2690
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 11, November, 2005
Koroleva et al.
3
2
Table 6. Degree of cleavage of the [5´ꢀ P]ꢀlabeled 96ꢀmer fragꢀ
ment of RNA HIV 1 with conjugates of DABCO derivatives with
tetrapeptides of the R series (K series) after 6 and 19 h*
tane/1ꢀbromodecane (20 mmol) were added. The reaction mixꢀ
ture was refluxed for 1 day. Triethylamine hydrobromide that
precipitated was filtered off. The solution was washed succesꢀ
sively with water, a 2% citric acid solution, water, a saturated
NaHCO solution, and water. The organic phase was dried with
Peptide
Cleavage (%)
3
anhydrous Na SO and the solvent was removed in vacuo. The
2
4
6
h
19 h
resulting oily compound was used in subsequent experiments
without additional purification.
1
1
1
2
2
7 (22)
8 (23)
9 (24)
0
62 (89)
77 (49)
69 (82)
40
82 (98)
99 (93)
99 (98)
82
NꢀHydrosuccinimide esters of protected amino acids were
synthesized according to a known general procedure.1
BocꢀDeprotection (general procedure). A Bocꢀprotected
amino acid or peptide (1 mmol) was dissolved in a 1 : 1
TFA—CH Cl mixture (5 mL). The reaction mixture was kept
2
1 (25)
79 (6)
98 (15)
2
2
at ~20 °C for 1.5 h, the solvent was removed in vacuo, and ethanol
was added to, and distilled from, the residue (3×10 mL).
*
The reaction conditions are given in the note to Table 5; C (conꢀ
–
4
–1
jugates with tetrapeptides) = 10 mol L . The values for the
K series are given in parentheses.
Peptide synthesis (general procedure). A solution of
Nꢀhydrosuccinimide esters of a protected amino acid (1.2 mmol)
in ethyl acetate (5 mL) was added with stirring to a solution of
TFA salt of an Nꢀnonprotected amino acid or peptide (1 mmol)
and triethylamine (2 mmol, 278 µL) in ethyl acetate (10 mL).
The reaction mixture was stirred until the amino compoꢀ
nent was completely consumed (TLC control) and then
N,N´ꢀdimethylethylenediamine (1 mmol, 110 µL) was added.
The reaction mixture was stirred for 30 min and washed with a
Catalytically active tetrapeptide 16, which was used for
the synthesis of conjugate 25, also exhibited the lowest
activity. Apparently, the pꢀaminobenzoic linker used in
the present study hinders the optimum arrangement of
the catalytically active groups with the respect to the riꢀ
bose phosphate backbone of RNA.
Thus, based on the results of the present study, it can
be concluded that the development of structureꢀfunction
models of catalytic sites of natural enzymes is an efficient
approach to the design of artificial ribonucleases. The
addition of catalytically active groups to molecules posꢀ
sessing high affinity for RNA increases the efficacy of the
substrate cleavage.
2
% citric acid solution (20 mL), water (20 mL), a saturated
NaHCO solution (20 mL), and water (20 mL). The organic
3
layer was dried with anhydrous Na SO4 and the solvent was
2
removed in vacuo. The solution of the protected tetrapeptide in
ethyl acetate (15 mL), after Bocꢀdeprotection, was washed with
a saturated NaHCO solution (10 mL) and water (10 mL) and
3
then dried with anhydrous Na SO . The solvent was removed
2
4
in vacuo.
N,OꢀDeprotection of tetrapeptides (general procedure). Afꢀ
ter Bocꢀdeprotection, tetrapeptides (0.1 mmol) were dissolved
in methanol (10 mL) and subjected to hydrogenolysis in the
presence of 5% Pd/C (50 mg) (TLC control). After completion
of the reaction, the catalyst was filtered off and the solvent was
removed in vacuo.
Synthesis of peptide conjugates with 1,4ꢀdiazabicycloocꢀ
tane derivatives (general procedure). A protected tetrapeptide
(0.1 mmol), after Bocꢀdeprotection, was dissolved in distilled
DMF (1 mL). Then triethylamine (0.1 mmol, 13.9 µL) was
added. A suspension of 1ꢀtetradecylꢀ4ꢀ(4ꢀnitrophenoxycarꢀ
bonyl)methylꢀ1,4ꢀdiazoniabicyclo[2.2.2]octane dibromide
(0.1 mmol, 64.9 mg) in DMF (1 mL) was added to the reaction
mixture and the mixture was stirred at ~20 °C for 48 h. The
product was precipitated with 10 volumes of diethyl ether. The
white flakeꢀlike precipitate was separated by centrifugation,
washed with diethyl ether, and dried in vacuo.
Experimental
In the present study, we used dicyclohexylcarbodiimide,
Nꢀhydroxysuccinimide, Bocꢀβꢀalanine, diꢀtertꢀbutyl pyroꢀ
carbonate, Nꢀethyldiisopropylamine, N ꢀNO ꢀLꢀarginine
ω
2
α
(
Fluka, Germany), NꢀBocꢀglycine, N ꢀBocꢀOꢀBzlꢀLꢀserine,
α
α
e
N ꢀBocꢀOꢀBzlꢀLꢀthreonine, N ꢀBocꢀN ꢀ(2ClꢀCbz)ꢀLꢀlysine,
NꢀBocꢀOꢀBzlꢀLꢀglutamic acid, and NꢀBocꢀLꢀphenylalanine
(
FisherBiotech USA). Other chemical reagents and solvents (of
reagent grade and special purity grade; manufactured in Russia)
1
1
were purified, if necessary, according to standard procedures.
ꢀTetradecylꢀ4ꢀ(4ꢀnitrophenoxycarbonyl)methylꢀ1,4ꢀdiazoniaꢀ
bicyclo[2.2.2]octane dibromide was kindly provided by D. A.
Konevets (Institute of Chemical Biology and Fundamental
Medicine of the Siberian Branch of the Russian Academy of
Sciences).
1
Thinꢀlayer chromatography was performed on DCꢀAlufolien
Kieselgel 60 F254 plates (Merck, Germany) using the
CH Cl —methanol (9 : 1) (A) and ethyl acetate—methanol
This study was financially supported by the Russian
Foundation for Basic Research (Project No. 04ꢀ04ꢀ
48566ꢀа) and the US Civilian Research and Developꢀ
ment Foundation (CRDF, Grant RECꢀ008).
2
2
(
3 : 1) (B) solvent systems.
1
The H NMR spectra were recorded on a Bruker WPꢀ200ꢀSY
spectrometer (200 MHz) with Me Si as the internal standard.
4
The chemical shifts are given on the δ scale. The MALDIꢀTOF
mass spectra were obtained on a Bruker FLEX III spectrometer
References
(
Bruker Analytical Systems).
α
N ꢀBocꢀAmino acid octyl/decyl esters (general procedure).
1. I. L. Kuznetsova, N. S. Zhdan, M. A. Zenkova, V. V. Vlassov,
and V. N. Sil´nikov, Izv. Akad. Nauk, Ser. Khim., 2004, 435
[Russ. Chem. Bull., Int. Ed., 2004, 53, 455].
α
N ꢀBocꢀAmino acid (20 mmol) was dissolved in ethyl acꢀ
etate (15 mL). Triethylamine (22 mmol) and 1ꢀbromoocꢀ
Koroleva
