Towards a general method for the stepwise solid-phase synthesis of peptide-oligonucleotide conjugates

Article abstract of DOI:10.1016/S0040-4039(01)02185-2

Three peptide-oligonucleotide phosphorothioate hybrids were synthesized using a new approach for stepwise solid-phase synthesis of conjugates. This method utilizes commercially available N_α-Fmoc amino acids for peptide synthesis and a new solid support. Three specific modifications of the solid-phase were made after the peptide and before the oligonucleotide assembly.

Full text of DOI:10.1016/S0040-4039(01)02185-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 527–530
Towards a general method for the stepwise solid-phase synthesis
of peptide–oligonucleotide conjugates
Maxim Antopolsky, Elena Azhayeva, Unni Tengvall and Alex Azhayev*
Department of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland
Received 10 October 2001; revised 8 November 2001; accepted 14 November 2001
Abstract—Three peptide–oligonucleotide phosphorothioate hybrids were synthesized using a new approach for stepwise solid-
phase synthesis of conjugates. This method utilizes commercially available Na-Fmoc amino acids for peptide synthesis and a new
solid support. Three specific modifications of the solid-phase were made after the peptide and before the oligonucleotide assembly.
© 2002 Elsevier Science Ltd. All rights reserved.
No general method for the stepwise synthesis of any
peptide–oligonucleotide phosphorothioate conjugate
(POPC) on a single solid support has been reported so
far.1 The sequential assembly leads to a limited number
of POPCs due to the incompatibility of the peptide (P)
and the oligonucleotide (ON) protecting groups. As a
result, the use of various base-labile peptide side-chain
protecting groups have been reported.2–7
Described herein, is a procedure for further developing
a general method for the stepwise solid-phase synthesis
of POPCs. This procedure also uses commercially avail-
able Na-Fmoc amino acids for P synthesis along with
certain specific modifications after the P and before the
ON assembly.
In this work a suitable solid support was prepared,
containing a Fmoc-protected amino-function for easy
standard peptide chain elongation, a Boc-protected
amino function, which was stable during the process of
peptide synthesis but easily removable for subsequent
modification and, finally, a base-labile bridge to a solid
matrix–long chain alkylamino controlled pore glass
(lcaa-CPG). The synthesis of solid support SS-1 is
outlined in Scheme 1. Initially, a long chain alkylamino
controlled pore glass (CPG) was derivatized by the
N-hydroxy-5-norbornene-endo-2,3-dicarboxamide ester
of 2-(4%,4¦-dimethoxytriphenylmethyloxy) acetic acid
Previously we8 described the synthesis of three conju-
gates containing 10- or 16-mer Ps, which incorporated
two or three arginine residues. The required Ps were
assembled on the support by using common Na-Fmoc
amino acids, but with Fmoc-Orn(Mtt)-OH as a precur-
sor of -Arg-. Solid phases were then modified to offer
doubly protected guanidine functions of arginines.
Finally, ONs were assembled and POPCs were cleaved
from the support and deprotected with aqueous
ammonia.
O
O
O
OH
NH2
NB
ODMTr
ODMTr
+
CPG
i
CPG
NH
3
O
NH
CPG
HN
1
2
O
ii
O
NHFmoc
N
O
NB =
NHBoc
CPG
SS-1
O
O
Scheme 1. (i) DCA; (ii) Fmoc-Lys(Boc)-OH/TPS-Cl.
* Corresponding author. Tel.: +358-17-162204; fax: +358-17-162456; e-mail: alex.azhayev@uku.fi
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)02185-2

528
M. Antopolsky et al. / Tetrahedron Letters 43 (2002) 527–530
(1)9 togivematrix2. AftertheremovaloftheDMTrgroup
with 2% dichloroacetic acid (DCA) in dichloromethane,
Fmoc-Lys(Boc)-OH was linked to the matrix 3 in the
presence of 2,4,6-triisopropylbenzenesulfonyl chloride
(TPS-Cl) to give solid support SS-1. The resulting SS-1
contained 35 mmol of Fmoc-groups per gram of CPG.10
assembly. In this procedure, standard Fmoc-peptide
chemistry and commercially available Na-Fmoc amino
acids11 were employed to assemble two Ps on our initial
SS-1 matrix to give phases SS-2. These Ps were either H
-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys-
(Acm)-OH (P-1) or (H-Met-His-Ile-Glu-Ser-Leu-Asp-
Ser-Tyr-Thr-Cys(Acm)-OH (P-2). After the P assembly,
all acid-labile protecting groups were removed from Ps,
attached to the solid-phase, which resulted in SS-3
supports.
Scheme 2 demonstrates the preparation of solid support
SS-5, incorporating the required P with base-labile
side-chain protecting groups and a linker ready for ON
Boc
N
Support
i
Support
SS-1
OtBu COOBzl
BocHN
NHFmoc
BocHN
P e p t i d e
SS-2
NH
NHFmoc
ii
H
N
NHTfa
N
Support
OH
P e p t i d e
NHTfa
COOBzl
N
Tr
H2N
NH
NHFmoc
H
N
SS-3
iii
N
Support
OH
COOBzl
NH
NH
P e p t i d e
SS-4
Ac
N
NH
NHFmoc
O
iv
NHTfa
N
Support
DMTrO
OAc
COOBzl
NH
NH
P e p t i d e
NH
NHFmoc
O
SS-5
NHTfa
N
DMTrO
N
Ac
Scheme 2. (i) Standard Fmoc-peptide synthesis; (ii) 40% TFA; (iii) 1; (iv) Ac2O/N-methylimidazole/2,6-lutidine.
Ac
N
Support
OAc COOBzl
i
P e p t i d e
NH
SS-5
NH
NHFmoc
O
NHTfa
N
O
N
P e p t i d e
Ac
ii
NH2
O
Protected oligo
NH
Oligonucleotide
phosphorothioate
HO
O
5'
NH
O
Scheme 3. (i) Standard oligonucleotide phosphorothioate synthesis; (ii) 0.4 M NaOH, rt, 17 h.

M. Antopolsky et al. / Tetrahedron Letters 43 (2002) 527–530
529
give rise to SS-5 supports, ready for ON assembly.
Both SS-5 matrices contained 32 mmol of DMTr
groups per gram of CPG.12
ON phosphorothioates (5%-TGGCGTCTTCCATTT-3%,
O-1 and 5%-TATGATCTGTCACAGCTTGA-3%, O-2)
were assembled on SS-5 supports using standard proto-
cols (Scheme 3). Protected POPCs were cleaved from
the support and deprotected with aqueous sodium
hydroxide to give rise to the crude conjugates, O-1-P-1,
O-2-P-1 and O-2-P-2. The chromatographic profile of
crude desalted O-2-P-1 (product peak at tR about 18
min) is shown in Fig. 1 as an illustrative example. After
purification by IE HPLC7 and desalting, the purity of
the POPCs was higher than 95%, as determined by RP
HPLC. Typically 20–30 AU of pure POPCs were
obtained when starting from 1 mmol of SS-5 solid
supports. Fig. 2 demonstrates a typical 15% PAGE of
conjugate O-2-P-1 and oligomer O-2 as an illustrative
example. In all cases, POPCs migrated considerably
slower than the corresponding ONs. Final characteriza-
tion was made by ESI-MS (Table 1). The measured and
calculated average molecular masses of the conjugates
were in good agreement, with the difference between
the calculated and measured Mr being less than 0.03%.
Figure 1. Ion-exchange HPLC traces of crude conjugate O2-
P1.
Acknowledgements
We thank Dr. James Callaway (University of Kuopio)
for reading the manuscript. Financial support from the
Technology Development Center of Finland is grate-
fully acknowledged.
References
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Figure 2. Photograph of 15% denaturing PAGE. Lane 1,
O2-P1; lane 2, contaminated O2-P1; lane 3, O2.
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set, I.; Pedroso, E.; Grandas, A. Tetrahedron 1999, 55,
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Table 1. Measured and theoretically calculated average
molecular masses of synthesized conjugates.
7. Antopolsky, M.; Azhayev, A. Helv. Chim. Acta 1999, 82,
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Conjugate
Calculated mass
Measured mass (Mr)
O1-P1
O2-P1
O2-P2
6404.8
8067.1
8046.0
6403.1
8067.3
8044.8
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9. Glycolic acid (22 mmol, 1.67 g) was dissolved in 100 ml
of dry pyridine. DMTr-Cl (20 mmol, 6.76 g), dissolved in
50 ml of dry THF, was added and the mixture was stirred
at room temperature overnight. The reaction was
quenched by the addition of ice. The mixture was concen-
trated, dissolved in ethyl acetate and extracted with
water. The organic layer was dried, concentrated and
The primary amino group of SS-3 was then selectively
derivatized with compound 1 to give SS-4, incorporat-
ing a DMTr-oxy function. The remaining unprotected
side-chain functional groups were finally acetylated to

530
M. Antopolsky et al. / Tetrahedron Letters 43 (2002) 527–530
finally purified by flash chromatography on silica gel.
CHꢀCH); 1.66 (d, 1H, J=8.85, (CH-ChaHb-CH); 1.51 (d,
1H, J=8.85, (CH-ChaHb-CH). Anal. calcd for
C32H29NO7: C, 71.23; H, 5.42; N, 2.60; O, 20.76. Found:
C, 71.49; H, 5.51; N, 2.32%.
Concentration in vacuo gave 5.88 g (78%) of a pale yellow
oil. This oil (11 mmol, 4.4 g) and N-hydroxy-5-norbornene-
endo-2,3-dicarboxamide (1.97 g, 11 mmol) were dissolved
in 50 ml of dry acetonitrile, the solution was cooled down
to −20°C and a solution of N,N%-dicyclohexylcarbodiimide
(11 mmol, 2.31 g) in 10 ml of acetonitrile was added. The
reaction mixture was left at +4°C overnight. Solids were
filtered off and washed with cold acetonitrile. The filtrate
and washings were combined and evaporated to dryness.
Crystallization of the residue from 2-propanol gave 3.8 g
(65%) of 1 as white crystals; mp 52–54°C; 1H NMR
(CD3CN): l 7.43–6.85 (m, 13H, arom.); 6.11 (s, 2H,
CHaꢀCHb); 4.04 (s, 2H, ChaHbCO); 3.75 (s, 6H, 2×
CH3OC6H4); 3.34 (s, 2H, 2×COCH); 3.32 (s, 2H, 2×CH-
10. Novabiochem Catalog & Peptide Synthesis Handbook, 1999,
p. 43.
11. Na-Fmoc amino acids used in this work were: Fmoc-Met-
OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Glu-
(OBzl)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Asp-
(OBzl)-OH, Fmoc-Lys(OTfa)-OH, Fmoc-Cys(Acm)-OH,
Fmoc-His(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-
OH.
12. Atkinson, T.; Smith, M. In Oligonucleotide Synthesis. A
Practical Approach; Gait, M. J., Ed.; IRL Press: Oxford,
1984; pp. 35–81.