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A. Wiemann et al. / Tetrahedron 56 (2000) 1331–1337
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CH2Ph), 5.11 (s, 2 H, CH2Ph), 4.74 (m, 1 H, Ha), 4.67 (m, 1
H, Hg), 4.1 (m, 8 H, Et), 2.43 (m, 2 H, Hb), 1.3 (m, 12 H,
Et). 13C NMR: 171.1 (CHCOOBzl), 156.1 (NHCOOBzl),
136.4 (Car), 135.4 (Car), 128–129 (CHar), 69.5 (d,
1J(C,P)172 Hz, Cg), 67.3 (CH2, Et), 66.9 (CH2, Et), 64.5
(CH2Ph), 63.3 (CH2Ph), 51.9 (Ca), 33.4 (Cb), 16.4 (CH3,
Et), 16.0 (CH3, Et). 31P NMR: 20.2 (d, 1J(P,P)22 Hz, phos-
25 Hz, phosphate, second diastereomer), Ϫ2.4 (d, J(P,P)
22 Hz, phosphate, first diastereomer).
Introduction of the Fmoc group into 12a and 12b
The Fmoc group was introduced into 590 mg of 12a and
into 191 mg of 12b as described for 7c (see above). The
products were isolated by preparative HPLC with gradients
from 50% methanol in water to 100% methanol (containing
0.5% TFA), with elution of the compounds at 90–95%
methanol. Yields: 280 mg (30%) and 57 mg (6%) of the
separated diastereomers of 13a and 173 mg (60%) of the
mixture of diastereomers of 13b.
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phonate), 0.3 (d, J(P,P)22 Hz, phosphate).
Preparation of the isopropyl protected phosphate
phosphonate 11b
To a suspension of 600 mg sodium hydride (60% in mineral
oil, 15 mmol) in 30 mL THF were added 2.5 mL (15 mmol)
diisopropyl phosphite at 0ЊC. The mixture was stirred at
20ЊC until gas development had stopped. The solution was
added in portions of 3 mL to 1.13 g (2.9 mmol) acid
chloride 10 in 30 mL THF at 20ЊC. When the solution
turned slightly reddish, 30 mL of 1 M phosphate buffer
pH 7 was added and the mixture was extracted with ethyl
acetate. The product 11b was purified from the organic layer
by preparative HPLC with a gradient from 50% methanol in
water to 100% methanol, with elution at 85–90% methanol.
Yield 549 mg (0.82 mmol, 28%); 383 mg of 9 (37%) were
also recovered.
Compound 13a: 1H NMR: 7.73 (d, 2 H, Fmoc), 7.60 (dd, 2
H, Fmoc), 7.38 (dd, 2 H, Fmoc), 7.29 (dd, 2 H, Fmoc), 6.20
(d, 1 H, NH), 4.92 (m, 1 H, Hg), 4.60 (m, 1 H, Ha), 4.34 (m,
2 H, Fmoc), 4.20 (m, 9 H, Fmoc, Et), 2.42 (m, 2 H, Hb), 1.3
(m, 12 H, Et). 13C NMR: 172.4 (COOH), 155.7 (Fmoc),
143.8 (Fmoc), 141.3 (Fmoc), 127.7 (CHar, Fmoc), 127.1
(CHar, Fmoc), 125.3 (CHar, Fmoc), 120.0 (CHar, Fmoc),
70.0 (d, 1J(C,P)172 Hz, Cg), 67.2 (CH2, Fmoc), 65.2
(CH2, Et), 63.7 (CH2, Et), 50.5 (CH, Fmoc), 47.1 (Ca),
34.9 (Cb), 16.4 (CH3, Et), 16.0 (CH3, Et). 31P NMR
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(CDCl3, 162 MHz): 20.3 (d, J(P,P)20 Hz, phosphonate,
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first diastereomer), 19.9 (d, J(P,P)23 Hz, phosphonate,
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second diastereomer), 0.1 (d, J(P,P)23 Hz, phosphate,
1H NMR: 7.33 (m, 10 H, Har), 6.34 (d, 1 H, NH), 5.18 (s, 2
H, CH2Ph), 5.09 (s, 2 H, CH2Ph), 4.67 (m, 6 H, Ha, Hb,
iPr), 2.43 (m, 2 H, Hb), 1.32 (m, 24 H, iPr). 13C NMR: 171.1
(CHCOOBzl), 156.1 (NHCOOBzl), 136.3 (Car), 135.3 (Car),
128-129 (CHar), 73.2 (CH, iPr), 72.1 (CH, iPr), 69.7 (d,
1J(C,P)174 Hz, Cg), 67.0 (CH2Ph), 66.6 (CH2Ph), 51.2
(Ca), 33.3 (Cb), 23.3–23.7 (CH3, iPr). 31P NMR: 18.4 (d,
1J(P,P)29 Hz, phosphonate), Ϫ1.2 (d, 1J(P,P)29 Hz,
phosphate).
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second diastereomer), Ϫ0.2 (d, J(P,P)20 Hz, phosphate,
first diastereomer).
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Compound 13b: H NMR: 7.74 (d, 2 H, Fmoc), 7.59 (m, 2
H, Fmoc), 7.39 (dd, 2 H, Fmoc), 7.29 (dd, 2 H, Fmoc), 6.10
(d, 1 H, NH), 4.73 (dm, 6 H, Ha, Hg, iPr), 4.38 (d, 2 H,
Fmoc), 4.21 (t, 1 H, Fmoc), 2.45 (m, 2 H, Hb), 1.34 (m, 24
H, iPr). 13C NMR: 174.4 (COOH), 156.6 (Fmoc), 143.7
(Fmoc), 141.3 (Fmoc), 127.8 (CHar, Fmoc), 127.1 (CHar,
Fmoc), 125.1 (CHar, Fmoc), 120.0 (CHar, Fmoc), 75.2
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(CH, iPr), 74.2 (CH, iPr), 70.2 (d, J(C,P)185 Hz, Cg),
Removal of the Z- and Bn-groups from 11a and 11b
67.7 (Fmoc), 50.7 (Ca), 47.0 (CH, Fmoc), 33.7 (Cb), 23.4
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(CH3, iPr). 31P NMR: 16.9 (d, J(P,P)26 Hz, phospho-
Hydrogenations were carried out in glacial acetic acid with
palladium hydroxide on carbon for 3 h. After filtering off the
catalyst the solvent was removed by lyophylization and
traces of acetic acid were removed by repeated (at least
three times) lyophylizations from dioxane to yield 12a
and 12b quantitatively.
nate), Ϫ3.3 (d, 1J(P,P)27 Hz, phosphate).
Peptide assembly
The peptide sequences Ac-AAEGGSXNVFSK-amide (P1)
and RRRRAAXVA-amide (P2) (with X representing the
new derivatives or regular serine phosphate) were synthe-
sized manually according to the Fmoc/tBu method in scales
of 10–20 mmol. The amino acids were coupled in DMF
according to standard protocols in a three-fold excess with
activation by 1 equiv. of TBTU and 2 equiv. of diisopropyl-
ethylamine. Coupling time was 1 h. After each coupling and
before the removal of the Fmoc group the amount of free
amino groups in the synthesis resin was determined accor-
ding to the bromophenol blue method as described.23 The
new derivatives and the following amino acid were double-
coupled before the removal of the Fmoc group. Cleavage of
the Fmoc group was carried out with 20% piperidine in
DMF for 10 min. Cleavage of the peptides from the resin
and deprotection of the side chains (except for the isopropyl
phosphate protection) was carried out by a 3 h treatment
with TFA/triisobutylsilane/water in a ratio of 95:3:2.
Crude peptides were obtained by precipitation with
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Compound 12a: H NMR: 7.7 (br. s), 5.14 (m, 1 H, Ha),
4.13 (m, 8 H, Et), 3.85 (m, 1 H, Hg), 2.42 (dm, 2 H, Hb),
1.27 (m, 12 H, Et). 13C NMR: 172.7 (COOH), 70.3 (d,
1J(C,P)169 Hz, Cg), 64.6 (CH2, Et), 63.4 (CH2, Et), 50.8
(Ca), 32.7 (Cb), 16.3 (CH3, Et), 15.9 (CH3, Et). 31P NMR:
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20.7 (d, J(P,P)22 Hz, phosphonate, first diastereomer),
20.3 (d, 1J(P,P)20 Hz, phosphonate, second diastereomer),
0.6 (d, 1J(P,P)20 Hz, phosphate, first diastereomer), Ϫ0.6
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(d, J(P,P)22 Hz, phosphonate, second diastereomer).
Compound 12b: 1H NMR: 4.93 (m, 1 H, Ha), 4.68 (m, 4 H,
iPr), 4.10 (m, 1 H, Hg), 2.50 (br. d, 2 H, Hb), 1.31 (m, 24 H,
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iPr). 13C NMR: 172.1 (COOH), 70.2 (d, J(C,P)169 Hz,
Cg), 74.1 (CH, iPr), 72.8 (CH, iPr), 50.3 (Ca), 32.1 (Cb),
23–24 (CH3, iPr). 31P NMR: 18.1 (d, 1J(P,P)25 Hz,
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phosphonate, first diastereomer), 17.6 (d, J(P,P)25 Hz,
phosphonate, second diastereomer), Ϫ1.2 (d, 1J(P,P)