Enzymatic acylation of nucleosides-Novel route to nucleopeptides

Article abstract of DOI:10.1016/j.molcatb.2009.09.006

An efficient and straightforward proteolytic enzyme-catalyzed approach towards the regioselective synthesis of nucleopeptides was developed. Appropriate reaction conditions were investigated and certain peptide-modified nucleosides were obtained with 70-9

Full text of DOI:10.1016/j.molcatb.2009.09.006

Journal of Molecular Catalysis B: Enzymatic 62 (2010) 105–107
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Letter
Enzymatic acylation of nucleosides—Novel route to nucleopeptides
a r t i c l e i n f o
a b s t r a c t
Keywords:
Nucleoside
Enzyme
Catalysis
Regioselectivity
Nucleopeptide
An efficient and straightforward proteolytic enzyme-catalyzed approach towards the regioselective syn-
thesis of nucleopeptides was developed. Appropriate reaction conditions were investigated and certain
peptide-modified nucleosides were obtained with 70–90% yields. The obtained compounds could be
efficiently used for medicinal diagnostic kits and therapeutic treatment.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
2.2. Enzymatic reactions
Nucleopeptides and peptide-oligonucleotide conjugates have
been of great interest to researchers over the last decade due
to their unique combination of outstanding properties. Relevant
application in many fields of high technology can be found for
nucleotides and oligonucleotides containing peptidyl moieties:
organic synthesis of natural products, pharmaceuticals, drugs, etc.
They are used for native DNA/RNA labeling in order to estab-
lish the dimensional structure and hybridization properties of
nucleic acids [1,2] and as markers for synthetic polynucleotides [3].
Synthetic oligonucleotides bearing arginine-containing peptidyl
modified centers are reported to be immune-stimulating agents
and anti-cancer reagents [4]. Shvachkin and coworkers briefly
reported anti-nociceptive activity of synthetic nucleopeptides [5].
Recently the applicability of a peptide-nucleotide dual vaccine for
inhibition of human telomerase reverse transcriptase (hTERT) was
described; therefore nucleopeptides bear out significant promise
as anti-cancer therapeutic agents [6]. It was also demonstrated
that nucleopeptides could be efficiently used for enhanced targeted
delivery of therapeutic agents, antisense and siRNA into living cells
for medical treatments [7–9].
2.2.1. Representative experimental procedure for the synthesis of
3ꢀ-O-(Dnp-AlaAlaLeu)-dT
A mixture of Subtilisin-72 (10 ␮g) and 2ꢀ-deoxythymidine 1a
(10 ␮mol) was dissolved in 500 ␮l of phosphate buffer (pH 7.4).
The resulting solution was frozen and lyophilized. The lyophilisate
obtained was dissolved in 120 ␮l of phosphate buffer (pH 7.4) and
digested dropwise on the surface of Silochrom-C-80 in a reaction
vial. The sorbent was dried in high vacuum over phosphorus pen-
toxide. Next, Dnp-AlaAlaLeu-OMe 2 (20 ␮mol) was dissolved in THF
(1 ml), pyridine (50 ␮l) was added and the resulting solution was
added to a reaction vial with dried sorbent, the vial was capped
and the whole reaction mixture was stirred at room temperature
(approx. 18–23 ◦C) for 96 h. After the reaction was complete, the
solvent was separated from the solid phase and evaporated. Ace-
tonitrile (3 ml) was added to the residue, undissolved precipitate
was discarded. The solution obtained was purified with RP-HPLC
(30–60% acetonitrile in water + 0.1% TFA gradient) and the tar-
get compound 3ꢀ-O-(Dnp-AlaAlaLeu)-dT 3a was obtained in 89%
yield as a fine yellow powder. SALDI-MS: 664.3 [M+H]+; 1H NMR
(DMSO-d6, 300 MHz) ı: 1.1(s), 1.4–1.6 (dd), 1.8–2.2(m), 2.4–2.5(m),
3.5–3.8(m), 4.3–4.4(m), 4.9(m), 5.8(t), 6.7–6.8(dd), 7.6(s), 8.0(2d),
8.3–8.9 (d + s), 10.1(s) ppm.
Therefore, we set out to devise a straightforward and efficient
synthetic approach in the hope that it could be amenable for the
preparation of novel nucleopeptides.
2.3. Analytical methods
2. Materials and methods
The aminolysis reaction was monitored by RP-HPLC analysis
carried out in a system (Beckman System Gold) composed of a col-
umn (Beckman C18 5 ␮M, 250 mm × 4.6 mm) and a UV/vis detector
(Beckman System Gold, modul167, ꢀ = 220 nm). The compounds
were eluted by gradient elution with an eluent system of 5%
acetonitrile/H2O (A) and 95% acetonitrile/H2O (B), both containing
0.05% of trifluoroacetic acid (see Table 1).
2.1. Enzyme and chemicals
Subtilisin-72 was isolated and purified by common meth-
ods from Bacillus subtilis strain 72. All other chemicals used
were of analytical grade and purchased from Sigma–Aldrich and
Serva.
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.09.006

106
Letter / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 105–107
Table 1
Table 2
Gradient elution for HPLC analysis.
Starting materials and yields of novel nucleopeptides 3a–d.
Time, min
Flow rate, ml/min
% A
% B
#
Nucleoside
Nucleopeptide
Yield, %
0
10
15
20
1
1
1
1
80
40
40
80
20
60
60
20
3a
3b
3c
3d
dT
dC
dA
dG
3ꢀ-O-(Dnp-AlaAlaLeu)-dT
3ꢀ-O-(Dnp-AlaAlaLeu)-dC
3ꢀ-O-(Dnp-AlaAlaLeu)-dA
3ꢀ-O-(Dnp-AlaAlaLeu)-dG
89
87
71
77
3. Results and discussion
3.1. The reaction parameters
In order to run enzymatic reactions in non-conventional media
(organic solvent) it is necessary to prevent the enzyme inactiva-
tion. We used the enzyme that had been adsorbed on the surface
of Silochrom-C-80. Adsorbtion, followed by lyophilization, leads
to formation of a monolayer of enzyme with incorporated water
molecules. Thus, incorporated water supplies enough humidity for
enzyme to stay active in organic solvent.
Scheme 1.
3.2. Choice of enzyme
Subtilisin-72, which is closely related to the well character-
ized subtilisin Carlsberg, has a rather broad specificity. A number
of reactions for peptide bond synthesis catalyzed by Subtilisin-72
were described. Moreover, we have shown that the enzyme’s Sꢀ1-
subsite is capable of accepting different nucleophilic components,
not only N-nucleophiles (amines, amino acids and peptides) but
also O-nucleophiles (alcohols, sugars).
lation of unprotected nucleosides. We employed the combined
methods of Wong and Fang [15] and Rich and Dordick [16], and
have modified and improved these methods to achieve the desired
results. It was found that experiments with unchanged methods
(i.e. without lyophilization and sorption) do not lead to the desired
target molecule but only a peptide hydrolysis product. It was found
that the acylation reaction between non-activated esters (pep-
tidyl methyl ester) and free nucleosides (3a–d, Table 2) proceeded
smoothly at room temperature in high yields when an acylating
agent was lyophilized together with the enzyme and deposited on
the surface of Silochrom-C-80 to prepare the catalyst. The general
procedure is shown in Scheme 1.
In the course of our investigations we have tried different sol-
vents, namely, acetonitrile, THF and 1,4-dioxane. It was shown that
reactions in acetonitrile and THF produced the target compounds in
almost the same yields, while no reaction occurred if dioxane was
used as a solvent. Also pyridine was added to the reaction medium
for better solubility of starting nucleoside.
It was shown that coupling of 1 with peptide 2 proceeded regios-
electively. The formation of 3ꢀ-O-acylation products was observed
while no N-substitution of peptido-nucleosides took place. Reac-
tion products are shown in Table 2, NMR-spectra and appearance
are shown in Table 3.
Surprisingly, further experiments demonstrated that under
the described conditions only acylation of deoxyribonucleosides
occurred. All attempts to modify ribonucleosides, ribo- or deoxyri-
bonucleotides with the peptide failed. We believe that in the case
of these compounds no coupling between target hydroxyl and
the enzyme’s active center took place due to sterically hindered
phosphate groups of nucleotides and ribo-hydroxyl groups. The
obtained novel compounds are shown in Tables 2 and 3 with
appearance, reaction yields and spectroscopic data.
3.3. Known synthetic procedures
Different approaches to the nucleopeptide synthesis have been
suggested, but generally these methods are too complicated. The
most used synthetic pathways to the nucleopeptides are those
based on solid-phase synthesis [10], and pure chemical prepara-
tions, exemplified by different authors, for example EDCI/HOBt
coupling [11–13]. Basically, all the synthetic procedures described
so far are based on multi-step synthesis involving employment
of protective groups. Also enzymatic catalysis is employed, using
lipases from different species (i.e. Candida antarctica, Pseudomonas
sepacia). These lipases show pretty good results, but lipase-
catalysis is widely investigated and is broadly used. The novelty
of our work is that we prove the use of the proteolytic enzyme
Subtilisin-72 with an uncommon acyl-acceptor of non-amino acid
nature.
3.4. Results for peptidoacylation of nucleosides
Recently we reported the efficient enzyme-catalyzed synthe-
sis of various peptidyl-amides using Subtilisin-72/Silochrom as
a catalyst [14]. Here we desired to develop a more efficient
synthetic route by using the solid-phase-supported enzyme in
non-conventional medium to catalyze the reaction of direct acy-
Table 3
Physical data of novel nucleopeptides 3a–d.
#
Color
1H NMRa ı ppm
MSb
3a
3b
3c
3d
Yellow solid
Yellow solid
Yellow solid
Yellow solid
1.1(s), 1.4–1.6(dd), 1.8–2.2(m), 2.4–2.5(m), 3.5–3.8(m), 4.0(s), 4.3–4.4(m), 4.9(m), 5.8(t), 6.7–6.8(t), 7.6(s), 8.0(2d), 8.3–8.9(d), 10.1(s)
664.3
649.7
673.3
689.6
1.1(s), 1.3–1.6(dd), 1.8–2.1(m), 2.4–2.6(m), 3.4–3.7(m), 4.0(s), 4.3–4.4(m), 5.8(t), 6.7–6.9(t), 7.3(s), 8.0(dd), 8.3(d), 8.9(s)
1.2(s), 1.4–1.6(dd), 1.7–1.9(m), 2.1(s), 2.3–2.6(m), 3.5–3.7(m), 4.0(s), 4.3–4.5(m), 5.8(t), 6.6–6.9(t), 8.0(dd), 8.1(s), 8.3(d), 8.7(s), 8.9(s)
1.0(s), 1.3–1.5(dd), 1.7–1.8(m), 2.1(s), 2.5–2.8(m), 3.4–3.8(m), 4.0(s), 4.2–4.4(m), 5.9(t), 6.7–6.9(t), 7.9–8.1(dd), 8.4(d), 8.9(s)
a
1H NMR was registered in DMSO-d6 at 300 MHz.
SALDI-MS: [M+H]+.
b

Letter / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 105–107
107
4. Conclusions
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[12] V. Marchan, A. Grandas, Curr. Protoc. Nucleic Acid Chem. 4 (2007) 4.32.
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(5) (1995) 573.
[14] T.L. Voyushina, M.E. Sergeev, G.G. Chestukhina, J. Peptide Sci. 14 (8) (2008) 57.
[15] C.-H. Wong, J.-M. Fang, Synlett 06 (1994) 393.
[16] J.O. Rich, J.S. Dordick, J. Am. Chem. Soc. 119 (1997) 3245.
A novel and highly efficient method was developed for the
synthesis of peptide-modified deoxynucleosides under mild con-
ditions under proteolytic enzyme catalysis. Regioselectivity of the
reaction was investigated; formation of 3ꢀ-O-acylation products
was established. Biological evaluation of the synthesized peptides
is ongoing.
Maxim E. Sergeev∗
Tatiana L. Voyushina
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∗ Corresponding author. Tel.: +7 495 315 37 38;
fax: +7 495 315 05 01.
E-mail addresses: mesergeev@mail.ru,
maxsergeev@iskra-net.ru (M.E. Sergeev)
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25 June 2009
Available online 22 September 2009