PNA
Synthesis Using
Peptide nucleic acid (PNA), a nucleic acid analogue, is attractive as a gene-sensing component for diagnostic, biological,
and pharmaceutical applications. Panagene has developed self-activated Bts PNA monomers suitable for bulk production,
Novel
Self-Activated
which yield high purity of PNA oligomers.
Monomers
1
Hyunil Lee, Ph.D.
[email protected]
Panagene Inc. www.panagene.com
816 Tamnip-dong, Yuseong-gu, Daejeon 305-510, Korea
Peptide nucleic acid (PNA), a nucleic acid analogue, is attractive as a gene-sensing component for diagnostic, biological,
and pharmaceutical applications. Panagene has developed self-activated Bts PNA monomers suitable for bulk production,
which yield high purity of PNA oligomers.1
Peptide nucleic acid (PNA) is a nucleic acid analogue,
which was first reported by Nielsen et al. in 1991.2 PNA has
received great attention due to many favorable properties
including chemical and thermal stability, resistance to
nucleases and proteases, stronger and faster binding affinity
to the complementary nucleic acid,3 hybridization under low
salt concentration,4 and higher specificity and sensitivity to a
single mismatch. PNA has been used for many diagnostic,
molecular biological, and pharmaceutical applications. PNA
has been prepared by well-established solid phase synthesis
by using Fmoc or Boc monomers. However, these methods
have serious drawbacks due to harsh reaction conditions and
side reactions during either monomer synthesis and/or PNA
oligomer synthesis.
Self-activated PNA monomers by Bts strategy
Benzothiazole-2-sulfonyl (Bts) is an amine-protecting group
of amino acid, of which sulfonamide is stable and
deprotection conditions are mild.5 Due to the strong electronwithdrawing effect of the sulfonyl group, the acyl group of
acylsulfonamide is easily attacked by nucleophiles after
alkylation. Bts was applied in the safety-catch strategy6 or
the synthesis of a peptide thioester7 for native chemical
ligation of the peptide. Utilizing these characteristics,
Panagene developed self-activated cyclic PNA monomers.
In these PNA monomers the Bts group plays an important
role not only as a protecting group of the amine of the PNA
backbone but also as an activating group for the coupling
reaction. Bts activates the carbonyl of piperazinone to be
easily attacked by the primary amine of PNA, thus enables a
simple coupling process without any coupling reagent or
pre-activation process.
Synthesis of self-activated Bts PNA monomers
The cyclic Bts PNA monomers were synthesized according
to the method described in Fig 1. A Bts-protected PNA
monomer backbone (2) was prepared from the reaction of
ethyl N-(2-aminoethyl)glycinate (1)8 with benzothiazole-2sulfonyl chloride (BtsCl). Compound 2 was coupled with a
corresponding nucleobase acetic acid (3a-d) in the coupling
reagent, followed by hydrolysis and cyclization to give selfactivated Bts PNA monomers (5a-d). In spite of activated
structure, these monomers are sufficiently stable even to be
stored in DMF solution at room temperature for more than a
week.
Fig 1. Synthesis of cyclic Bts PNA monomers
PNA oligomers synthesis using Bts PNA monomers
PNA oligomers are prepared by the solid-phase synthesis of
which protocols are composed of three steps: deprotection,
coupling, and capping steps as shown in Fig 2. As an
example, HPLC analysis of a 15-mer PNA synthesized using
Bts PNA monomers showed the 87 area% purity. The
estimated average coupling yield per base is over 99%.
Fig 2. Solid Phase Synthesis of PNA Oligomer by cyclic Bts Monomer
condition is five- to eight times less than that in the Fmoc
deprotecting conditions as shown in Fig 3.
Normal and trans-acylated PNA trimers were synthesized
from the corresponding monomer and the reactivity of Bts or
Fmoc monomers towards normal and trans-acylated PNA
were compared as described in Fig 4. Bts monomer showed
no reactivity with trans-acylated PNA but Fmoc monomer
showed significant reactivity although both monomers
showed good reactivity with normal PNA.
Fig 4. Reactivity of Bts or Fmoc monomer with normal and transacylated PNA trimer
In Summary
Suppressed side reaction in PNA synthesis by Bts
monomers
During PNA oligomer synthesis using Fmoc monomers,
trans-acylation9 of the nucleobase acetyl moiety is a common
side reaction, because the 2-ethylamino group in PNA is
more reactive than that of -amino acid due to the increased
basicity and the favorable geometry. The side reaction
lowers the yield and purity of synthesized PNA oligomers.
The trans-acylated by-products show strongly altered
hybridization properties and very difficult to purify because
of the same molecular weight and other similar properties.
Bts monomers show no reactivity with trans-acylated PNA
in addition to much lower rate of trans-acylation. And thus
Bts monomers produce high yield of the intended PNA
oligomers with limited amount of aborted PNA oligomers,
which are of smaller molecular weight and can be easily
separated.
Trans-acylation of PNA trimers (H-XTT-NH2) under Bts
or Fmoc deprotecting condition was monitored with HPLC.
The amount of trans-acylated PNA in the Bts deprotecting
Fig 3. Trans-acylation ratio of PNA trimer under Bts (A) or Fmoc (B)
deprotecting condition
Panagene has developed a novel strategy for the synthesis of
PNA oligomers. Self-activated Bts monomers are suitable
for bulk production. The Bts strategy provides excellent
purity of PNA oligomers and does not require anhydrous
reaction conditions or the use of harsh coupling reagents. It
allows a large number of PNA modifications such as
peptide-PNA conjugates, bis-PNA, fluorescently labeled
PNA, etc. It will enable the widespread use of PNA in
biological, diagnostic and pharmaceutical applications.
Reference
1. H. Lee et al., Org. Lett., 2007, 9(17), 3291.
2. P.E. Nielsen, et al., Science 1991, 254, 1497.
3. S.K. Kim et al., J. Am. Chem. Soc. 1993, 115, 6477; B.
Hyrup et al., J. Am. Chem.Soc. 1994, 116, 7964 ; M.
Egholm et al., Nature 1993, 365, 566 ; M. Egholm et al.,
Nucleic Acids Res. 1995, 23, 217 ; P. Wittung et al., J.
Am. Chem. Soc. 1996, 118, 7049 ; M. Leijon et al.,
Biochemistry 1994, 33, 9820.
4. H. Orum et al., BioTechniques 1995, 19, 472 ; S. Tomac
et al., J. Am. Chem. Soc. 1996, 118, 5544.
5. E. Vedejs et al., J. Am. Chem. Soc. 1996, 118, 9796 ; E.
Vedejs et al, J. Org. Chem. 2000, 65, 2309.
6. L. Yang et al., Tetrahedron Lett. 1990, 40, 8197.
7. B. Backes et al., J. Am. Chem. Soc. 1996, 118, 3055.
8. D.W. Will et al., Tetrahedron 1995, 51, 12069.
9. R. Casale et al., 1999, Peptide Nucleic Acids, P.E.
Nielsen, M. Egholm (Eds), Horizon Scientific Press,
Norfolk, pp 39-50 ; S.A. Thomsom et al., Tetrahedron,
1995, 51, 6179-6194 ; M. Eriksson et al., New J. Chem.,
1998, 22(10), 1055-1059.