The synthesis of a lipophilic derivative of RGD peptide

Article abstract of DOI:10.1023/B:RUBI.0000023094.72361.c7

Bishexadecyl ester of RGD peptide was synthesized in solution by the conventional methods of peptide chemistry in a total yield of 48%.

Full text of DOI:10.1023/B:RUBI.0000023094.72361.c7

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 2, 2004, pp. 111–113. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 2, 2004, pp. 126–129.
Original Russian Text Copyright © 2004 by Stepanenko, Sebyakin.
The Synthesis of a Lipophilic Derivative of RGD Peptide
E. G. Stepanenko and Yu. L. Sebyakin1
Lomonosov State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 117571 Russia
Received October 18, 2002; in final form, February 20, 2003
Abstract—Bishexadecyl ester of RGD peptide was synthesized in solution by the conventional methods of
peptide chemistry in a total yield of 48%.
Key words: RGD peptide, nonviral transfection
1
INTRODUCTION
affinity to the integrin receptors on the lung surface and
can easily penetrate into a cell through the receptor-
associated endocytosis mechanism [5], was observed.
The authors noted that the cationic liposomes can prob-
ably shield the RGD sequence and make it unavailable
for the integrins of cell surface when the mass ratio
between DNA, peptide, and lipid was 1 : 5 : 24 and pre-
sumed that the transfection mainly proceeds through
the nonspecific mechanism of the peptide penetration
into cell. Nevertheless, the enhancement of transfection
efficiency by the additional introduction of peptide
fragments that favors activation of the receptor-associ-
ated endocytosis or the energy-independent mechanism
of fusion with the cell membrane into the complex of
DNA with cationic liposomes is recognized as promis-
ing. Therefore, the further search for new systems
increasing the efficiency of cell transfection with the
reporter genes is topical.
The problem of direct transfer of functional genes
and therapeutic agents (transfection) into cells has cur-
rently assumed great importance in connection with the
2
development of clinical protocols of gene therapy. In
addition to viral vectors widely used as the delivery sys-
tems, aviral systems and the methods of BAS transfer
into cells and tissues with the use of cationic liposomes
attract growing attention of researchers [1].
Cationic liposomes have a number of advantages
over viral vectors. They protect DNA and mRNA mol-
ecules and oligonucleotides from inactivation by cellu-
lar enzymes, are noninfectious and nonimmunogenic,
and can be directly transported to the cells of definite
types. Cationic liposomes can easily be prepared, are
stable on storage, and are economically available. Nev-
ertheless, at present, they transfer genes less effectively
than viral vectors [2].
Cationic conjugates of guanidine with cholesterol
were synthesized and studied in order to search for
effective systems of BAS delivery. Guanidine groups
were shown to be capable of hydrogen bonding to phos-
phate groups of polynucleotides, protect them from
degradation in the late endosomes and lysosomes, and
are promising for the transfection purposes [3].
The efficiency of nonviral transfection of the epithe-
lial cells of lung air channels with plasmid DNA using
a complex of cationic liposomes on the basis of com-
mercially available Lipofectamine and cyclic peptides
containing polylysine and RGD and RGE motifs was
studied by Colin et al. [4]. They demonstrated that the
presence of the peptides substantially enhanced the
RESULTS AND DISCUSSION
In this connection, we synthesized a lipophilic
derivative of RGD peptide. We plan further studies of
complexes of this lipopeptide with DNA, its additional
inclusion into the complexes of cationic liposomes with
DNA, and a comparative study of the efficiency of non-
viral transfection of the prepared complexes.
Bishexadecyl ester of the Arg-Gly-Asp tripeptide
V) was synthesized by the successive elongation of
peptide chain using active esters [6] according to the
scheme.
(
The product of the N-succinimide ester of tert-butyl-
transfection efficiency in comparison with the control oxycarbonylglycine (II) reaction with bishexadecyl
experiments without them. However, no expected ester of aspartic acid (I) was treated with trifluoroacetic
increase in the transfection efficiency in the case of the acid, and the resulting TFA salt of (III) was converted
RGD-containing peptide, which has a predominant into the free base using triethylamine. The structure of
1
(
III) was confirmed by H NMR and IR spectroscopy,
1
Corresponding author; phone: +7 (095) 434-8544; e-mail:
httos.mitht@g23.relcom.ru
Abbreviations: BAS, biologically active substances and MALDI
MS, matrix-assisted laser desorption and ionization mass spec-
trometry.
and mass spectrometry. Further, Boc-Arg(Boc) -OH
2
2
[7] and its N-succinimide ester (IV) were synthesized
and coupled with (III). The protective groups were
removed from the product. The total yield of (V) after
1
068-1620/04/3002-0111 © 2004 MAIK “Nauka /Interperiodica”

1
12
STEPANENKO, SEBYAKIN
H NCHCOO
2
CH COO
2
(I)
1
. Boc-Gly-ONSu
II)
. CF COOH
(
2
3
3
. Et N
3
H NCH CONHCHCOO
2
2
CH COO
2
(III)
1
2
. Boc-Arg(Boc )-ONSu
2
(IV)
. CF COOH
3
CF COOH · H NCHCONHCH CONHCHCOO
3
2
2
(
CH2)3
NH
CF COOH · H N C NH
CH COO
2
(V)
3
2
Scheme
the three stages was 48%. The structure of the lipotri-
Substances were detected on TLC plates by treat-
peptide synthesized was confirmed by spectral meth- ment with iodine vapor, by heating at 350°ë, or by
ods.
Thus, the procedure of synthesis of bishexadecyl
ester of arginylglycylaspartic acid was developed, and
sufficient quantities of the lipotripeptide were prepared
for further biochemical studies.
treatment with chlorine vapors with subsequent spray-
ing with a solution of tolidine (160 mg) and KI (1 g) in
an acetic acid (30 ml) and water (470 ml) mixture [9].
The substances with free amino group were detected by
the treatment with 5% ninhydrin solution in a 30 : 1
acetone–acetic acid mixture with subsequent heating.
N-Succinimide esters were detected by a successive
spraying with the following solutions: 14% NH OH ·
EXPERIMENTAL
2
HCl in water (20 ml), 14% NaOH in water (8.5 ml),
N-Hydroxysuccinimide and N,N'-dicyclohexylcar-
bodiimide were of domestic production and of “kh. ch.”
reagent grade; tert-butyloxycarbonylglycine N-succin-
imide ester was from Merck (Germany). Bishexadecyl
ester of L-aspartic acid was prepared according to [8].
and, after 2 min, 5% FeCl in 1.2 N HCl [6].
3
Glycylaspartic acid bishexadecyl ester (III). A
solution of bishexadecyl ester of L-aspartic acid (0.8 g,
1
1
.38 mmol) in THF (5 ml) and Na CO (0.15 g,
.38 mmol) were added to a solution of tert-butyloxycar-
bonylglycine N-succinimide ester (0.41 g, 1.51 mmol) in
2 3
1H NMR spectra were recorded on a Bruker WM-
00 pulse NMR spectrometer (Germany) operating at
2
THF (5 ml). The reaction mixture was stirred for 8 h at
4
working frequency of 200 MHz. Hexamethyldisiloxane
was used as an internal standard. IR spectra were regis-
tered on a Shimadzu IR-435 spectrophotometer
5°ë with TLC monitoring and evaporated in a vac-
uum. The residue was acidified to pH 6 with 0.1 N HCl,
and products were extracted with ethyl acetate. The
(
Japan). Mass spectra were recorded on aVISION 2000
extract was dried by MgSO , evaporated in a vacuum,
4
time-of-flight mass spectrometer (UK) by the MALDI
method.
and the residue was chromatographed on a column with
silica gel in the mixture of hexane and ethyl acetate
Melting points were determined on a Boetius device (4 : 1). Bishexadecyl ester of glycylaspartic acid (III)
Germany). For TLC, silufol plates UV-245 (Czech was obtained in yield of 0.59 g (60%); R 0.54 (D); mp
(
f
Republic) were used in the following chromatographic
systems: (A) 9 : 1 chloroform–methanol, (B) 3 : 4 : 1
chloroform–ethanol–ammonia, (C) 8.5 : 0.5 : 0.2 chlo-
roform–methanol–acetic acid, and (D) 4 : 1 petroleum
ether–ethyl acetate. Column chromatography was car-
ried out on silica gel L40/100 µm (Czech Republic).
20
3
9–41°ë; [α ] + 12.3° (Ò 1, ëHCl ); IR (Vaseline oil,
D
3
–
1
νmax, cm ): 3292 (NH), 2902 (CH), 2833 (CH), 1727
C=O), 1647 (NHCO), 1545 (NHCO), 1460 (CH),
375 (CH), 1289 (C=O), 1215 (CO), 1175 (CO), 1090
(
1
1
(CH), 710 (CH); H NMR (CDCl , δ, ppm): 0.83 (6 H,
3
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004

THE SYNTHESIS OF A LIPOPHILIC DERIVATIVE OF RGD PEPTIDE
113
t, ëç ), 1.26 (52 H, s, CH ), 1.43 [9 H, s, C(CH ) ], (52 H, s, CH ), 1.56 [27 H, s, C(CH ) ], 1.50 (4 H, m,
3
2
3 3
2
3 3
1
.62 (4 H, m, βëç ), 2.86 (2 H, m, ëç ëç), 3.78 (2 H, βëç ), 2.2–2.6 (6 H, m, ëç ), 2.86 (2 H, m, ëç ëç),
2 2 2 2 2
s, CH ), 4.06 (4 H, m, αëç ), 4.79 (1 H, m, CH), 5.18 3.89 (2 H, s, ëç ); 4.03 (1 H, m, CH), 4.08 (4 H, m,
2
2
2
(1 H, m, NHBoc), and 7.18 (1 H, d, NH).
αëç ), 4.28 (1 H, m, CH), 5.3 (1 H, m, NHBoc), 6.8
2
(
1 H, d, NH), 7.80 (1 H, d, NH), and 11.15 (2 H, m,
The substance (0.45 g, 0.61 mmol) was dissolved in
NHBoc).
trifluoroacetic acid (5 ml); the mixture was kept for
3
0 min (TLC monitoring) and evaporated in a vacuum.
The residue was dissolved in anhydrous chloroform
5 ml), and triethylamine was added dropwise to pH 7–
. The reaction product was washed with water and
dried in a vacuum. The yield of (III) was 0.36 g (94%);
The resulting compound (0.10 g, 0.091 mmol) was
treated with trifluoroacetic acid (2 ml) for 30 min with
TLC monitoring, and the reaction mixture was evapo-
rated in a vacuum. The residue was recrystallized from
ethanol. Chromatographically pure bistrifluoroacetate
(
8
2
0
of (V) was obtained in yield of 0.05 g (70%); R 0.39
R 0.36 (A); mp 66–68°ë; [α ] + 8.3° (Ò 1, ëHCl ); IR
f
f
D
3
2
D
0
–
1
1
(
Vaseline oil, νmax, cm ): 3349 (NH), 2936 (CH), 2832 (A), [α ] + 9.6° (c 1, CHCl ); H NMR (CDCl , δ,
3 3
(
1
7
CH), 1740 (C=O), 1649 (NHCO), 1604 (NHCO),
ppm): 0.83 (6 H, t, ëç ), 1.28 (52 H, s, CH ), 1.61 (4 H,
3
2
485 (CH), 1298 (CH), 1312 (CO), 1279 (CO), 802,
m, βëç ), 1.6–2.4 (6 H, m, ëç ), 2.87 (2 H, m,
2
2
1
10 (CH); H NMR (CDCl , δ, ppm): 0.89 (6 H, t,
3
ëç ëç), 3.8 (2 H, s, CH ), 4.03 (1 H, m, CH), 4.1–4.3
2 2
CH ), 1.26 (52 H, s, CH ), 1.60 (4 H, m, βCH ), 2.70
3
2
2
(5 H, m, αëç , CH), 5.3 (1 H, m, NH), 7.0–7.8 (2 H,
2
(2 H, m, NH ), 2.84 (2 H, m, CH CH), 3.56 (2 H, s,
2 2
m, NH), 10.6 (1 H, m, NH); mass spectrum, m/z: 795.1
CH ), 4.00 (4 H, m, αCH ), 4.80 (1 H, m, CH), 6.90
+
2
2
[M] , M 795.20.
calc
+
(
6
1 H, d, NH); mass spectrum, m/z: 638.9 [M] , Mcalc
39.01.
Arginylglycylaspartic acid bishexadecyl ester
ACKNOWLEDGMENTS
bistrifluoroacetate (V). A solution of di-tert-butyl
pyrocarbonate (2.2 g, 10 mmol) in a 2 : 1 isopropanol–
water mixture (15 ml) was added to a solution of L-argi-
nine (0.5 g, 2.87 mmol) and potash (0.59 g, 4.31 mmol)
in a 2 : 1 isopropanol–water mixture (30 ml). The reac-
tion mixture was stirred for 24 at room temperature and
pH 11–12 and carefully acidified with citric acid to pH
This study was supported by the Russian Founda-
tion for Basic Research, project no. 01-03-32410 and
by the Inter-High-School Scientific and Technical Pro-
gram Fundamental Studies in the Area of Chemical
Technologies, project no. TOO-9.3.-2053.
3
. The product was extracted with ethyl acetate, and the
REFERENCES
extract was dried and evaporated in a vacuum. Boc-
Arg(Boc )-OH was obtained in yield 0.94 g (70%); Rf
1. Podobed, O.V. and Zhdanov, R.I., Vopr. Biol. Med.
Farm. Khim., 1999, no. 4, pp. 7–15.
2
0
.68 (B) [7].
2
. Maslov, M.A., Sycheva, E.V., Morozova, N.G., and
Cooled (0°ë) solutions of N-hydroxysuccinimide
Serebrennikova, G.A., Izv. Ross. Akad. Nauk, Ser. Khim.,
(
0.23 g, 2.03 mmol) in ethyl acetate (5 ml) and dicyclo-
2
000, no. 3, pp. 385–400.
hexylcarbodiimide (0.38 g, 1.85 mmol) in ethyl acetate
(
(
5 ml) were added to a solution of Boc-Arg(Boc) -OH
3. Pitard, B., Oudrhiri, N., Vigneron, J., Hauchecorne, M.,
Aguerre, O., Toury, R., Airiau, M.R., Scherman, R.,
Crouzeti, J., Lehn, J., and Lehn, P., Proc. Natl. Acad. Sci.
USA, 1999, vol. 96, pp. 2621–2626.
2
0.8 g, 1.69 mmol) cooled to 0°C. The reaction mixture
was kept at 5–10°ë for 24 h with TLC monitoring. The
precipitated solids were filtered off and washed with
anhydrous ethyl acetate. The combined filtrate was
evaporated in a vacuum, and the raw product was
4. Colin, M., Harbottle, R.P., Knight, A., Kornprobst, M.,
Cooper, R.G., Miller, A.D., Tragnan, G., Capeau, J.,
Coutelle, C., and Brahimi-Horn, M.C., Gene Therapy,
recrystallized from ethanol. Boc-Arg(Boc) -ONSu
2
1
998, vol. 5, pp. 1488–1498.
(
IV) was prepared in yield 0.73 g (76%); R 0.38 (C);
f
mp 56–58°ë (lit. mp 64–65°C [7]).
5. Ruoslahti, E., Annu. Rev. Cell Del. Biol., 1996, vol. 12,
pp. 697–715.
A solution of (III) (0.16 g, 0.25 mmol) in THF
(
0
5 ml) was added to a solution of (IV) (0.172 g,
6. Lapidot,Y., Rappoport, S., and Wolman,Y., J. Lipid Res.,
.301 mmol) in THF (5 ml). The reaction mixture was
1967, vol. 8, pp. 142–145.
vigorously stirred for 10 h with TLC monitoring and
evaporated. The residue was fractionated on a column
with silica gel in a 4 : 1 hexane–ethyl acetate mixture.
7
. Pozdnev, V.F., Bioorg. Khim., 1986, vol. 12, pp. 1013–
022.
1
α
G
G'
8. Hachisako, H., Yamazaki, T., Ihara, H., Hirayama, C.,
and Yamada, K., J. Chem. Soc., Perkin Trans. 2, 1994,
pp. 1671–1680.
Bishexadecyl ester of (N ,N ,N -tris-tert-butyloxycar-
bonylarginyl)glycylaspartic acid was prepared in yield
2
0
0
1
.135 g (49%); R 0.59 (D); [α ] + 14.9° (Ò 1, ëHCl3);
f
D
9
. Nitecki, D.E. and Goodman, J.W., Biochemistry, 1966,
H NMR (CDCl , δ, ppm): 0.86 (6 H, t, ëç ), 1.26
vol. 5, pp. 665–673.
3
3
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004