Comparative synthesis of the (34–42) fragment of ꢁ-amyloid
1–42) peptide
(41.3 mg, 0.109 mmol), HOBt (15 mg, 0.109 mmol) and DIEA
(19 µl, 0.109 mmol) for 40 min. All coupling and deprotection
(
20
steps were monitored by means of the Kaiser test. The resin
was washed with DMF (5 × 15 ml), methanol (5 × 15 ml) and
ether (5 × 15 ml), and dried under vacuum. Fmoc-protection of
the target peptidyl resin was removed and the resin was washed
with DMF (5 × 15 ml), methanol (5 × 15 ml) and ether (5 × 15
ml), and dried under vacuum. The NR2B 23 residue peptide
was cleaved from the resin by suspension of the peptidyl resin in
Reagent K. The solution was filtered and the filtrate concen-
trated under reduced pressure. The peptide was precipitated by
adding ice-cold ether and the precipitate was washed with ether
until the scavengers were removed, and dried under vacuum to
yield 95% of the crude peptide. HPLC analysis of the peptide
was carried out by injection of a small amount of peptide
dissolved in water into a C-18 RPC and eluting using a gradient
of solvent A (nanopure water containing 0.5% TFA) and
solvent B (80% acetonitrile in nanopure water containing 0.5%
TFA). Amino acid analysis: Leu, 1.01 (1); Arg, 2.97 (3); Glu,
The synthesis of β-amyloid (34–42) fragment LMVGGVVIA
was carried out manually using Fmoc-Ala-HMPA-4%
PS-TRPGGDA resin (150 mg, 0.025 mmol), Fmoc-Ala-
HMPA-Merrifield resin (135 mg, 0.025 mmol) and Fmoc-Ala-
R
Novasyn KA 125 resin (180 mg, 0.025 mmol). C-Terminal
Fmoc-Ala was anchored to these supports using the MSNT
method using Fmoc-Ala–MSNT–MeIm in the ratio 2:2:1.5.
Fmoc-protection of these amino acid bound resins was
removed with 20% piperidine in DMF and the resins were
washed thoroughly with DMF (5 × 15 ml). For each acylation
cycle the respective Fmoc-amino acids (0.086 mmol), mixed
with HBTU (33 mg, 0.086 mmol), HOBt (12 mg, 0.086 mmol)
and DIEA (15 µl, 0.086 mmol) dissolved in the minimum
amount of DMF, were added to the Fmoc-deprotected
resin. Coupling solutions were made at three times the indicated
scale and apportioned equally to the three parallel syntheses.
The coupling reaction was carried out for 40 min at room
temperature. After incorporation of all amino acids, Fmoc-
protection of the target peptidyl resin was removed and the
resin was washed with DMF (5 × 15 ml), methanol (5 × 15 ml)
and ether (5 × 15 ml), and dried under vacuum. The target
peptides were cleaved from the polymer supports by suspending
the peptidyl resin in Reagent K at room temperature for 4 h.
The solution was filtered and the filtrate concentrated under
reduced pressure. The peptide was precipitated by adding
ice-cold ether and the precipitate was washed with ether until
the scavengers were removed, and dried under vacuum. HPLC
analysis of the peptide was carried out by injecting a small
amount of peptide dissolved in water into a C-18 RPC and
eluting using a gradient of solvent A (nanopure water contain-
ing 0.5% TFA) and solvent B (80% acetonitrile in nanopure
water containing 0.5% TFA). Amino acid analysis of the
peptide from PS-TRPGGDA resin: Leu, 1.01 (1); Met, 0.81 (1);
Val, 3.02 (3); Gly, 2.02 (2); Ile 0.98 (1); Ala, 1.04 (1). MALDI-
1
.97 (2); Tyr, 0.82 (1); Ser, 1.59 (2); Asp, 3.98 (4); Thr, 1.63 (2);
Phe, 1.05 (1), Val, 1.00 (1); His, 0.91 (1); Lys, 4.11 (4); Ala, 1.01
ϩ
(
1). MALDI-TOF-MS: m/z 2821.17 (M ϩ H) , C120H199O N
36 43
ϩ
requires M 2820.06.
Acknowledgements
The authors thank the CSIR, New Delhi, for the award of a
junior research fellowship to P. G. Sasikumar and also the Rajiv
Gandhi Center for Biotechnology, Thiruvananthapuram and
DBT New Delhi for their support in this project.
References
1
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39
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2؉
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Asn-Lys-Leu-Arg-Arg-Gln-His-Ser-Tyr-Asp-Thr-Phe-Val
1
1
2
% PS-TRPGGDA-HMPA resin (350 mg, 0.035 mmol) was
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1
0 M. Renil and M. Meldal, Tetrahedron Lett., 1996, 34, 6185.
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2ϩ
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1
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1
1
3
0 min and dried under vacuum. The amino capacity of the
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Ϫ1
resin: 0.095 mmol g .
5
871.
Fmoc-Val-HMPA-PS-TRPGGDA resin (325 mg, 0.031
mmol) was placed in a manual peptide synthesiser and allowed
to swell in DMF for 1 h. Fmoc group protection was removed
with 20% piperidine in DMF (20 ml × 30 min), and the resin
was washed with DMF (5 × 15 ml). The remaining amino acids
of the target peptide sequence were successively incorporated
by using the respective amino acid (0.109 mmol), with HBTU
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1
1
2
8 J. C. Hendrix, K. J. Halverson, J. T. Jarrett and P. T. Lansbury Jr.,
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