C. Dose, O. Seitz / Bioorg. Med. Chem. 16 (2008) 65–77
75
4.9.9. a-N-(Boc-aminoethyl)-(e-N-Alloc)-lysine methylest-
er (5). To a stirred solution of Boc-glycinal (0.29 g,
1.82 mmol) in dry methanol (50 mL) were added 4
(1.53 g, 5.46 mmol) and NaBH3CN (0.14 g, 2.18 mmol)
at 0 ꢁC. The reaction mixture was stirred for 14 h at
room temperature and the solvent was evaporated under
reduced pressure. The residue was purified by silica gel
flash chromatography (ethyl acetate/cyclohexane = 5:1)
(3 · 50 mL). The combined organic phases were dried
(MgSO4) and the solvent was evaporated under reduced
pressure. The residue was purified by silica gel flash
chromatography (ethyl acetate/methanol/formic acid,
94:5:1) to afford 0.42 g (87%) of 2 as colorless powder.
C31H42N6O10; Rf = 0.15 (ethyl acetate/methanol/formic
25
D
acid, 94:5:1); mp = 74–77 ꢁC; ½aꢂ ꢁ13.1ꢁ; 1H NMR
(300 MHz, DMSO-d6, 25 ꢁC) both rotamers: d 1.22–
1.29 (m, 2H), 1.38–1.45 (m, 11H), 1.67–2.01 (m, 2H),
2.92–3.05 (m, 2H), 3.17–3.47 (m, 4H), 4.32–4.45 (m,
3H), 4.72, 4.81 (s, 2H), 5.13–5.29 (m, 4H), 5.83–5.96
(m, 1H), 7.01–7.03 (d, J = 7.3, 1H), 7.34–7.43 (m, 5H),
7.95–7.97 (d, J = 7.3, 1H) ppm; 13C NMR (75 MHz,
DMSO-d6, 25 ꢁC) both rotamers: d 23.05 (CH2), 28.10
(CH3), 28.19 (CH2), 29.10 (CH2), 38.99 (CH2), 39.94
(CH2), 45.72 (CH2), 49.83 (CH2), 50.15 (CH2), 58.77
(CH), 64.03 (CH2), 66.40 (CH2), 77.97 (Cq), 93.72 (CH),
109.46 (Cq), 116.77 (CH2), 127.85 (CH), 128.07 (CH),
128.40 (CH), 133.81 (CH), 150.96 (CH), 153.12 (Cq),
154.89 (Cq), 155.63 (Cq), 155.82 (Cq), 158.34 (Cq),
163.10 (Cq), 167.17 (Cq), 172.12 (Cq) ppm; HR-FTMS:
M+H+/pos.: Anal. calcd: 659.3035, found: 659.3037.
to afford 0.52 g (74%) of
C18H33N3O6; Rf = 0.2 (ethyl acetate/cyclohexane, 5:1);
5
as colorless oil.
25
D
½aꢂ ꢁ1.9ꢁ; 1H NMR (300 MHz, CDCl3, 25 ꢁC): d
1.34–1.71 (m, 15H), 2.52–2.60 (m, 1H) and 2.71–2.79
(m, 1H), 3.13–3.25 (m, 5H), 3.71 (s, 3H), 4.53 (d,
J = 5.4, 2H), 5.17–5.31 (ddd, J = 0.9, J = 10.4, J = 1.4,
J = 17.2, 2H), 5.84–5.97 (m, 1H) ppm. 13C NMR
(75 MHz, CDCl3, 25 ꢁC):
d 22.80 (CH2), 28.37
(3 · CH3), 29.57 (CH2), 32.74 (CH2), 40.15 (CH2),
40.64 (CH2), 47.47 (CH2), 51.85 (CH3), 60.79 (CH),
65.39 (CH2), 79.23 (Cq), 117.54 (CH2), 132.92 (CH),
156.06 (Cq), 156.22 (Cq), 175.36 (Cq) ppm; HR-FTMS:
M+H+/pos.: Anal. calcd: 388.2442, found: 388.2445.
4.9.10. a-N-(Boc-aminoethyl)-a-N-(2-(4-Cbz-cytosine-1-
yl)-acetyl)-(e-N-Alloc)-lysine methylester (6). To a solu-
iCys-cctac((CH2)4NH-TMR)ag-GlyGlyCONH
2
4.9.12.
tion
of
4-Cbz-1-carboxymethylcytosine
(0.37 g,
(Nu3). Starting from Fmoc–glycine loaded MBHA-Resin
7 (loading 0.3 mmol/g, 8.5 mg) the linear solid phase
PNA synthesis was accomplished according to the Boc/Z
strategyasdescribed previously. The resin was washedwith
argon saturated DCM (5 · 1 mL) and the Alloc group was
deprotected by adding 1 equiv Pd(PPh3)4 and 6 equiv
Me2NHÆBH3 in argon saturated DCM (2 · 1 mL) to the re-
sin for 30 min. Subsequently the resin was washed with
DCM (5·), DMF (5·), dioxane/H2O (2·, 9:1), methanol
(2·), DMF (5·), DCM (5·), and DMF (5·). After preacti-
vation of 4 equiv 5-carboxytetramethylrhodamine (final
concentration 0.1 M in DMF) for 3 min using 4 equiv Py-
BOP and 6 equiv N-methylmorpholine, the solution was
added to the resin for 60 min. The resin was washed follow-
ing the general washing procedure. Cleavage from the solid
support was accomplished following the general cleavage
protocol according to the Boc/Z strategy described previ-
ously. Synthesis yield was determined by measuring the
1.23 mmol) in dry DMF/CH3CN (10 mL) were added
N-methylmorpholine (0.54 mL, 4.92 mmol) and piva-
loylchloride (0.17 mL, 1.35 mmol) at ꢁ15 ꢁC (ice/metha-
nol). After 20 min 5 (0.32 g, 0.82 mmol) in dry DMF
(5 mL) was added dropwise and the reaction mixture
was stirred for 12 h at room temperature. Ethyl acetate
(50 mL) was added and the organic phase was washed
with 0.1 N HCl (3 · 20 mL). The combined organic
phases were dried (MgSO4) and the solvent evaporated
under reduced pressure. The residue was purified by sil-
ica gel flash chromatography (ethyl acetate) to afford
0.22 g (39%) of 6 as colorless powder. C32H44N6O10;
25
Rf = 0.2 (ethyl acetate); mp = 64–66 ꢁC; ½aꢂ ꢁ21.5ꢁ;
D
1H NMR (300 MHz, CDCl3, 25 ꢁC) both rotamers: d
1.28–1.62 (m, 15H), 1.96 (q, J = 7.36, J = 14.4, 2H),
3.13–3.39 (m, 5H), 3.70 (s, 3H), 4.16 (t, 1H), 4.52 (d,
J = 5.4, 2H), 4.62–4.86 (dd, J = 15.7,J = 15.5), 5.15–
5.30 (m, 4H), 5.40 (s, 1H), 5.58 (s, 1H), 5.82–5.96 (m,
1H), 7.22 (d, J = 6.2, 1H), 7.34–7.38 (m, 5H), 7.63 (d,
J = 6.8, 1H) ppm; 13C NMR: (75 MHz, CDCl3, 25 ꢁC)
both rotamers: d 23.31 (CH2), 28.08 (CH2), 28.42
(CH3), 29.32 (CH2), 39.27 (CH2), 40.34 (CH2), 47.70
(CH2), 50.44 (CH2), 52.57 (CH3), 60.45 (CH), 65.31
(CH2), 68.05 (CH2), 77.24 (Cq), 79.87 (Cq), 95.18
(Cq), 95.25 (Cq), 117.37 (CH2), 128.23 (2· CH),
128.28 (CH), 128.67 (2· CH), 133.17 (CH), 134.91
(Cq), 150.30 (CH), 156.05 (Cq), 156.54 (Cq), 162.50
(Cq), 162.57 (Cq), 167.17 (Cq), 171.61 (Cq), 171.66
(Cq), 181.40 (Cq), 187.84 (CH) ppm; HR-FTMS:
M+H+/pos.: Anal. calcd: 673.3200, found: 673.3198.
UV absorption at 565 nm (e565nm = 91.000 L molꢁ1 cmꢁ1
)
in phosphate buffer (100 mM NaH2PO4, pH 7.4) at 25 ꢁC.
C110H135N47O27S; yield: OD260 = 8.6, 88 nmol, 3.5%;
MALDI-TOF/MS (m/z): Anal. calcd for [M+H]+: 2580,
found: 2579; HPLC: tR = 16.2 min (Gradient B).
4.9.13.
iCys-cctac((CH2)4NH-AEEA-TMR)ag-Gly-
GlyCONH (Nu4). The synthesis was accomplished
according to the previously described procedure using
pre-activated Fmoc-aminoethyloxyethyloxyacetic acid
as building block for the coupling reaction after Alloc-
deprotection. C116H146N48O30S; yield: OD260 = 10.8,
110 nmol, 4.4%; MALDI-TOF/MS (m/z): Anal. calcd
for [M+H]+: 2726, found: 2726; HPLC: tR = 16.6 min
(Gradient B).
2
4.9.11. a-N-(Boc-aminoethyl)-a-N-(2-(4-Cbz-cytosine-1-
yl)-acetyl)-(e-N-Alloc)-lysine (2). To a solution of 6
(0.49 g, 0.73 mmol) in THF (10 mL) was added drop-
wise 1 N LiOH (4 mL) at 0 ꢁC. After stirring for 4 h at
room temperature the solution was acidified by adding
1 N HCl to pH 2–3. Water (20 mL) was added and the
aqueous phase was extracted with ethyl acetate
4.9.14.
ðCH2Þ
AcHNctcttcððCH2Þ NH-FAMÞcccac-Gly-S
4
(El2). Starting from Fmoc–glycine loaded
SO3H
2
MBHA-Resin 7 (loading 0.3 mmol/g, 8.5 mg) the linear
solid phase PNA synthesis was accomplished according
to the Boc/Z strategy as described previously. The resin