A novel approach to the preparation of peptide-oligonucleotide conjugates

Article abstract of DOI:10.1055/s-0029-1217812

A novel approach to the synthesis of peptide-oligonucleotide conjugates (POC) based on the oxathiaphospholane chemistry has been developed. Peptide and oligonucleotide fragments, which were separately prepared, were linked postsynthetically by the functio

Full text of DOI:10.1055/s-0029-1217812

LETTER
2269
A Novel Approach to the Preparation of Peptide–Oligonucleotide Conjugates
Preparation of
Pe
e
ptide–Olig
n
C
a
onjugates ta Kaczmarek, Janina Baraniak,* Wojciech J. Stec
Department of Bioorganic Chemistry, Center of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112,
90-363 Łódż, Poland
Fax +48(42)6815483; E-mail: baraniak@bio.cbmm.lodz.pl
Received 18 May 2009
ward. The chemistries used on peptide and DNA synthe-
Abstract: A novel approach to the synthesis of peptide–oligonucle-
sizers are not fully compatible with each other. Thus,
otide conjugates (POC) based on the oxathiaphospholane chemistry
many research groups are actively involved in searching
new routes for chemical synthesis of POC, and in the re-
has been developed. Peptide and oligonucleotide fragments, which
were separately prepared, were linked postsynthetically by the func-
tionalization of the peptide either by attachment of the oxathiaphos- cent years significant advancements have been made in
this field.8 Despite a number of papers describing the syn-
pholane residue directly at the N-terminus or at the hydroxy group
of the linker connected to N-terminus. The conjugation reaction,
thesis of POC there is not a general strategy for peptide–
based on DBU-assisted nucleophilic attack of hydroxy group of oli-
oligonucleotide conjugation.
gonucleotide on phosphorus atom in the oxathiaphospholane deriv-
In this communication we present a novel approach to the
synthesis of POC-containing phosphoramidate or phos-
phorodiester linkage between the peptide and oligonucle-
otide fragments. Among a variety of chemical linkages
commonly used in conjugations of oligonucleotides with
peptides there are only two references in chemical litera-
ture concerning phosphoramidate type of linkage (i.e., N-
acylphosphoramidate9 and N-alkylphosphoramidate10
linkages, respectively).
atives of peptide, furnished the desired POC.
Key words: conjugation, peptides, oligonucleotides, phosphoryla-
tions, solid-phase synthesis
Synthetic oligonucleotides and their modified analogues
constitute a class of potential therapeutic agents to modu-
late the function of specific genes.1 The reason for the lack
of an effective oligonucleotide-based therapy for treat-
ment of gene-related diseases is predominantly due to
poor cellular uptake of naked oligonucleotides, their insta-
bility in intracellular fluid, and low binding affinity to the
target.2 There have been concerted efforts, using different
approaches, to surpass all, or at least most, of these barri-
ers.3 Among those reported are enhanced penetration
(both into cells and into the nuclei) or targeted delivery by
administration of noncovalent complexes4 or covalent
conjugates of an oligonucleotide with suitable peptide(s).5
The latter give more consistent products in which the de-
sired properties can be carefully controlled by structural
variations.
Our strategy was based on the oxathiaphospholane chem-
istry, originally developed in our laboratory for the stereo-
controlled synthesis of P-chiral analogues of oligo-
nucleotides.11 We further synthesized nucleoside phos-
phorothioylated polyols mimicking the function of dinu-
cleoside polyphosphates12 and phosphorothioylated
amino acids13 by application of this chemistry. The suc-
cess in our synthesis of conjugates of amino acids with
nucleoside-5¢-O-phosphorothioates14 suggested that effi-
cient conjugation of peptide via phosphoramide bond for-
mation to oligonucleotide would be viable.
Since nucleoside 5¢-O-phosphoramidothioates have been
successfully synthesized by introduction of 2-thiono-
1,3,2-oxathiaphospholane to 5¢-hydroxy group, followed
by aminolysis with selected amines,15 replacement of
amines with amino acids in our model experiments would
be the most logical approach for our purposes. However,
no reaction occurred due to paucity of nucleophilicity of
the amino group of amino acids. We, therefore, changed
our strategy.
Linking two distinct classes of biologically important
biopolymers – peptides and oligonucleotides – by cova-
lent bonds in many possible ways, results in a new class
of compounds known as peptide–oligonucleotide conju-
gates (POC).6 A selected peptide can be bound either at
the base-, 3¢- or 5¢-position of the sugar unit, or at the
phosphate backbone of the oligonucleotide. Similarly, the
site of conjugation in a peptide can be either the C- or N-
terminus or the side chain. There are two different ap-
proaches, which have been studied extensively for prepar-
ing POC. One is total in-line synthesis, and the other is
fragment conjugation.7
Our synthesis of POC described herein consists of the fol-
lowing steps: (i) N-derivatization of the corresponding
peptides with oxathiaphosphitylating reagent, (ii) solid-
phase oligonucleotides synthesis, (iii) reaction of 2-
thiono-1,3,2-oxathiaphospholane moiety in peptide deriv-
atives with 5¢- hydroxy group of oligonucleotide attached
to solid support, (iv) cleavage of POC from the solid sup-
port.
Although both peptide and oligonucleotide syntheses are
well developed, preparation of POC are not as straightfor-
SYNLETT 2009, No. 14, pp 2269–2272
x
x
.x
x
.2
0
0
9
As a peptide component we chose L-alanyl-L-alanyl-L-
Advanced online publication: 07.08.2009
alanine methyl ester (Ala-Ala-Ala-OMe, 1) which was
DOI: 10.1055/s-0029-1217812; Art ID: G15609ST
© Georg Thieme Verlag Stuttgart · New York

2270
R. Kaczmarek et al.
LETTER
oxathiaphosphorylated by the reaction with 2-chloro- product 8 was obtained and purified by means of HPLC
1,3,2-oxathiaphospholane (2) in pyridine solution and in (45% yield18), and its identity was confirmed by means of
the presence of elemental sulfur as depicted in Scheme 1. MALDI-MS analysis {m/z = 1523 [M – 1]}.
Me
N
O
C
O
C
O
C
O
C
O
C
O
C
S
S
H
H
H
OMe
H2N CH
Me
CH
Me
N
CH
Me
OMe
H2N CH
Me
N
CH
Me
N
CH
Me
P
Cl
+
P
Cl
+
O
2
O
2
1
6
i
i
S
P
O
O
C
O
S
S
P
O
O
H
H
H
O
Me
S
H
H
N
CH
C
N
CH
N
CH C
OMe
N
CH
Me
C
CH
C
N
CH
Me
C
OMe
N
O
Me
Me
Me
O
Me
3
7
5'OH
/ ii
T
PS
(TPS)2TO
5'OH
1)
/ ii
T
PS
(TPS)2TO
4
4
2) iii
–S
O
HN
CH Me
P
N
R
C
O
O
C
O
O
O
H
Me
N
H
H2N
C
CH
Me
N
C
CH
Me
CH
Me
N
P
O
TPS(TPS)2TOH
5
S–
O
C
O
C
O
C
O
8
H
H
H
N
H2N
CH
Me
N
CH
Me
N
CH
Me
P
S–
O
TPS(TPS)2TOH
Scheme 2 Reagents and conditions: (i) S8, pyridine; (ii) DBU,
MeCN; (iii) NH4OH.
Scheme 1 Reagents and conditions: (i) S8, pyridine; (ii) DBU,
MeCN.
The oxathiaphospholane chemistry could also be used to
prepare POC with the N-nonmethylated peptide at the b-
position, albeit in this case introduction of a linker be-
tween the peptide and the oligonucleotide is required in
order to avoid formation of the cyclic product. Corre-
sponding linker attached to the N-terminus of the peptide
should contain the terminal hydroxy group susceptible to
the oxathiaphospholane derivatization. Hence a linkage
reagent 9 (Scheme 3) with suitable functional group being
able to react with amino group of the peptide was synthe-
sized according to the procedure described by Halambidis
et al.19
The reaction monitored by 31P NMR was complete within
12 hours, and the final product 3 was isolated in 83% yield
by silica gel column chromatography. The identity of
compound 3, obtained as the mixture of diastereomers
[31P NMR (81 MHz, CDCl3): d = 95.68, 95.48 ppm], was
confirmed by MS–FAB analysis {m/z = 382 [M – 1]}. The
modified peptide 3 was then coupled manually to resin-
bound tetrathymidine phosphorothioates (4), which was
earlier constructed according to the oxathiaphospholane
methodology.11 The reaction was carried out in the typical
way in acetonitrile solution in the presence of DBU.16
Treatment of p-nitrophenyl 3-[6-(4,4¢-dimethoxytrityl-
oxy)hexylcarbamoyl]propanoate (9, Scheme 3) with
commercially available methyl ester of L-leucyl-glycil-
glycine (10, NH2-Leu-Gly-Gly-OMe) in DMF and in the
presence of DMAP gave compound 11 in 39% yield
{MS–FAB: m/z = 759 [M – 1]}. Removal of the DMTr
group from 11 in the standard manner afforded compound
12 in which the hydroxy group was available for oxathia-
phospholane derivatization. Therefore treatment of 12
with 2-chloro-1,3,2-oxathiaphospholane (2) in pyridine
and in the presence of elemental sulfur gave derivative 13
{31P NMR (81 MHz, CDCl3): d = 104.52. MS–FAB: m/z =
595 [M – 1]} in 90% isolated yield after silica gel column
chromatography.
However, MS–FAB analysis of the crude material ob-
tained after standard ammonia cleavage from the solid
5¢HO
support revealed only the presence of
T TPSTPSTOH
PS
{m/z = 1201 [M – 1]}. Apparently, DBU removed the
acidic proton from b-NH in the N-(2-thiono-1,3,2-oxa-
thiaphospholane) derivative 3, and the resulting anionic
nitrogen attacked the phosphorus to form compound 5.
This intramolecular cyclization was confirmed in the in-
dependent experiment.17
In order to prevent dissociation at b-nitrogen in 3, we used
methyl ester of L-alanyl-N-methyl-L-alanyl-L-alanine (6,
Scheme 2) as the starting peptide, and its oxathiaphos-
pholane derivatives 7 were obtained {31P NMR (81 MHz,
CH3OD): d = 97.60, 97.40 ppm. MS–FAB: m/z = 396 [M
– 1]} in 71% isolated yield under conditions already de-
scribed for the synthesis of 3.
Conjugation of 13 was performed with tetrathymidine
phosphorothioates linked to LCA-CPG solid-phase sup-
port (4), by the procedure described already, and, after
completion of the reaction the desired POC 14 was
cleaved from the solid support by aqueous ammonia treat-
ment and purified by means of HPLC in 62% yield,18 and
Ring-opening condensation of 7 with tetrathymidine
phosphorothioates linked to sarcosinylated LCA-CPG
support 4 was performed in the presence of DBU. From
the reaction mixture, after ammonia treatment, desired
Synlett 2009, No. 14, 2269–2272 © Thieme Stuttgart · New York

LETTER
Preparation of Peptide–Oligonucleotide Conjugates
2271
O
C
O
H
DMTO (CH2)6
N
(CH2)2
i
C
O
NO2
9
O
H
O
C
O
O
C
O
C
H
H
H
DMTO (CH2)6
N
C
(CH2)2
11
C
N
CH
CH2
CH
N
CH2
N
CH2
OMe
Me
ii
Me
O
O
O
O
O
H
H
H
H
HO (CH2)6
N
C
(CH2)2
C
N
CH
CH2
CH
C
N
CH2
C
N
CH2
C
OMe
12
Me
Me
iii
S
S
O
O
C
O
C
O
C
O
H
H
H
H
N
P
O
(CH2)6
N
C
(CH2)2
N
CH
CH2
CH
N
CH2
CH2
C
OMe
O
13
Me
Me
iv, v
O
C
O
O
O
C
O
C
O
H
H
H
N
H
H2N
CH2
N
C
CH2
N
C
CH
CH2
CH
(CH2)2
N
(CH2)6
O
P
O
TPS(TPS)2TOH
S–
14
Me
Me
Scheme 3 Reagents and conditions: (i) MeO-Gly-Gly-Leu-NH2 (10); (ii) PTSA; (iii) 2-chloro-1,3,2-oxathiaphospholane (2), S8; (iv) tetra-
thymidine phosphorothioate linked to sarcosinylated LCA-CPG support (4), DBU, MeCN; (v) NH4OH.
its identity was confirmed by MALDI-MS {m/z = 1723 and oligonucleotide the oxathiaphospholane moiety rep-
[M – 1]}.
resents a new type of such a group attached at the peptide
site of conjugation. Further studies on the application of
oxathiaphospholane chemistry for conjugation of longer
peptides with oligonucleotides are under way.
In conclusion, we developed a novel effective method for
the synthesis of peptide–oligonucleotide conjugates based
on the oxathiaphospholane chemistry. Two strategies
have been executed for the functionalization of the pep-
tide either by attachment of the oxathiaphospholane resi-
due directly at the N-terminus as in the case of 6 or at the
hydroxy group of the linker connected to N-terminus (for
12). These oxathiaphospholane intermediates are ob-
tained in a facile and efficient way and are stable com-
pounds that can be stored for a long time. The conjugation
reaction, based on DBU-assisted nucleophilic attack of
hydroxy group of oligonucleotide on phosphorus in the
oxathiaphospholane ring in 7 or 13 furnished the desired
POC in satisfactory yields. As it has been confirmed in
our earlier work13 concerning phosphorothioylation of
amino acids, the presence of DBU in the oxathiaphos-
pholane ring-opening reaction did not induce the racem-
ization of amino acids.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
Acknowledgment
These studies were supported financially by the Ministry of
Sciences and Higher Education, projects: N 204 075 32/2063 and
PBZ-MNiSW-07/I/2007. The authors are grateful to Dr. Kyoichi
A. Watanabe for careful reading of the manuscript.
References and Notes
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Our approach, so called the solid-phase fragment conjuga-
tion, is to link peptide and oligonucleotide fragments,
which are separately prepared postsynthetically. Among
reactive functional groups utilized to connect the peptide
Synlett 2009, No. 14, 2269–2272 © Thieme Stuttgart · New York

2272
R. Kaczmarek et al.
LETTER
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TEAB (pH 7.5); buffer B, 40% MeCN in 0.1 M TEAB;
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Synlett 2009, No. 14, 2269–2272 © Thieme Stuttgart · New York