl group, as is the case for (2-aminoethyl)gly-
cine-PNA building blocks in general. This method was
clearly superior to basic hydrolysis (removal of the Fmoc
group, attack on the oxetane) or hydrogenolytic cleavage of
a C-terminal benzyl ester with H2 over Pd/C (reductive
oxetane opening),[17] so lithium iodide-mediated dealky-
lation seems suitable as a general approach for acid-
sensitive (2-aminoethyl)glycine-PNA building blocks.
Figure 4. Synthesis of thymine oxetane acetic acid 8 through a Pa-
terno–Büchi reaction.
Oligomer Synthesis
The solid-phase peptide synthesis of oligomers 12–20
was performed manually in a plastic syringe on Nova-
Syn TG Sieberamide resin with use of a polyethylene glycol
spacer to improve the solubility and swelling properties of
the resin (Table 1). The resin was loaded to a low Fmoc-
glycine capacity (0.1–0.2 mmol·g–1); free amino termini
were acetylated with acetic anhydride. PNA oligomer syn-
thesis was performed with the commercially available
Fmoc/Bhoc nucleo amino acids, the synthesized building
blocks 1 and 6, and -Fmoc-Lys(Mtt)-OH by use of the
HATU/HOAt activation technique. The synthesis was fol-
lowed by UV monitoring of the dibenzofulvene released
during the Fmoc deprotection,[22] which allowed optimiza-
tion of the coupling conditions. In contrast with the incor-
poration of the flavin building block, which required double
The thymine oxetane acetic acid rac,exo-8 was synthe-
sized as a racemic mixture from thymineacetic acid 10 and
benzaldehyde (11) in a Paterno–Büchi reaction (Figure 4)
and was obtained in an exo configuration, as indicated by coupling and prolonged reaction times, the coupling of the
the coupling constant (3J(H-6,benzyl–H) = 6.6 Hz) and con- oxetane building block was readily achieved. Both the flavin
Eur. J. Org. Chem. 2007, 681–688
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T. Stafforst, U. Diederichsen
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building block and the Mtt-protected lysine were prone to thene cation.[10] Both problems were sufficiently solved by
acetylation during capping with acetic anhydride,[23] so a use of a HFIP-containing cleavage solution under con-
capping procedure based on Mmt chloride was used after tinuous flow conditions (Figure 5).
incorporation of one of these amino acids.
To this end a solution of TFA (1%), TES (4%) in DCM/
As expected, the solid-phase synthesis succeeded easily, HFIP (2:1) was passed continuously through the resin by
while the key problem turned out to be the cleavage of the use of a burette with a flow of 1 mLmin–1. The oligomers
oligomers. Cleavage under standard conditions (1% TFA, were readily dissolved in this solution and cleavage was
4% TES in DCM) failed even when the TFA content was complete within 30 min. Without use of continuous flow,
increased to 10%; this might have been due to extremely but under otherwise identical conditions, no cleavage was
reduced solubility of the oligomer after Bhoc and Mtt re- observed even after one day. All oligomers were synthesized
moval in the cleavage solution. Another problem might be on a 5 µmol scale and obtained in 5–10% yields after pre-
the reaction of the deprotected, nucleophilic oligomer with parative HPLC separation. A loss of about 20% in yield
the xanthene cation of the Sieber linker, which was sug- was due to acid-mediated hydrolysis of the oxetane moiety.
gested by the absence of the typical red color of the xan- The integrity of the oxetane moieties in oligomers 12–20
Table 1. Techniques used for manual solid-phase synthesis of bis-functionalized PNA by the Fmoc/Bhoc strategy on TG Sieberamide
resin on a 5 µmol scale.
Step
Deprotection 20% piperidine/NMP
Washing i. NMP, 5% DIPEA, ii. DCM, iii. NMP
Reagent
Comment
3ϫ3 min
i.–iii. 5ϫ
Fmoc removal
dibenzofulvene removal
in a separated vessel
Preactivation 4 equiv. monomer, 3.9 equiv. HATU, 4 equiv. HOAt[a] 5 min
in case of a second coupling 3 equiv. monomer,
2.9 equiv. HATU, 10 min for flavin and g
2ϫ120 min for flavin
Coupling
Washing
Capping
preactivation mixture
60 min
i. NMP, 5% DIPEA, ii. DCM, iii. NMP
i. Ac2O/lutidine/NMP 1:1:8, or
ii. 0.5  Mmt-Cl, 0.1  DMAP, 0.5  DIPEA, in
NMP/pyridine 4:1
i. 2ϫ5 min, ii. and iii. 5ϫ activated ester removal
i. 2ϫ5 min
ii. 1ϫ15 min
blocking of free N-termini
ii. starting with the incorporation of flavin or
Lys(Mtt)
Washing
i. NMP, 5% DIPEA, ii. DCM,
iii. tert-butyl methyl ether, iv. NMP
i.–iv. 5ϫ
[a] Fmoc-Lys(Mtt)-OH and Fmoc-Gly-OH were coupled by the HBTU/HOBt method.[24]
Figure 5. Preparative RP-HPLC analysis after cleavage of oligomer 12 with TFA (1%), TES (4%) in DCM/HFIP (2:1) under continuous
flow conditions. Synthesis was performed as described in Table 1. The absorption at 260 nm (nucleobases) and 450 nm (flavin) is given.
tR = retention time (10% B to 40% B in 25 min, B = acetonitrile/water 9:1 + 0.1% TFA, A = water + 0.1% TFA); PG = protecting
group; g = aeg-guanine.
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Acid-Sensitive N-(2-Aminoethyl)glycine-PNA Oligomers
FULL PAPER
Table 2. Bis-functionalized PNA oligomers 12–20 synthesized for excess electron transfer experiments. High-resolution mass spectroscopy
data (HR-ESI-MS) and the RP-HPLC retention times tR (analytical column, Vydac 25 cm, C-4, 5% Ǟ 40% B in 20 min, B = acetonitrile/
water 8:2 + 0.1% TFA, A = water + 0.1% TFA) are shown. Flavin = aeg-flavin, Ox = aeg-(thymine oxetane); Uro = N=C[N(CH3)2]2; t,
a, c, g = aeg-(nucleobase), K = lysine, G = glycine.
Sequence
HR-ESI-MS
species
HPLC
m/z found
m/z calcd.
tR [min]
Ac-Flavin-t2-Ox-t2-g2-c-g2-c-K2-G-NH2 (12)
Ac-Flavin-t5-g2-c-g2-c-K2-NH2 (13)
Uro-t5-g2-c–g2-c-K2-NH2 (14)
Ac-Flavin-t-Ox-t7-a-g-c-K2-G-NH2 (15)
Ac-Flavin-t2-Ox-t6-a-g-c-K2-G-NH2 (16)
Ac-Flavin-t4-Ox-t4-a-g-c-K2-G-NH2 (17)
Ac-Flavin-t6-Ox-t2-a-g-c-K2-G-NH2 (18)
Ac-Flavin-t8-Ox-a-g-c-K2-G-NH2 (19)
Ac-Flavin-t9-a-g-c-K2-G-NH2 (20)
[M]+
3857.5725
1232.5103
843.1183
1019.1737
1019.1737
1019.1737
1019.1737
1019.1737
992.6633
3857.5734
1232.5099
843.1183
1019.1738
1019.1735
1019.1736
1019.1738
1019.1737
992.6631
16.04
13.60
12.32
16.54
16.37
16.32
15.96
15.56
14.05
[M + 3H]3+
[M + 4H]4+
[M + 4H]4+
[M + 4H]4+
[M + 4H]4+
[M + 4H]4+
[M + 4H]4+
[M + 4H]4+
plete sets of NMR assignments with the aid of H,H-COSY, HSQC,
and HMBC experiments.
was confirmed by HR-ESI-MS (Table 2) and by subsequent
excess electron transfer experiments, which are described
elsewhere.[4a]
Fmoc-aeg-flavin-OH (1): Fmoc-aeg-flavin-OtBu (5, 230 mg,
0.34 mmol) was suspended in DCM (4 mL), and HCl (4 )/1,4-
dioxane (8 mL) was added slowly. After stirring overnight at room
temperature the resulting solution was mixed with tert-butyl methyl
ether (60 mL, 0 °C), and precipitation of 1 was complete within
30 min in an ice bath. The solid product was precipitated by centri-
Conclusions
The synthesis of (2-aminoethyl)glycine-PNA function-
alized both with a flavin and with an oxetane moiety was fugation and the liquid phase was separated. The precipitate 1 was
washed twice by suspension in tert-butyl methyl ether (60 mL) and
obtained as a yellow solid (200 mg, 0.32 mmol, 95%), which was
used for solid-phase synthesis without further purification and was
stored in the dark at –28 °C to prevent slow degradation of the
Fmoc group. RF (ethyl acetate/methanol 95:5) = 0.05. 1H NMR
(300 MHz, [D6]DMSO, 35 °C): δ = 11.34 (s, 1 H, NH flavin), 7.90
(d, 3JH,H = 7.2 Hz, 1 H, CH Fmoc), 7.88 (s, 0.5 H, CH flavin), 7.86
(s, 0.5 H, CH flavin), 7.83 (d, 3JH,H = 7.8 Hz, 1 H, CH Fmoc), 7.66
achieved by the Fmoc/Bhoc strategy on solid support. The
problem of the high acid sensitivity of the oxetane unit was
circumvented by careful optimization of the cleavage condi-
tions, and the resulting solid-phase peptide synthesis meth-
odology is applicable in general for the incorporation of
acid-sensitive functionalities into (2-aminoethyl)glycine-
PNA. The synthesis of the thymine oxetane building block
through dealkylation of an intermediate methyl ester has
been introduced as an advantageous alternative to the com-
3
(t, JH,H = 8.4 Hz, 2 H, CH Fmoc), 7.57 (s, 1 H, CH flavin), 7.39
(br. t, 2 H, CH Fmoc), 7.29 (br. d, 2 H, CH Fmoc), 5.68 (br. s, 1
mon approach for synthesis of Fmoc-protected acid-sensi- H, CH2 flavin), 5.51 (br. s, 1 H, CH2 flavin), 4.41–4.19 (m, 4 H,
tive building blocks.[2a,9a,14]
CH Fmoc, CH2 Fmoc, 1 H CH2COOH), 4.02 (s, 1 H, CH2COOH),
3.66 (br. t, 1 H, CH2CH2), 3.40 (br. t, 1 H, CH2CH2), 3.30 (br. t,
1 H, CH2CH2), 3.16 (br. t, 1 H, CH2CH2), 2.45 (s, 1.5 H, CH3),
2.41 (s, 1.5 H, CH3), 2.38 (s, 1.5 H, CH3), 2.36 (s, 1.5 H, CH3) ppm.
Experimental Section
13C NMR (150 MHz, [D6]DMSO, 35 °C):
δ = 171.4/170.1
General Methods: Infrared spectra were recorded on a Perkin–El-
mer FT-IR 1600 instrument, NMR spectra were recorded on a Var-
ian Unity 300 or a Varian Inova 600 spectrometer, and UV absorp-
tion spectra were collected with a Jasco V-550 spectrometer. Fluo-
rescence emission and excitation spectra were collected with a Jasco
FP 6200 instrument. ESI mass spectra were collected with a Finni-
gan LCQ iontrap spectrometer, while high-resolution mass spectra
(HR-ESI) were recorded on a Bruker FTMS-7 APEX® IV 70e FT-
ICR spectrometer. Reversed-phase HPLC was performed on a
Pharmacia Biotech Äkta Basic 900 system [eluents: A: deionized
water (18 MΩ·cm–1) + 0.1% TFA; analytical run: B: acetonitrile/
water 8:2 + 0.1% TFA; preparative run: B: acetonitrile/water 9:1 +
0.1% TFA]. Analytical HPLC was carried out on a Vydac
214TP54, 250ϫ4.6 mm, 5 µm, 300 Å, C-4 (butyl) column
(1 mLmin–1). Preparative HPLC was performed on a J’sphere
ODS-H80, 250ϫ20 mm ID, 8–4 µm, 8 nm, C18 (YMC) column
(10 mLmin–1).
(COOH), 165.3/165.0 (CO), 160.0, 156.1, 155.1, 150.1, 146.7 (C
flavin), 143.8 (2ϫC Fmoc), 140.6 (2ϫC Fmoc), 136.7, 135.8,
133.4, 131.1, 130.8 (CH flavin), 127.5 (2ϫCH Fmoc), 127.0
(2ϫCH Fmoc), 125.0 (2ϫCH Fmoc), 120.0 (2ϫCH Fmoc), 116.3
(CH flavin), 65.4 (CH2 Fmoc), 49.7, 47.4, 46.7 (CH Fmoc), 46.0,
38.0, 20.5 (CH flavin), 18.6 (CH flavin) ppm. IR (KBr): ν = 3400,
˜
3
3
1714, 1673, 1545, 1456, 1351, 1251, 1019, 867, 762, 741 cm–1. UV/
Vis (methanol/water 1:1): λmax = 445, 367, 266, 222 nm. Fluores-
cence: λexc = 450, 370 nm, λmax,em = 525 nm. ESI-MS: m/z (%):
645.2 (90) [M + Na]+, 621.1 (100) [M – H]–, 1243.1 (30)
[2M – H]–. HR-ESI-MS: m/z [M + H]+ = calcd. 623.2249 for
C33H30N6O7; found: 623.2253.
N-(4,5-Dimethyl-2-nitrophenyl)glycine (3):[15] 3,4-Dimethyl-2-nitro-
aniline (8.0 g, 48 mmol) and bromoacetic acid (7.6 g, 50 mmol)
were melted at 120 °C in an oil bath and stirred as long as the melt
was liquid (1 h). It was necessary to ensure that the temperature
never rose above 140 °C since product 3 tends to decarboxylate
explosively. After cooling to room temperature, the resulting solid
was dissolved in a mixture of ethyl acetate (300 mL) and aqueous
KOH (1 , 300 mL), the aqueous layer was separated, and the or-
ganic layer was extracted twice with aqueous KOH (1 , each
200 mL). The combined aqueous layers were acidified with HCl
Analytical Data: The NMR analysis of compounds 1, 4, 5, 6, and
9 suffered from high rotation barriers caused by the tertiary amide
bond of the N-acyl-(2-aminoethyl)glycine, which resulted in broad-
ening and partial doubling of 1H and 13C NMR signals.[14] In most
cases the rotamers have been obtained separately giving two com-
Eur. J. Org. Chem. 2007, 681–688
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(12 , pH 2) and the precipitated product was separated by fil-
tration with a Buchner funnel. The crude product was recrystallized
from acetic acid (70 mL) or ethanol and 3 was obtained as red
crystals (3.77 g, 17 mmol, 35%). M.p. 204 °C. 1H NMR (300 MHz,
CD3OD): δ = 7.90 (s, 1 H, aryl), 6.65 (s, 1 H, aryl), 4.12 (s, 2
H, CH2), 2.28 (s, 3 H, CH3), 2.19 (s, 3 H, CH3) ppm. 13C NMR
(50.3 MHz, [D6]DMSO, 35 °C): δ = 171.2 (COOH), 147.2 (C aryl),
142.7 (C aryl), 129.1 (C aryl), 124.9 (C aryl), 124.4 (CH), 114.8
HCOO]–. HR-ESI-MS: m/z [M + H]+ = calcd. 603.2813; found:
603.2813.
Fmoc-aeg-flavin-OtBu (5): Fmoc-aeg-(dimethylnitrophenylglycine)-
OtBu 4 (1.50 g, 2.5 mmol) was dissolved in glacial acetic acid
(30 mL) and the system was flushed with nitrogen. Pd/C (450 mg,
15 mol-%) was added and hydrogen was bubbled slowly through
the solution until the yellow color of the starting material had dis-
appeared (2 h). The reaction mixture was degassed with nitrogen
and filtered through celite into a brown glass flask, alloxane
(0.52 g, 3.2 mmol) and boric acid (50 mg) were added, the solution
was stirred overnight, and the solvent was removed in vacuo. The
residue was dissolved in ethyl acetate (200 mL), washed (3ϫ1 
aqueous HCl, 1ϫbrine, 3ϫ0.5  aqueous KHCO3, 2ϫbrine), and
dried with Na2SO4, and the obtained product was purified on silica
gel (ethyl acetate/methanol 95:5) to provide compound 5 (0.71 g,
1.05 mmol, 43%) as a yellow solid. RF (ethyl acetate, 2% methanol)
= 0.23. M.p. 174 °C. NMR: Two rotamers A and B with A/B =
1.33 were analyzed separately. Rotamer A: 1H NMR (600 MHz,
[D6]DMSO, 35 °C): δ = 11.33 (s, 1 H, NH), 7.89 (s, 1 H, CH aryl),
(CH), 44.16 (CH ), 19.6 (CH ), 17.5 (CH ) ppm. IR (KBr): ν =
˜
2
3
3
3364, 2917, 1636, 1588, 1508, 1392, 1221, 1147, 1057, 919, 762,
614 cm–1. UV/Vis (methanol): λmax = 434, 292, 235 nm. ESI-MS:
m/z (%) = 223.0 (70) [M – H]–, 447.0 (100) [2M – H]–, 225.1 (30)
[M + H]+. HR-ESI-MS: m/z [M + H]+ = calcd. 225.0870; found:
225.0869.
Fmoc-aeg-(dimethylnitrophenylglycine)-OtBu (4): N-(4,5-Dimethyl-
2-nitrophenyl)glycine (3, 1.35 g, 6.0 mmol) and HOBt (1.12 g,
8.3 mmol) were dissolved in DMF abs. (50 mL), a solution of
HBTU (2.20 g, 6.0 mmol) in DMF (5 mL) was added, and the sys-
tem was stirred for 2 min. DIPEA (2.90 mL) was added and the
solution was stirred for 2 min, after which Fmoc-aeg-OtBu·HCl (2,
2.00 g, 4.6 mmol) in DMF (2 mL) was added and the system was
stirred for a further 2 h. The solvent was removed in vacuo, the
crude product was extracted with ethyl acetate (200 mL), and the
organic layer was washed with aqueous HCl (0.1 , 4ϫ100 mL),
brine (100 mL), sat. NaHCO3 (4ϫ100 mL), and brine (100 mL)
and dried with Na2SO4. The title compound 4 (2.71 g, 4.37 mmol,
95%) was obtained after purification on silica gel (ethyl acetate/
hexane 1:1) as an orange foam. RF (ethyl acetate/hexane 1:1) =
0.25. M.p. 98 °C. NMR: Two rotamers, A and B with A/B = 1.35
were analyzed separately. Rotamer A: 1H NMR (300 MHz,
CDCl3): δ = 8.45 (br. s, 1 H, NH), 7.71 (s, 1 H, CH aryl), 7.55 (d,
3
7.82 (d, JH,H = 7.8 Hz, 2 H, CH Fmoc), 7.67–7.64 (m, 2 H, CH
Fmoc), 7.50 (br. t, 1 H, NH), 7.48 (s, 1 H, CH aryl), 7.40–7.35 (m,
3
2 H, CH Fmoc), 7.29 (t, JH,H = 7.8 Hz, 2 H, CH Fmoc), 5.67 (br.
s, 2 H, CH2 flavin), 4.41 (s, 2 H, CH2), 4.39 (d, covered, 2 H, CH2
Fmoc), 4.24 (br. t, 1 H, CH Fmoc), 3.65 (br. t, 2 H, CH2), 3.43
(m, 2 H, CH2), 2.40 (s, 3 H, CH3 flavin), 2.36 (s, 3 H, CH3 flavin),
1.36 [s, 9 H, C(CH3)3] ppm. 13C NMR (150 MHz, [D6]DMSO,
35 °C): δ = 169.1, 165.3, 159.6, 156.1, 155.1, 150.2, 146.7, 143.8
(2ϫC Fmoc), 140.7 (2ϫC Fmoc), 136.7, 135.8, 133.5, 131.2, 130.8
(CH flavin), 127.5 (2ϫCH Fmoc), 127.0 (2ϫCH Fmoc), 125.0
(2ϫCH Fmoc), 120.0 (2ϫCH Fmoc), 116.1 (CH flavin), 81.0
[C(CH3)3], 65.5 (CH2 Fmoc), 50.3, 47.5, 46.7 (CH Fmoc), 45.9,
38.9, 27.6 [C(CH3)3], 20.4 (CH3 flavin), 18.7 (CH3 flavin) ppm. Rot-
3
3JH,H = 7.5 Hz, 2 H, CH Fmoc), 7.41 (d, JH,H = 7.5 Hz, 2 H, CH
1
amer B: H NMR (600 MHz, [D6]DMSO, 35 °C): δ = 11.33 (s, 1
3
3
Fmoc), 7.26 (t, JH,H = 7.5 Hz, 2 H, CH Fmoc), 7.15 (t, JH,H
=
H, NH), 7.89 (s, 1 H, CH flavin), 7.85 (d, 3JH,H = 7.8 Hz, 2 H, CH
Fmoc), 7.67–7.64 (m, 2 H, CH Fmoc), 7.42 (s, 1 H, CH flavin),
7.5 Hz, 2 H, CH Fmoc), 6.24 (br. t, 3JH,H = 6.3 Hz, 1 H, NH), 6.16
(s, 1 H, CH aryl), 4.26 (d, JH,H = 7.2 Hz, 2 H, CH2 Fmoc), 4.04
(t, JH,H = 7.2 Hz, 1 H, CH2CH Fmoc), 4.04 (s, 2 H, CH2), 3.92
(s, 2 H, CH2), 3.53 (t, JH,H = 5.5 Hz, 2 H, CH2), 3.40 (m, 2 H,
3
3
7.40–7.35 (m, 2 H, CH Fmoc), 7.29 (t, JH,H = 7.8 Hz, 2 H, CH
3
Fmoc), 7.23 (br. t, 1 H, NH), 5.47 (br. s, 2 H, CH2 flavin), 4.30 (d,
3
3
3JH,H = 7.2 Hz, 2 H, CH2 Fmoc), 4.19 (t, JH,H = 7.2 Hz, 1 H,
CH2), 1.93 (s, 3 H, CH3 aryl), 1.79 (s, 3 H, CH3 aryl), 1.50–1.44
[m, 9 H, C(CH3)3] ppm. 13C NMR (75.5 MHz, CDCl3, 25 °C): δ =
169.4, 168.4, 156.8, 146.9, 143.2, 141.9 (2ϫC Fmoc), 140.9 (2ϫC
Fmoc), 130.0, 127.6 (2ϫCH Fmoc), 126.9 (2ϫCH Fmoc), 126.4
(CH aryl), 124.7, 124.4 (2ϫCH Fmoc), 119.7 (2ϫCH Fmoc),
114.0 (CH aryl), 82.8 [C(CH3)3], 67.2 (CH2 Fmoc), 49.6, 47.9, 46.8
(CH2CH Fmoc), 44.3, 38.9, 28.0 [C(CH3)3], 20.1 (CH3 flavin), 18.3
3
CH2CH Fmoc), 3.97 (s, 2 H, CH2), 3.37 (t, JH,H = 6.0 Hz, 2 H,
CH2), 3.14 (m, 2 H, CH2), 2.45 (s, 3 H, CH3), 2.36 (s, 3 H, CH3),
1.52 [s, 9 H, C(CH3)3] ppm. 13C NMR (150 MHz, [D6]DMSO,
35 °C): δ = 167.8, 165.1, 159.6, 156.5, 155.2, 150.1, 146.6, 143.8
(2ϫC Fmoc), 140.7 (2ϫC Fmoc), 136.8, 135.9, 133.4, 131.1, 130.9
(CH flavin), 127.5 (2ϫCH Fmoc), 127.0 (2ϫCH Fmoc), 125.0
(2ϫCH Fmoc), 120.0 (2ϫCH Fmoc), 116.4 (CH flavin), 82.4
[C(CH3)3], 65.4 (CH2 Fmoc), 48.9, 47.3, 46.7 (CH Fmoc), 45.4,
38.1, 27.7 [C(CH3)3], 20.4 (CH3 flavin), 18.6 (CH3 flavin) ppm. IR
(CH3 flavin) ppm. Rotamer B: 1H NMR (300 MHz, CDCl3): δ =
3
8.45 (br. s, 1 H, NH), 7.88 (s, 1 H, CH aryl), 7.70 (d, JH,H
=
3
7.5 Hz, 2 H, CH Fmoc), 7.54 (d, JH,H = 7.5 Hz, 2 H, CH Fmoc),
7.35 (t, JH,H = 7.5 Hz, 2 H, CH Fmoc), 7.24 (t, JH,H = 7.5 Hz, 2
H, CH Fmoc), 6.39 (s, 1 H, CH aryl), 5.49 (br. t, JH,H = 6.0 Hz,
(KBr): ν = 3407, 2977, 1715, 1673, 1582, 1546, 1242, 1155, 1017,
˜
3
3
861, 740 cm–1; UV/Vis (methanol/water 1:1): λmax = 445, 367, 266,
222 nm. Fluorescence: λexc = 450, 365 nm, λmax,em = 525 nm. ESI-
MS: m/z (%) = 701.2 (100) [M + Na]+, 1379.0 (90) [2M + Na]+,
677.2 (40) [M – H]–, 1355.2 (100) [2M – H]–. HR-ESI-MS: m/z [M
+ H]+ = calcd. 679.2875; found: 679.2871.
3
1 H, NH), 4.31 (d, 3JH,H = 7.2 Hz, 2 H, CH2 Fmoc), 4.13 (t, 3JH,H
= 7.2 Hz, 1 H, CH2CH Fmoc), 4.04 (s, 2 H, CH2), 3.92 (s, 2 H,
CH2), 3.60 (t, 3JH,H = 5.4 Hz, 2 H, CH2), 3.40 (m, 2 H, CH2), 2.19
(s, 3 H, CH3), 2.11 (s, 3 H, CH3), 1.50–1.44 [m, 9 H, C(CH3)3] ppm.
13C NMR (75.5 MHz, CDCl3, 25 °C): δ = 169.2, 168.3, 156.8,
147.2, 143.8, 142.5 (2ϫC Fmoc), 141.2 (2ϫC Fmoc), 130.4, 127.6
(2ϫCH Fmoc), 127.0 (2ϫCH Fmoc), 126.6 (CH aryl), 125.0,
124.4 (2ϫCH Fmoc), 119.9 (2ϫCH Fmoc), 114.3 (CH aryl), 83.6
Fmoc-aeg-(thymine oxetane)-OH (rac,exo-6): Fmoc-aeg-(thymine
oxetane)-OMe rac,exo-9 (660 mg, 1.05 mmol) was dissolved in
ethyl acetate (20 mL), lithium iodide (500 mg, 3.7 mmol) was
added, and the mixture was heated at reflux for 25 h. Ethyl acetate
[C(CH3)3], 66.8 (CH2 Fmoc), 50.5, 48.2, 47.1 (CH Fmoc), 44.5, (200 mL) was added and the solution was washed with NaHSO3
39.0, 28.0 [C(CH3)3], 20.5 (CH3 flavin), 18.5 (CH3 flavin) ppm. IR solution (3ϫ100 mL 0.5  aqueous NaHSO3/HCl, pH 2) and brine
(KBr): ν = 3423, 2976, 1722, 1633, 1567, 1509, 1397, 1237, 1155, (2ϫ100 mL sat. aqueous NaCl/HCl, pH 2) and dried with
˜
1042, 848, 742, 569 cm–1. UV/Vis (methanol): λmax = 430, 301, 258,
215 nm. ESI-MS: m/z (%) = 625.2 (30) [M + Na]+, 1226.9 (100)
[2M + Na]+, 647.0 (100) [M + HCOO]–, 1248.5 (60) [2M +
Na2SO4. The resulting crude product (654 mg) was purified on RP-
18 silica gel (600 mL, methanol/water 6:4) and compound 6
(550 mg, 0.90 mmol, 86%) was obtained as a white solid. RF (ethyl
686
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 681–688

Acid-Sensitive N-(2-Aminoethyl)glycine-PNA Oligomers
FULL PAPER
acetate/methanol/water/acetic acid 10:1:1:0.5) = 0.66. M.p. 196 °C
at 0 °C, and dried in vacuo. If the purity was not sufficient, the
3
(decomp.). 1H NMR (600 MHz, CD3OD): δ = 7.77 (d, JH,H
=
precipitation was repeated. M.p. 240 °C. 1H NMR (300 MHz, [D6]-
7.2 Hz, 2 H, Fmoc), 7.60 (m, 2 H, Fmoc), 7.43–7.21 (m, 9 H, 4 H DMSO, 35 °C): δ = 12.81 (s, 1 H, OH), 10.84 (s, 1 H, NH), 7.46–
3
Fmoc, 5 H aryl), 5.75–5.72 (d, JH,H = 6.6 Hz, 1 H, CH thymine),
7.31 (m, 5 H, CH aryl), 5.64 (d, 3JH,H = 6.6 Hz, 1 H, O–CH), 4.35
4.50–3.70 (m, 2 H, CH2 thymine), 4.32–4.25 (m, 2 H, CH2 Fmoc), (d, 3JH,H = 6.6 Hz, 1 H, H-6), 4.09 (d, 2JH,H = 17.7 Hz, 1 H, CH2),
2
4.20–4.10 (m, 2 H, CH Fmoc, CH thymine), 4.10–3.95 (m, 2 H,
CH2COOMe), 3.42–3.37 (m, 1 H, CH2–CH2), 3.34–-3.31 (m, 1 H,
CH2–CH2), 3.23–3.11 (m, 2 H, CH2–CH2), 1.72 (s, 1.5 H, CH3
3.80 (d, JH,H = 17.4 Hz, 1 H, CH2), 1.66 (s, 3 H, CH3) ppm. 13C
NMR (75.5 MHz, [D6]DMSO, 35 °C): δ = 170.3, 170.2, 151.3 (C-
2), 139.2 (C aryl), 128.5 (2ϫCH aryl), 128.3 (2ϫCH aryl), 126.4
thymine), 1.70 (s, 1.5 H, CH3 thymine) ppm. 13C NMR (150 MHz, (CH aryl), 85.5 (O–CH), 77.1 (C-5), 64.3 (CH-6), 48.2, 22.3 (CH3
CD3OD): δ = 2ϫ172.7 (C-4 thymine, COOH), 170.2 (C), 158.8 flavin) ppm. IR (KBr): ν = 3430, 1717, 1671, 1489, 1406, 1282,
˜
(CO-Fmoc), 153.4 (C-2 thymine), 145.3 (2ϫC Fmoc), 142.6 (2ϫC
Fmoc), 140.6 (C aryl), 129.7 (3ϫCH aryl), 128.8 (2ϫCH Fmoc),
128.1 (2ϫCH Fmoc), 127.2 (2ϫCH aryl), 126.2 (2ϫCH Fmoc),
120.9 (2ϫCH Fmoc), 87.4 (CH–O thymine), 79.0 (C thymine),
67.8 (CH2 Fmoc), 67.0 (CH thymine), 51.8 (CH2), 49.5 (CH2), 49.0
(CH2), 48.4 (CH Fmoc), 39.6 (CH2), 23.1 (CH3 thymine) ppm. IR
1237, 886, 774 cm–1; UV/Vis (methanol/water 1:1): λmax = 225 nm.
ESI-MS: m/z (%): 602.9 (100) [2M + Na]+, 289.1 (100) [M – H]–,
578.9 (90) [2M – H]–. HR-ESI MS: m/z [M + H]+ = calcd.
291.0976; found: 291.0977.
Fmoc-aeg-(thymine oxetane)-OMe (rac,exo-9): Thymine oxetane
acetic acid (rac,exo-8, 600 mg, 2.07 mmol), Fmoc-aeg-OMe·HCl (7,
734 mg, 1.88 mmol), HBTU (784 mg, 2.07 mmol), and HOBt
(508 mg, 3.76 mmol) were dissolved at 0 °C in DMF (25 mL), DI-
PEA (1.45 mL) was added, and the resulting solution was stirred
(14 h) at room temperature. The solvent was removed in vacuo and
the residue was dissolved in ethyl acetate (100 mL), washed
(KBr): ν = 3422, 1715, 1475, 1276, 1216, 759, 551 cm–1. UV/Vis
˜
(methanol): λmax = 214, 263, 289, 299 nm. ESI-MS: m/z (%): 613.1
(30) [M + H]+, 1247.1 (80) [2M + Na]+; 611.1 (70) [M – H]–, 1223.1
(100) [2M – H]–. HR-ESI-MS: m/z [M + H]+ = calcd. 613.2293 for
C33H32N4O8; found: 613.2292.
Fmoc-aeg-OMe·HCl (7): Fmoc-aeg-tBu·HCl (2, 500 mg, (3ϫ100 mL 0.5  aqueous NaHCO3, 100 mL brine, 3ϫ100 mL
1.15 mmol) was suspended in DCM (10 mL) and dissolved slowly
by addition of HCl (4 ) in 1,4-dioxane (15 mL). After complete
conversion (TLC), the solvent was removed in vacuo, and the resi-
due was dissolved in HCl in methanol (1.25 , 20 mL) and heated
at reflux after addition of a small amount of mol. sieves and tolu-
ene (20 mL) for 4 h without stirring. The mol. sieves were filtered
off, the solvent was removed in vacuo, the residue was partitioned
between DCM (100 mL) and aqueous NaHCO3 (0.5 , 100 mL),
the aqueous layer was extracted with DCM (3ϫ70 mL), and the
combined organic layers were washed with aqueous NaHCO3
(0.5 , 2ϫ100 mL) and brine (100 mL) and dried with Na2SO4.
After addition of HCl in MeOH (1.25 , 2 equiv.) the solvent was
removed and 7 (438 mg, 1.12 mmol, 97%) was obtained as a white
1  aqueous HCl, 100 mL brine), and dried with Na2SO4. The
crude product was purified on silica gel (300 mL, ethyl acetate),
and compound 9 (780 mg, 1.24 mmol, 66%) was obtained as a
white solid. RF (ethyl acetate) = 0.40. M.p. 104 °C. NMR: Two
1
rotamers A and B with A/B = 3:1. H NMR (300 MHz, CD3OD):
δ = 7.77 (d, 3JH,H = 7.5 Hz, 2 H, Fmoc), 7.60 (m, 2 H, Fmoc), 7.43–
7.21 (m, 9 H, 4ϫH Fmoc, 5ϫH aryl), 5.75 (d, 3JH,H = 6.3 Hz, 0.75
3
H, CH thymine), 5.73 (d, JH,H = 6.3 Hz, 0.25 H, CH thymine),
4.50–3.80 (m, 2 H, CH2 thymine), 4.32–4.25 (m, 2 H, CH2 Fmoc),
4.20–4.15 (m, 1 H, CH Fmoc), 4.15–4.05 (m, 3 H, CH thymine,
CH2COOMe), 3.69 (s, 2.25 H, OCH3), 3.61 (s, 0.75 H, OCH3),
3.36–3.32 (m, 2 H, CH2–CH2), 3.21–3.11 (m, 2 H, CH2–CH2), 1.72
(s, 3 H, CH3 thymine) ppm. 13C NMR (75.5 MHz, CD3OD): δ =
172.7 (C-4 thymine), 171.4 (COOMe), 170.2 (C), 158.8 (CO Fmoc),
solid. RF (ethyl acetate/methanol 7:1) = 0.43 (11), 0.62 (6), 0.25
1
(Fmoc-aeg-OH). M.p. 100 °C. H NMR (300 MHz, CD3OD): δ = 153.3 (C-2 thymine), 145.3 (2ϫC Fmoc), 142.6 (2ϫC Fmoc),
3
3
7.77 (d, JH,H = 7.5 Hz, 2 H, Fmoc), 7.63 (d, JH,H = 7.5 Hz, 2 H,
140.6 (C aryl), 129.8 (3ϫCH aryl), 128.8 (2ϫCH Fmoc), 128.2
(2ϫCH Fmoc), 127.2 (2ϫCH aryl), 126.2 (2ϫCH Fmoc), 120.9
Fmoc), 7.37 (t, 3JH,H = 7.5 Hz, 2 H, Fmoc), 7.29 (t, 3JH,H = 7.5 Hz,
3
2 H, Fmoc), 4.38 (d, JH,H = 6.3 Hz, 2 H, CH2 Fmoc), 4.19 (t, (2ϫCH Fmoc), 87.3 (CH benzyl thymine), 79.2 (C thymine), 67.7
3JH,H = 6.3 Hz, 1 H, CH Fmoc), 4.00 (s, 2 H, CH2COOMe), 3.81
(CH2 Fmoc), 67.0 (CH thymine), 52.8 (CH3O), 50.4 (CH2), 49.2
3
(s, 3 H, OCH3), 3.45 (t, JH,H = 5.5 Hz, 2 H, CH2–CH2), 3.20 (CH2), 48.8 (CH2), 48.4 (CH Fmoc), 39.9 (CH2), 23.0 (CH3 thy-
(d, JH,H = 5.5 Hz, 2 H, CH2–CH2) ppm. 13C NMR (75.5 MHz,
mine) ppm. IR (KBr): ν = 3412, 1716, 1473, 1276, 1212, 759,
˜
3
CD3OD): δ = 168.0 (COOMe), 159.4 (CO-Fmoc), 145.1 (2ϫC 530 cm–1. UV/Vis (MeOH): λmax = 214, 263, 289, 299 nm. ESI-MS:
Fmoc), 142.6 (2ϫC Fmoc), 128.8 (2ϫCH Fmoc), 128.1 (2ϫCH
Fmoc), 126.1 (2ϫCH Fmoc), 120.9 (2ϫCH Fmoc), 68.1 (CH2
m/z (%) = 649.3 (25) [M + Na]+, 1275.0 (100) [2M + Na]+, 1251.0
[2M – Na]–. HR-ESI-MS: m/z [M + H]+ = calcd. 627.2450; found:
Fmoc), 53.5 (CH3O), 49.2 (CH2), 48.2 (CH Fmoc), 48.1 (CH2), 627.2449.
38.1 (CH ) ppm. IR (KBr): ν = 3462, 2953, 1743, 1539, 1450, 1255,
˜
2
Solid-Phase Peptide Synthesis: Oligomers 12–20 were synthesized
manually on a 5 µmol scale in a 2 mL plastic syringe. Commercially
available nucleo amino acids Fmoc-aeg-thymine-OH, Fmoc-aeg-
guanine(Bhoc)-OH, Fmoc-aeg-adenine(Bhoc)-OH, Fmoc-aeg-cy-
tosine(Bhoc)-OH (purchased from Applied BioSystems), amino ac-
ids Fmoc-lysine(Mtt)-OH, Fmoc-glycine-OH (purchased from No-
vaBiochem), and the synthesized buildings blocks Fmoc-aeg-flavin-
OH (1) and Fmoc-aeg-oxetane-OH (6) were used. The syntheses
742, 583 cm–1. UV/Vis (MeOH): λmax = 214, 263, 299 nm. ESI-MS:
m/z (%) = 355.1 (100) [M + H]+, 708.7 (45) [2M + H]+. HR-ESI-
MS: m/z [M + H]+ = calcd. 355.1652; found: 355.1653.
Thymine Oxetane Acetic Acid (rac,exo-8): Thymineacetic acid[25]
(10, 4.00 g, 21.7 mmol) and benzaldehyde (11, 10 mL, 95 mmol)
were suspended in acetonitrile (240 mL) in a Pyrex flask. With vig-
orous stirring, water was added (40 mL) until a clear solution was
obtained, and the solution was flushed with argon. The reaction
mixture was stirred and exposed to the unfiltered output of a
300 W Hg-lamp for 50 h under argon. The solvent was removed,
the residue was dissolved in ethyl acetate (500 mL) and filtered, the
organic layer was extracted with aqueous KHCO3 (0.5 ,
5ϫ100 mL), the combined aqueous layers were acidified with HCl
(12 , pH 2) at 0 °C, and the precipitated title compound 8 (1.81 g,
6.24 mmol, 28%) was filtered off, washed with aqueous HCl (0.1 )
were
performed
on
NovaSyn TG
Sieberamide
resin
(0.15 mmol·g–1), which was swollen in DCM (1 h) before the first
amino acid was attached. The ordinary amino acids Fmoc-
Lys(Mtt)-OH and Fmoc-Gly-OH were coupled by the HBTU/
HOBt method with four equivalents relative to the resin. All nucleo
amino acids were coupled with four equivalents relative to the resin
by the HATU/HOAt technique, which required the preactivation
(5 min) of the monomers in a separating vessel. The removal of
Eur. J. Org. Chem. 2007, 681–688
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
687

T. Stafforst, U. Diederichsen
FULL PAPER
[6]
[7]
[8]
[9]
Peptide Nucleic Acids, Protocols and Application (Eds.: P. E.
Nielsen, M. Egholm), Horizon Scientific Press, 1999, Norfolk
UK.
T. Koch, in: Peptide Nucleic Acids, Protocols and Application
(Eds.: P. E. Nielsen, M. Egholm), Horizon Scientific Press,
1999, Norfolk UK, Sec. 2.1, 21–38.
the Fmoc group was followed by UV monitoring of the released
dibenzofulvene at 290 nm. For incorporation of flavin, coupling g
on g and t on g, double coupling (2ϫ3 equiv.) and prolonged coup-
ling times (2ϫ120 min) were used.
Synthesis Procedure: Deprotection: 2ϫ4 min 20% piperidine in
NMP; washing: 5ϫNMP, 2ϫDCM, 5ϫNMP; coupling of ordi-
nary amino acids: 4 equiv. amino acid, 4 equiv. HBTU/HOBt,
10 equiv. DIPEA, in NMP, final concentration of amino acid 0.2 ,
45 min; coupling of nucleo amino acids: 4 equiv. amino acid,
3.9 equiv. HATU/HOAt, 0.20  DIPEA, 0.30  lutidine in NMP,
final concentration of amino acid 0.1 , 5–10 min preactivation,
60 min coupling, for double coupling 3 equiv. amino acid, for flavin
2 h coupling time; washing: 5ϫ5% DIPEA in NMP, 2ϫ5 min
NMP (for removal of active ester); capping: before incorporation
of flavin or Lys(Mtt) 2ϫ5 min 10% Ac2O/20% lutidine in NMP,
after incorporation of flavin or Lys(Mtt) 1ϫ15 min 0.5  Mmt-Cl,
0.1  DMAP, 0.5  DIPEA in NMP/pyridine, 4:1; washing: 5ϫ5%
DIPEA in NMP, 5ϫtert-butyl methyl ether, 2ϫNMP.
R. Casale, I. S. Jensen, M. Egholm, in: Peptide Nucleic Acids,
Protocols and Application (Eds.: P. E. Nielsen, M. Egholm),
Horizon Scientific Press, 1999, Norfolk UK, Sec. 2.2, 39–50.
a) G. Breipohl, J. Knolle, D. Langer, G. O’Melley, E. Uhlmann,
Bioorg. Med. Chem. Lett. 1996, 6, 665–670; b) E. Uhlmann, B.
Greiner, G. Breipohl, in: Peptide Nucleic Acids, Protocols and
Application (Eds.: P. E. Nielsen, M. Egholm), Horizon Scien-
tific Press, 1999, Norfolk UK, Sec. 2.3, 51–70; c) G. Kovacs,
Z. Timar, Z. Kupihar, Z. Kele, L. Kovacs, J. Chem. Soc., Perkin
Trans. 1 2002, 1266–1270; d) Z. Timar, L. Kovacs, G. Kovacs,
Z. Schmel, J. Chem. Soc., Perkin Trans. 1 2000, 19–26.
The high basicity of the unprotected oligomer might also buffer
the trifluoroacetic acid and, therefore, prevent the cleavage. P.
Sieber, Tetrahedron Lett. 1987, 28, 2107–2110.
In this context the application of the hycron linker could also
be of interest. O. Seitz, Tetrahedron Lett. 1999, 40, 4161–4164.
a) R. Kuhn, H. Rudy, Ber. Dtsch. Chem. Ges. 1935, 68, 300–
302; b) R. Kuhn, F. Weygand, Ber. Dtsch. Chem. Ges. 1935, 68,
1281–1288; c) D. J. Brown, in: The Chemistry of Heterocyclic
Compounds (Ed.: E. C. Taylor), Wiley & Sons, New Jersey,
1988, vol. 24(3), p. 139ff.
a) M. Gladys, W. Knappe, Chem. Ber. 1974, 107, 3658–3673;
b) S. J. Gumbley, L. Main, Aust. J. Chem. 1976, 29, 2753–2755;
c) N. Choy, K. C. Russel, J. C. Alvarez, A. Fider, Tetrahedron
Lett. 2000, 41, 1515–1518; d) H. W. Rothkopf, D. Wöhrle, R.
Müller, G. Koßmehl, Chem. Ber. 1975, 108, 875–886; e) T. Car-
ell, R. Epple, V. Gramlich, Angew. Chem. 1996, 108, 676–679;
Angew. Chem. Int. Ed. Engl. 1996, 35, 620–623; f) A. Schwögler,
V. Gramlich, T. Carell, Helv. Chim. Acta 2000, 83, 2452–2463.
S. A. Thomson, J. A. Josey, R. Cadilla, M. D. Gaul, C. F. Hass-
man, M. J. Luzzio, A. J. Pipe, K. L. Reed, D. J. Ricca, R. W.
Wiethe, S. A. Noble, Tetrahedron 1995, 51, 6179–6194.
Please take care to monitor the temperature with a contact
thermometer, since the product tends to decarboxylate explos-
ively above 160 °C: J. Plöchl, Ber. Dtsch. Chem. Ges. 1886, 19,
6–11.
[10]
[11]
[12]
Cleavage Procedure: Washing: 5ϫ10 min NMP, 5ϫDCM, 5ϫtert-
butyl methyl ether, 5ϫswitching between DCM and tert-butyl
methyl ether; swelling: 30 min DCM; cleavage: HFIP/DCM, 1:2
10 min for removal of Mmt and Mtt groups; 1% TFA, 4% TES in
HFIP/DCM, 1:2, slowly dropping over the resin from a burette
(1 mLmin–1), 20–30 min, color change of the resin to deep red
within 5 min; drying in vacuo, pay attention to the instability of
the oxetane under the cleavage conditions (20% degradation in
45 min). Purification: Solvation of the crude product in 0.5–1 mL
1% acetic acid, microfiltration, purification by preparative HPLC
10% B to 40% B in 25 min tR ca. 21 min (B = acetonitrile/water
9:1 + 0.1% TFA, A = water + 0.1% TFA), lyophilization and stor-
age in brown caps at –28 °C.
[13]
[14]
[15]
Abbreviations: aeg = (2-aminoethyl)glycine, Bhoc = benzhydryloxy-
carbonyl, Boc = tert-butyloxycarbonyl, Fmoc = 9-fluorenylme-
thoxycarbonyl, HFIP = 1,1,1,3,3,3-hexafluoropropan-2-ol, HATU
= O-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate, HBTU = O-(1H-benzotriazol-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate, HOAt = 1-hydroxy-7-azab-
enzotriazole, HOBt = 1-hydroxybenzotriazole, Mmt = monometh-
oxytrityl, Mtt = monomethyltrityl, TES = triethylsilane, TFA =
trifluoroacetic acid, TFMSA = trifluoromethanesulfonic acid, Z =
benzyloxycarbonyl.
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3
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Acknowledgments
This work was supported by the Volkswagen-Stiftung and the
Deutsche Forschungsgemeinschaft (GRK 782). We are grateful for
a fellowship from the Fonds der Chemischen Industrie for T. S.
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Published Online: November 27, 2006
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