BRANCHED ANTIMICROBIAL PEPTIDES
547
Boc-Leu-Ala-Arg(NO2)-OCH3 (II). A solution of 0.30 and minors 0.10 and 0.36 in 8 : 2 : 0.2 chloroform–
dipeptide (I) (0.87 g, 2.15 mmol) in 5 ml of TFA was methanol–hexane system. In addition, (VIII) was chro-
kept for 10 min at room temperatureand evaporated in matographed in the same system on a preparative
a vacuum of water-jet pump. The residue was dissolved Merck plate (Cat. Number 11798.0001), yield 0.08 g
in acetonitrile (25 ml), and triethylamine was added to (28%), Rf 0.30.
neutral reaction. Boc-Leu-OPfp (0.85 g, 2.14 mmol)
and triethylamine to pH ≈ 9 were added to the solution.
The mixture was kept for 3 h at room temperature and
treated as described in the synthesis of dipeptide. Ethyl
acetate was distilled off, and the residue was triturated
with ether. The resulting tripeptide (II) was crystallized
from ethyl acetate–hexane to yield 0.86 g (78%),
mp 104–106°C, Rf 0.50 (9 : 1 chloroform–methanol).
Boc-Arg(NO2)-Leu-Ala-Arg(NO2)-OCH3 (III).
Boc-group of tripeptide (II) (1.09 g, 2.11 mmol) was
removed with the help of TFA, the residue was dis-
solved in DMF (20 ml), triethylamine was added to
neutral reaction, and then Boc-Arg(NO2)-ONSu [14]
(1.04 g, 2.5 mmol) and triethylamine to pH ≈ 9. The
solution was kept for 4 h at room temperature and evap-
orated in a vacuum. The residue was dissolved in boil-
ing acetonitrile (5 ml), ethyl acetate was added to tur-
bidity, and the solution was kept in a refrigerator for
20 h. Yield of amorphous product (III) was 0.68 g
(45%), Rf 0.85 (on alumina in 95 : 5 chloroform–metha-
nol system).
Boc-Glu(OPfp)-OPfp (V). Pentafluorophenol
(3.9 g, 21 mmol) was added to a solution of Boc-Glu-
OH (2.47 g, 10 mmol)) in acetonitrile (20 ml), the solu-
tion was cooled with ice water and DCC (4.5 g,
22 mmol) was added. The mixture was kept for 16 h at
4–5°ë, filtered, and evaporated in a vacuum. The resi-
due was crystallized from ethyl acetate–hexane to get
diester (V); yield 3.6 g (88%), mp 128–132°ë. Rf 0.65
(9 : 1 : 10 chloroform–methanol–hexane).
Boc-Glu[Arg(NO2)-Leu-Ala-Arg(NO2)-OCH3]2
(VI). TFA (5 ml) was added to tetrapeptide (III) (0.6 g,
0.84 mmol), the mixture was kept for 10 min, evapo-
rated in a vacuum, the residue was dissolved in acetoni-
trile (15 ml), and triethylamine was added to neutral
reaction. Then diester (VI) (0.24 g, 0.42 mmol) and tri-
ethylamine to pH ≈ 9 were added to the reaction mix-
ture. The solution was kept for 3 h at room temperature
and evaporated in a vacuum. The residue was washed
with ethyl acetate, filtered, the crystals were washed
with ethyl acetate and ether. Yield of (V) 0.55 g (92%),
amorphous, Rf 0.52 in 8 : 2 : 0.2 chloroform–methanol–
hexane system.
Boc-Lys(Z)-Leu-Ala-Arg(NO2)-OCH3 (X). Tripep-
tide (II) (0.39 g, 0.75 mmol) was deblocked with the
help TFA. After the TFA removal in a vacuum, the res-
idue was dissolved in DMF (10 ml), triethylamine was
added to neutral reaction and, then, Boc-Lys (Z)-OPfp
(0.41 g, 0.75 mmol) and triethylamine to pH ≈ 9. The
solution was kept for 4 h at room temperature, ethyl
acetate (50 ml) was then added and the reaction was
treated as described in the synthesis of dipeptide. The
resulting tetrapeptide was crystallized from ethyl ace-
tate–hexane; yield 0.51 g (88%), mp 114–116°ë, Rf 0.50
(95 : 5 chloroform–methanol).
Boc-Glu[Lys(Z)-Leu-Ala-Arg(NO2)-OCH3]2 (XII)
was obtained as nonapeptide (VI) starting from (X)
(0.50 g, 0.64 mmol) and active ester (V) (0.185 g,
0.32 mmol); yield 0.47 g (92%); Rf 0.10 in 95 : 5 chlo-
roform–methanol and 0.71 in 9 : 1 chloroform–metha-
nol.
Boc-Glu{Glu[Lys(Z)-Leu-Ala-Arg(NO2)-OCH3]2}2
(XIV) was synthesized similarly to (VIII) starting from
nonapeptide (XII) (0.49 mmol) and ester (V)
(0.24 mmol). The resulting product was dissolved in
hot acetonitrile and crystallized at cooling; yield 0.32 g
(40%); Rf 0.45(9 : 1 chloroform–methanol).
ç-Arg-Leu-Ala-Arg-OCH3
ç-Glu[Arg-Leu-Ala-Arg-OCH3]2 · 5HCl (VII),
H-Glu{Glu[Arg-Leu-Ala-Arg-OCH3]2}2 9HCl
·
3HCl (IV),
·
(IX), H-Lys-Leu-Ala-Arg-OCH3 · 3HCl (XI), ç-
Glu[Lys-Leu-Ala-Arg-OCH3]2 · 5HCl (XIII), and H-
Glu{Glu[Lys-Leu-Ala-Arg-OCH3]2}2 · 9HCl (XV).
Deblocking of the protected peptides (III), (VI),
(VIII), (X), (XII), and (XIV) was carried out in two
stages by standard procedures: nitro and benzyloxycar-
bonyl groups were deleted by catalytic hydrogenation
over Pd in acetic acid, and Boc-group by treatment with
10% HCl for 1 h [15]. The peptides were purified by
preparative HPLC and characterized by ESI MS. The
molecular masses of the peptides, calculated from the
registered m/z values of series of multicharged ions are
given in Table 2.
REFERENCES
1. Travis, J., Science, 1994, vol. 264, pp. 360–362.
2. Neu, H.C., Science, 1992, vol. 257, pp. 1064–1073.
3. Hancock, R.E.W. and Scott, M.G., Proc. Natl. Acad. Sci.
USA, 2000, vol. 97, pp. 8856–8861.
Boc-Glu{Glu[Arg(NO2)-Leu-Ala-Arg(NO2)-
OCH3]2}2 (VIII). Nonapeptide (VI) (0.30 g, 0.21 mmol)
was deprotected with the help of TFA, TFA was deleted
in a vacuum, the free peptide was dissolved in DMF
(5 ml), triethylamine was added to neutral reaction,
then ester (V) (0.06 g, 0.1 mmol) and triethylamine to
pH ≈ 9 were added and the mixture also left overnight.
Water was added to the solution, crystals were filtered,
and washed with ester. Yield of (VIII) 0.23 g (79%), Rf
4. Zasloff, M., Nature, 2002, vol. 415, pp. 389–395.
5. Tossi, A., Sandri, L., and Giangaspero, A., Biopolymers,
2000, vol. 55, pp. 4–30.
6. Op de Kamp,Y.A.F., Annu. Rev. Biochem., 2001, vol. 48,
pp. 47–71.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007