Modular nucleic acid surrogates. Solid phase synthesis of α-helical peptide nucleic acids (αPNAs)

Article abstract of DOI:10.1021/ol990627q

(equation presented) The synthesis and characterization of prototype α-helical peptide nucleic acid (αPNA) modules 1-3 as well as disulfide dimers 4 and 5 are reported. These molecules combine an α-helical peptidyl scaffold with well-defined nucleobase molecular recognition patterns and could serve as a basis for novel antisense and/or antigene agents. Structure assignments for these αPNAs were supported by MALDI-TOF mass spectrometry, and the α-helical nature of 4 in water was confirmed by circular dichroism (CD) spectroscopy.

Full text of DOI:10.1021/ol990627q

ORGANIC
LETTERS
1999
Vol. 1, No. 3
403-405
Modular Nucleic Acid Surrogates. Solid
Phase Synthesis of r-Helical Peptide
Nucleic Acids (rPNAs)
Philip Garner,* Subhakar Dey, Yumei Huang, and Xiao Zhang
Department of Chemistry, Case Western ReserVe UniVersity,
CleVeland, Ohio 44106-7078
ppg@po.cwru.edu
Received April 30, 1999
ABSTRACT
The synthesis and characterization of prototype r-helical peptide nucleic acid (rPNA) modules 1−3 as well as disulfide dimers 4 and 5 are
reported. These molecules combine an r-helical peptidyl scaffold with well-defined nucleobase molecular recognition patterns and could
serve as a basis for novel antisense and/or antigene agents. Structure assignments for these rPNAs were supported by MALDI-TOF mass
spectrometry, and the r-helical nature of 4 in water was confirmed by circular dichroism (CD) spectroscopy.
In most DNA-regulatory proteins, R-helical subunits serve
as the molecular scaffolding for presentation of key amino
acid side chains to their specific nucleic acid binding sites.1
Sequence-specific binding of these R-helical binding domains
to dsDNA occurs in the major groove as a consequence of
multiple interactions arrived at (combinatorially) through
evolutionary selection. Despite work toward the elucidation
of Nature’s “code” for molecular recognition of dsDNA,2 it
is not yet feasible to rationally design a peptide structure
that will bind to any particular DNA duplex.3 On the other
hand, molecular recognition of single-stranded nucleic acids
via Watson-Crick (W-C) base pairing is quite predictable
and provides the basis for antisense approaches to genetic
manipulation.4 Nielsen’s pioneering discovery that the (2-
deoxy)ribose phosphate framework of nucleic acids could
be replaced by a much simpler polyamide backbone has
spawned considerable interest in “peptide nucleic acids”
(PNAs)5 and related constructs.6 However, the merger of an
R-helical peptidyl scaffold with well-defined nucleobase
molecular recognition patterns (W-C base pairing for
ssRNA and Hoogsteen base pairing for dsDNA) has not been
explored. A particular advantage of such hybrid structures,
which we term as R-helical peptide-based nucleic acids or
RPNAs,7 would be the opportunity to functionalize the
peptide scaffold itself to enhance transport, hybridization,
and other properties.
We now report the synthesis and characterization of
prototype RPNA modules Ac-C(Acm)-G-ST-D-A-E-ST-A-
A-K-ST-A-A-E-ST-A-Aib-A-ST-K-G-NH2 (1), Ac-Aib-G-ST-
D-A-E-ST-A-A-K-ST-A-A-E-ST-A-Aib-A-ST-K-C(Acm)-
NH2 (2), and Ac-C(Acm)-G-SC-D-A-E-SC-A-A-K-SC-A-A-
E-SC-A-Aib-A-SC-K-G-NH2 (3), as well as the tail-to-tail
dimer [Ac-C-G-ST-D-A-E-ST-A-A-K-ST-A-A-E-ST-A-Aib-
A-ST-K-G-NH2]2 (4) and head-to-head dimer [Ac-Aib-G-ST-
D-A-E-ST-A-A-K-ST-A-A-E-ST-A-Aib-A-ST-K-C-NH2]2 (5).8
(1) Cf. Steitz, T. A. Q. ReV. Biophys. 1990, 23, 205.
(2) (a) Desjarlais, J. R.; Berg, J. M. Proc. Natl. Acad. Sci. U.S.A. 1992,
89, 7345. (b) Desjarlais, J. R.; Berg, J. M. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 2256. (c) Rebar, E. J.; Pabo, C. O. Science 1994, 263, 671. (d)
Jamieson, A. C.; Kim, S.-H.; Wells, J. A. Biochemistry 1994, 33, 5689. (e)
Choo, Y.; Klug, A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11163.
(3) For an alternative approach to dsDNA recognition via the minor
groove, see: Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1996, 382,
559.
(5) PNA reviews: (a) Dueholm, K. L.; Nielsen, P. E. New J. Chem.
1997, 21, 19. (b) Eriksson, M.; Nielsen, P. E. Q. ReV. Biophys. 1996, 29,
369.
(4) Antisense Research and Application; Crooke, S. T., Ed.; Springer-
Verlag: Berlin, 1998.
10.1021/ol990627q CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/17/1999

The primary amino acid sequences of these prototype RPNA
modules 1-3 incorporate residues known to favor R-helix
formation.9 Specifically, these peptide backbones include
hydrophobic amino acids (A and Aib), internal salt bridges
(E-(aa)3-K-(aa)3-E), a macrodipole (D-(aa)15-K), and an
N-acetyl cap.10 The C-termini of these RPNA modules end
in a carboxamide function to preclude any potential intramo-
lecular end effects. Cysteine residues allow for the imple-
mentation of a “disulfide stitchery” strategy to connect the
RPNA modules at either termini.11 Finally, each RPNA
module incorporates five properly spaced nucleobases for
Watson-Crick base-pairing to a target nucleic acid sequence.
These nucleobases are attached via a methylene link to serine
hydroxyls in order to preserve the N-glycoside (O-C-N)
substructure found in nucleic acids.12 A molecular model of
4 bound to B-helical dA10 suggests that, despite some
expected deformation after energy minimization (Insight II/
CVFFssee Supporting Information for details), this RPNA
design is geometrically reasonable (Figure 1).
Scheme 1. General Protocol for RPNA Synthesis
step.14 The nucleobase-modified serine derivatives 14 and
15 were prepared by the route shown in Scheme 2. The acid-
labile Rink amide linker15 was employed so that release of
a fully extended peptide amide from the resin and removal
of the acid-labile PGs could be achieved at the same time.
(6) (a) Weller, D. D.; Daly, D. T.; Olsen, W. K.; Summerton, J. E. J.
Org. Chem. 1991, 56, 6000. Huang, S.-B.; Nelson, J. S.; Weller, D. D. J.
Org. Chem. 1991, 56, 6007. (These papers predate Nielsen’s original
publication.) (b) Garner, P.; Yoo, J. U. Tetrahedron Lett. 1993, 34, 1275.
(c) Lewis, I. Tetrahedron Lett. 1993, 34, 5697. (d) Almarsson, O.; Bruice,
T. C.; Kerr, J.; Zuckermann, R. N. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
7518. (e) Lenzi, A.; Reginato, G.; Taddei, M. Tetrahedron Lett. 1995, 36,
1713. Lenzi, A.; Reginato, G.; Taddei, M.; Trifilieff, E. Tetrahedron Lett.
1995, 36, 1717. See also: Ceulemans, G.; Khan, K.; Van Schepdael, A.;
Herdewijn, P. Nucleosides Nucleotides 1995, 14, 813. (f) Loy, E.; Kessler,
H. Liebigs Ann. 1996, 201. (g) Jordon, S.; Schwemler, C.; Kosch, W.;
Kretschmer, A.; Schwenner, E.; Stropp, U.; Mielke, B. Bioorg. Med. Chem.
Lett. 1997, 7, 681. See also: Gangamani, B. P.; Kumar, V. A.; Ganesh, K.
N. Tetrahedron 1996, 52, 15017. (h) Lowe, G.; Vilaivan, T. J. J. Chem.
Soc., Perkin Trans. 1 1997, 539; 547; 555. (i) Begmeier, S. C.; Fundy, S.
L. Bioorg. Med. Chem. Lett. 1997, 7, 3135. (j) Goodnow, R. A., Jr.; Richou,
A.-R.; Tam, S. Tetrahedron Lett. 1997, 38, 3195. Goodnow, R. A., Jr.;
Tam, S.; Pruess, D. L.; McComas, W. W. Tetrahedron Lett. 1997, 38, 3199.
(k) Altmann, K.-H.; Chiesi, C. S.; Garc´ıa-Echeverr´ıa, C. Biorg. Med. Chem.
Lett. 1997, 7, 1119. Garc´ıa-Echeverr´ıa, C.; Hu¨sken, D.; Chiesi, C. S.;
Altmann, K.-H. Biorg. Med. Chem. Lett. 1997, 7, 1123. Kuwahara, M.;
Arimitsu, M.; Sisido, M. J. Am. Chem. Soc. 1999, 121, 256. (l) Cantin, M.;
Schu¨tz, R.; Leumann, C. J. Tetrahedron Lett. 1997, 38, 4211. (m) Howarth,
N. M.; Wakelin, L. P. G. J. Org. Chem. 1997, 62, 5441. (n) Tsantrizos, Y.
S.; Lunetta, J. F.; Boyd, M.; Fader, L. D.; Wilson, M.-C. J. Org. Chem.
1997, 62, 5451.
(7) Wakelin (ref 6m) has proposed the term “R-PNA” to describe peptide
nucleic acids made up of R-amino acid building blocks. We feel that his
usage of the R-designation is somewhat redundant (since a peptide is, by
definition, made up of R-amino acids) and less descriptive (it could be
applied to many of the systems described in ref 6).
(8) Amino acid abbreviations: aa ) generic amino acid, A ) Ala, Aib
) 2-aminoisobutyric acid, C ) Cys, D ) Asp, E ) Glu, G ) Gly, K )
Lys, ST ) 1-[(Ser)methyl]thymine, SC ) 1-[(Ser)methyl]cytosine.
(9) Our prototype RPNA design was based on the amphiphilic helix
sequence contained in Baltzer’s SA-42 helix-loop-helix: Olofsson, S.;
Johansson, G.; Baltzer, L. J. Chem. Soc., Perkin Trans. 2 1995, 2047.
(10) Cf. Regan, L.; DeGrado, W. F. Science 1988, 241, 976.
Figure 1. Ribbon representation of energy-minimized molecular
model of RPNA(T10) “tail-to-tail” dimer 4 bound to B-helical
(dA)10.
The general synthetic route to dimeric RPNAs is depicted
in Scheme 1 and consists of three distinct stages: (1) solid-
phase peptide synthesis (SPPS) of the resin-bound B5 module
7, (2) cleavage these peptides from the resin with concomi-
tant deprotection of all aa residues except cysteine to give
thiol-protected RPNA module 8, and (3) thiol deprotection
and disulfide bond formation to produce the B5 dimer 9.
Because the modified serine residue is potentially both acid-
and base-labile, the strategic and tactical issues associated
with the SPPS of these RPNAs resemble those associated
with glycopeptides,13 making the endeavor nontrivial. Ac-
cordingly, we chose the base-labile Fmoc protecting group
(PG) for the N-terminal amines and acid-labile PGs (BOC
and tert-butyl ester) for all side chain functionality excepting
C. The I2-labile acetamidomethyl (Acm) PG was chosen for
the C residue to facilitate a separate disulfide formation
(11) Park, C.; Campbell, J. L.; Goddard, W. A., III. J. Am. Chem. Soc.
1995, 117, 6287.
(12) Since our original publication on peptide-based nucleic acid
surrogates (ref 6b), we discovered that a synthesis of racemic Bz-Ser[CH2U]-
OMe had been reported in the Russian chemical literature: Timoshchuk,
V. A.; Olimpieva, T. I. Zh. Obsh. Khim. 1988, 58, 2404.
(13) Cf. Paulsen, H.; Schleyer, A.; Mathieux, N.; Meldal, M.; Bock, K.
J. Chem. Soc., Perkin Trans. 1 1997, 281.
(14) Veber, D. F.; Milkowski, J. D.; Varga, S. L.; Denkewalter, R. G.;
Hirschmann, R. J. Am. Chem. Soc. 1972, 94, 5456.
404
Org. Lett., Vol. 1, No. 3, 1999

Scheme 2
Figure 2. CD spectra of tail-to-tail dimer 4 (21 µM in H2O) at
30, 40, 50, 60, 70, and 80 °C.
R-helix to random coil transition. The helical content of 4
at 20 °C in water was estimated to be 26%.20 In conclusion,
a practical solid-phase synthesis of prototype R-helical
peptide nucleic acids (RPNAs) has been achieved and their
propensity toward R-helix formation confirmed. Binding
studies are now underway in our laboratories. Preliminary
UV melting experiments show that RPNAs do form com-
plexes with complementary DNA targets (4‚dA10, Tm ) 51
°C; 5‚dA10, Tm ) 39 °C), thus validating the general concept.
Finally, DBU16 was used for Fmoc deprotection and Carpi-
no’s HATU17 reagent was used for all peptide couplings.
Our optimized experimental protocol could be applied to
either the manual or semiautomated SPPS of RPNAs and
provided peptides 1 and 2 in 16% and 15% overall yields,
respectively, from commercially available Fmoc-Rink-
MBHA resin after purification by gradient reverse-phase
HPLC. Subsequent I2-mediated Acm deprotection/disulfide
formation18 of T5 monomer 1 provided the tail-to-tail RPNA
dimer 4 in 84% yield after HPLC purification. Similar
treatment of module 2 resulted in a 49% purified yield of
the head-to-head dimer 5. Structure assignments for the T5
modules 1 and 2, C5 module 3, and the homodimers 4 and
5 were supported by MALDI-TOF mass spectrometry:
([1‚H]+, calcd mass 2685.12, found 2683.06; [2‚H]+, calcd
mass 2714.1627, found 2713.15; [3‚H]+, calcd mass
2714.1627, found 2713.15; [4‚H]+, calcd mass 5225.15,
found 5222.40; [5‚H]+, calcd mass 5281.21, found 5282.26).
The R-helical nature of 4 in water was confirmed by
circular dichroism (CD) spectroscopy (cf. Figure 2). The
double minimum at 220 and 206 nm and maximum at 193
nm are characteristic of an R-helix19 while the isodichroic
point at 202 nm was suggestive of a temperature-dependent
Acknowledgment. This work was supported by the
National Institutes of Health under Grant GM54796. Special
thanks to the Orlando Research Group (Complex Carbo-
hydrate Research Center, University of Georgia) for per-
forming the MALDI-TOF mass spectrometry on compounds
1-5. The mass spectrometer was obtained with funds from
NIH Grant S10 RR12013.
Supporting Information Available: Experimental pro-
cedures and characterization data for compounds 1-10 as
well as UV melting experiments on 4‚dA10 and 5‚dA10. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL990627Q
(20) Helical content was estimated by taking the ratio of ([θ]219 - [θ]0)/
[θ]100, where [θ]219 is the mean residual helicity at 219 nm () -14850 deg
cm2 dmol-1) and [θ]0 is the “background” mean residue ellipticity at 0%
helicity () -5368 deg cm2 dmol-1) as determined by a melting experiment
(Figure 2, inset). To account for the length dependence of CD for an R-helix,
(15) (Rink amide linker ) 4-(2′,4′-dimethoxyphenylaminomethyl)-
phenoxyacetamido) Rink, H. Tetrahedron Lett. 1987, 28, 3787.
(16) (DBU ) 1,8-diazabicyclo[5.4.0]undec-7-ene.) Wade, J. D.; Bedford,
J.; Sheppard, R. C. Tregear, G. W. Pept. Res. 1991, 4, 194. A control
experiment with Fmoc-Ser[CH2T]-OMe indicated that the use of 20%
piperidine led to the elimination of free thymine.
the formula [θ]100 ) [θ]∞(1 - k/n) was used to calculate [θ]100 according
H
to the following: Chen, Y.-H.; Yang, J. T.; Chau, K. H. Biochemistry 1974,
13, 3350. (We have not rigorously excluded the possibility that thymine
chromophores might be contributing to the R-helix circular dichroism at
219 nm. For a discussion of analogous aromatic side-chain effects on CD,
see: Chakrabartty, A.; Kortemme, T.; Padmanabhan, S.; Baldwin, R. L.
Biochemistry 1993, 32, 5560.)
(17) (HATU ) O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate.) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
(18) Kamber, B.; Hartmann, A.; Eisler, K.; Riniker, B.; Rink, H.; Sieber,
P.; Rittel, W. HelV. Chim. Acta 1980 63, 899.
(19) Holzwarth, G.; Doty, P. J. Am. Chem. Soc. 1965, 87, 218.
Org. Lett., Vol. 1, No. 3, 1999
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