α-PNA: A Novel Peptide Nucleic Acid Analogue of DNA

Article abstract of DOI:10.1021/jo970111p

Peptide nucleic acid (PNA) analogues of DNA have attracted interest as potential pharmacological regulators of gene expression since they have the capacity to invade duplex DNA forming Watson-Crick base paired PNA:DNA heteroduplexes. Unfortunately, strand invasion is limited to homopurine and homopyrimidine sequences and there is the need to explore further PNA analogues for the purpose of expanding the strand invasion alphabet. Accordingly, we have designed a true peptide mimic of DNA (designated α-PNA) involving novel L-α-amino acids, with side chains comprising the four DNA bases attached via an ethylene linkage, interspaced with glycine. The four base-containing amino acids have been synthesized from N-Boc-L-homoserine, via alkylation of the appropriate base with the key intermediate (S)-2-(N-Boc-amino)-4-bromobutyric acid methyl ester followed by hydrolysis. These amino acids have been incorporated into α-PNA oligomers using both solution and solid phase methods.

Full text of DOI:10.1021/jo970111p

J . Org. Chem. 1997, 62, 5441-5450
5441
r-P NA: A Novel P ep tid e Nu cleic Acid An a logu e of DNA
Nicola M. Howarth and Laurence P. G. Wakelin*
Cancer Drug Discovery, Department of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland
Received J anuary 22, 1997X
Peptide nucleic acid (PNA) analogues of DNA have attracted interest as potential pharmacological
regulators of gene expression since they have the capacity to invade duplex DNA forming Watson-
Crick base paired PNA:DNA heteroduplexes. Unfortunately, strand invasion is limited to
homopurine and homopyrimidine sequences and there is the need to explore further PNA analogues
for the purpose of expanding the strand invasion alphabet. Accordingly, we have designed a true
peptide mimic of DNA (designated R-PNA) involving novel L-R-amino acids, with side chains
comprising the four DNA bases attached via an ethylene linkage, interspaced with glycine. The
four base-containing amino acids have been synthesized from N-Boc-L-homoserine, via alkylation
of the appropriate base with the key intermediate (S)-2-(N-Boc-amino)-4-bromobutyric acid methyl
ester followed by hydrolysis. These amino acids have been incorporated into R-PNA oligomers
using both solution and solid phase methods.
In tr od u ction
recognition problem since duplex formation is governed
by the universal Watson-Crick hydrogen bonding scheme.
Unfortunately, strand displacement for mixed purine-
pyrimidine PNAs remains elusive and thus, to date, PNA
binding to duplex DNA is confined to the same recogni-
tion alphabet as oligonucleotide triple helices.9 One
approach to extending the strand invasion alphabet to
mixed sequences is to enhance the intrinsic stability of
PNA/DNA heteroduplexes. However, the PNA backbone
itself is not readily amenable to facile chemical modifica-
tions which would allow systematic exploration of struc-
tural features that may promote higher helix-to-coil
transition temperatures. Principal among these must be
electrostatic charge density, hydrophobicity, and back-
bone rigidity and topology. The importance of charge
density on duplex stability has recently been illustrated
in the case of oligonucleotides.10-12
The concept of peptide-based DNA surrogates is itself
not new. De Konig and Pandit13 prepared nucleopeptides
of the type [HNCH((CH2)4B)CO]n and [HNCH((CH2)4B)-
CONHCHRCO]n but found that the polyuridine deriva-
tive, in the former configuration, complexed only weakly
with polyadenylic acid. Buttrey et al.14 made polymers
of D-, L-, and DL-â-(thymin-1-yl)alanine and similarly
found no interaction with polyadenylic acid. Weller and
co-workers15 reported the synthesis of nucleopolyamides
of the form [NHCH(CH2B)CH2CH2CO]n, Garner et al.16
described the preparation of nucleotide amino acids
derived from serine and their subsequent incorporation
into a peptide structure with glycine spacers, and Lewis17
reported the synthesis of peptides containing nucleotide
amino acids derived from â-alanine interspaced with
Selective manipulation of gene expression will make
a major impact on the therapy of human disease and
much research is now focused on the design and synthesis
of ligands that bind to DNA in a sequence specific manner
so as to inhibit transcription. Nielsen et al.1,2 have
developed polyamide nucleic acid (PNA) mimics of DNA
in which the entire deoxyribose-phosphate backbone has
been exchanged with a structurally homomorphous un-
charged polyamide backbone composed of N-(2-amino-
ethyl)glycine units (Figure 1). These compounds contain
the same number of backbone bonds between the bases
(i.e. 6) and the same number of bonds from the backbone
to the base (i.e. 3) as in DNA.3 PNA binds with high
affinity and sequence specificity to both complementary
RNA and DNA, and a number of template functions are
inhibited on forming PNA/DNA and PNA/RNA com-
plexes.4-6 Thus PNA offers both antisense and antigene
strategies for regulating gene expression. Homopyrimi-
dine PNAs have been found to invade double-stranded
DNA by displacing the noncomplementary strand to form
(PNA)2/DNA triplexes and a displaced strand analogous
to a D-loop.1,4,6,7 Homopurine PNAs also invade double-
stranded DNA but fail to form triplexes, and their
invasion complexes are less stable.8
Strand invasion is of great importance because, in
principle, it provides a general solution to the molecular
* To whom all correspondence should be addressed. Tel: +353-1-
7062821. Fax: +353-1-7062821. E-mail: lwakelin@ollamh.ucd.ie.
X Abstract published in Advance ACS Abstracts, J uly 15, 1997.
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 254, 1497-1500.
(2) Nielsen, P. E.; Egholm, M.; Buchardt, O. Bioconjugate Chem.
1994, 5, 3-7.
(9) Sun, J .-S.; Garestier, T.; Helene, C. Curr. Opin. Struct. Biol.
1996, 6, 327-333.
(3) Hyrup, B.; Egholm, M.; Nielsen, P. E.; Wittung, P.; Norden, B.;
Buchardt, O. J . Am. Chem. Soc. 1994, 116, 7964-7970.
(4) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Nucleic
Acid Res. 1993, 21, 197-200.
(10) Fathi, R.; Huang, Q.; Coppola, G.; Delaney, W.; Teasdale, R.;
Krieg, A. M.; Cook, A. F. Nucleic Acids Res. 1994, 22, 5416-5424.
(11) Horn, T.; Chaturvedi, S.; Balasubramaniam, T. N.; Letsinger,
R. L. Tetrahedron Lett. 1996, 37, 743-746.
(12) (a) Dempcy, R. O.; Browne, K. A.; Bruice, T. C. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 6097-6101. (b) Browne, K. A.; Dempcy, R. O.;
Bruice, T. C. Proc. Natl. Acad. Sci. USA 1995, 92, 7051-7055.
(13) De Koning, H.; Pandit, U. K. Rec. Trav. Chim. 1971, 90, 1069-
1080.
(5) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Anti-Cancer
Drug Des. 1993, 8, 53-63.
(6) Hanvey, J . C.; Peffer, N. J .; Bisi, J . E.; Thomson, S. A.; Cadilla,
R.; J osey, J . A.; Ricca, D. J .; Hassman, C. F.; Bonham, M. A.; Au, K.
G.; Carter, S. G.; Bruckenstein, D. A.; Boyd, A. L.; Noble, S. A.; Babiss,
L. E. Science 1992, 258, 1481-1485.
(7) Peffer, N. J .; Hanvey, J . C.; Bisi J . E.; Thomson, S. A.; Hassman,
C. F.; Noble, S. A.; Babiss, L. E. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 10648-10652.
(8) Nielsen, P. E.; Christensen, L. J . Am. Chem. Soc. 1996, 118,
2287-2288.
(14) Buttrey, J . D.; J ones, A. S.; Walker, R. T. Tetrahedron 1975,
31, 73-75.
(15) Huang, S.-B.; Nelson, J . S.; Weller, D. D. J . Org. Chem. 1991,
56, 6007-6018.
(16) Garner, P.; Yoo, J . U. Tetrahedron Lett. 1993, 34, 1275-1278.
(17) Lewis, I. Tetrahedron Lett. 1993, 34, 5697-5700.
S0022-3263(97)00111-4 CCC: $14.00 © 1997 American Chemical Society

5442 J . Org. Chem., Vol. 62, No. 16, 1997
Howarth and Wakelin
F igu r e 1. Comparison of DNA, PNA, and R-PNA structures.
Sch em e 1. Syn th esis of
(S)-2-(N-Boc-a m in o)-4-br om obu tyr ic Acid Meth yl
Ester
containing amino acids from L-homoserine and their
subsequent incorporation into a glycine-spaced tetrapep-
tide R-PNA using solution phase techniques. In addition,
we outline a solid phase strategy for the preparation of
R-PNA using the 20-mer H-RCLysRTGlyRCGlyRTLysR-
20
CGlyRCGlyRTLysRTGlyRTGlyRTLys-NH2 as an ex-
ample.
During the course of our work, Lenzi et al.21,22 pre-
sented a preliminary report on the preparation of an
R-PNA in which the base-amino acids are derived from
D-glutamic acid.21 This results in an R-PNA of opposite
chirality (i.e. D-) to that described here.
Resu lts a n d Discu ssion
Syn t h esis of Key In t er m ed ia t e (S)-2-(N-Boc-
a m in o)-4-br om obu tyr ic Acid Meth yl Ester (3). (S)-
2-(N-Boc-amino)-4-bromobutyric acid methyl ester (3)
(Scheme 1) is the key intermediate in the alkylation of
all four nucleotide bases in our synthetic pathways,
giving rise to the base-containing amino acids. This
compound was prepared from N-Boc-L-homoserine (1) in
three steps as shown in Scheme 1. Firstly, the carboxylic
acid group in 1 was protected as a methyl ester; this
involved the initial formation of the corresponding dicy-
clohexylammonium salt followed by treatment of the salt
with methyl iodide. Compound 2 was given in an overall
yield of 75%. The methyl ester 2 was then converted into
the desired bromo derivative 3 according to the method
developed by Tius et al.23 for transformation of primary
hydroxyl groups into their corresponding bromides. Thus,
a solution of 2 in dichloromethane (DCM) was added to
the resulting complex formed on reacting triphenylphos-
phine with NBS. After purification by flash chromatog-
raphy, 3 was afforded in a 75% yield.
proline. No DNA binding studies have been reported for
any of these compounds, and they all have different
interbase and base-to-backbone bonding topologies com-
pared to nucleic acids.
We have designed a true peptide nucleic acid analogue
of DNA, designated R-PNA, where the base amino acid
moieties are derived from homoserine and in which the
base amino acids are interspaced with glycine (Figure
1). This results in the bases being separated from, and
positioned along, the backbone by the same numbers of
bonds as found in DNA. Since the synthesis of R-PNA
is compatible with solid phase peptide chemistry it will
be relatively straightforward to modify the backbone
electrostatic charge, hydrophobicity, and ridigity by
replacing glycine residues with appropriate amino acids.
We have briefly reported a molecular model of an R-PNA:
DNA duplex and progress in the synthesis toward
R-PNA.18,19 Here, we describe in detail the synthesis of
the thymine, cytosine, adenine, and guanine base-
Der iva tiza tion of th e P yr im id in e Ba ses. Alkyla-
tion of thymine 4 with 3 was achieved by heating a
mixture of 3, thymine, and anhydrous potassium carbon-
ate in anhydrous DMSO at 80 °C for 22 h (Scheme 2).
Following an aqueous workup and purification by flash
(18) Howarth, N. M.; Searcey, M.; Topham, C. M.; Wakelin, L. P.
G. Ir. J . Med. Sci. 1995, 164, 244.
(19) Howarth, N. M.; Topham, C. M.; Searcey, M.; Wakelin, L. P.
G. Ir. J . Med. Sci. 1996; 165 (suppl. no. 3); 23.
(20) We employ the following nomenclature: For the oligomer
H-RTLysRCGlyRAGlyRGLys-NH2,
H denotes the free N-terminal
amino group; RT, RC, RA, and RG the thymine, cytosine, adenine, and
guanine base-containing amino acids; Gly and Lys refer to glycine and
lysine, respectively, and NH2 indicates a C-terminal amide. Addition-
ally we propose the same designation to be used for the base-containing
amino acids i.e. Boc-RT-OH for (S)-2-(N-Boc-amino)-4-(thymin-1-yl-
)butyric acid.
(21) Lenzi, A.; Reginato, G.; Taddei, M. Tetrahedron Lett. 1995, 36,
1713-1716.
(22) Lenzi, A.; Reginato, G.; Taddei, M.; Trifilieff, E. Tetrahedron
Lett. 1995, 36, 1717-1718.
(23) Tius, M. A.; Fauq, A. H. J . Am. Chem. Soc. 1986, 108, 1035-
1039.

A Novel Peptide Nucleic Acid Analogue of DNA
J . Org. Chem., Vol. 62, No. 16, 1997 5443
Sch em e 2. Syn th esis of P yr im id in e Mon om er s
Der iva tiza tion of th e P u r in e Ba ses. For the syn-
thesis of the adenine monomer, the N6-amino group of
adenine likewise needs to be protected, and once again a
Cbz-protecting group was chosen. However here, unlike
for the cytosine monomer, protection of the exocyclic
amino group was performed after alkylation of adenine
11 with 3 in order to ensure that substitution occurred
mainly at the N-9 position. Although alkylation of
adenine is notoriously nonregiospecific, it has been
reported that alkylation of sodium adenide in DMF gives
rise primarily to N-9 substituted products;25 therefore
alkylation of adenine 11 with 3 was carried out by firstly
generating sodium adenide in situ in anhydrous DMF,
followed by addition of a solution of 3 in anhydrous DMF
(Scheme 3). This procedure yielded only one isolable
compound, which was the desired adenine monomer 12
(79% yield). N-9 alkylation was confirmed by comparison
of the 1H and 13C NMR spectra of 12 with reported
spectral data for N-7 and N-9 alkylated derivatives of
1
adenine.26,27 From the H NMR spectra of N-7 and N-9
substituted adenines, it has been shown that ∆δ values
between H2 and H8 signals in N-7 isomers are much
greater than those for N-9 isomers (cf. N7-(1,1-dimeth-
ylpropargyl)adenine δ 8.66 (H2) and 7.76 (H8) i.e. ∆δ
0.90;26 N9-(1,1-dimethylpropargyl)adenine δ 8.21 (H2) and
8.11 (H8) i.e. ∆δ 0.1026) and the NH2 signals in N-9
isomers are shifted upfield from those in N-7 isomers (cf.
N9-(dimethyl)propargyladenine δ 7.24 (NH2);26 N7-(di-
methyl)propargyladenine δ 8.09 and 7.99 (NH2)26). The
shifts observed for H2, H8, and NH2 in the 1H NMR
spectrum of 12 were in agreement with that of N-9
substitution (δ 8.12 (H2), 8.02 (H8) and 7.19 (NH2). On
examining the 13C NMR spectra of N-7 and N-9 substi-
tuted adenines, it has been found that the C5 signal in
N-9 isomers is shifted downfield from that in N-7 isomers
(cf. 7-methyladenine δ 111.7;27 9-methyladenine δ 118.727)
and that the C4 signal is shifted upfield (cf. 7-methyl-
adenine δ 159.8;27 9-methyladenine δ 149.927). The
corresponding signals in the 13C NMR spectrum of 12
were more consistent with N-9 substitution than with
N-7 substitution (δ 123.08 (C5) and 153.81 (C4)).
Rapoport et al. have reported on the Cbz-protection of
trimethylsilyl-protected adenine28,29 and found that treat-
ment with benzyl chloroformate under all the usual
conditions did not give clean or efficient acylation of the
N6-amino group. However, when 1-(benzyloxycarbonyl)-
3-ethylimidazolium tetrafluoroborate [PhCH2OCOIm+Et‚
BF4-, “Rapoport’s reagent”] was used instead, acylation
was markedly improved. A similar observation was
reported by Nielsen et al.24 for the preparation of their
adenine monomer. Thus, the exocyclic amino group in
12 was protected by treatment with a 6 molar excess of
freshly prepared Rapoport’s reagent in DCM (Scheme 3).
After purification, 13 was obtained in an 82% yield.
Final removal of the methyl ester by hydrolysis gave the
Cbz-protected adenine monomer 14 in a 72% yield
(Scheme 3).
chromatography, 5 was obtained in a 71% yield. Finally,
the methyl ester was removed by hydrolysis, giving the
thymine monomer 6 in a 92% yield (Scheme 2).
For the synthesis of the cytosine monomer, introduc-
tion of a protecting group for the N4-amino group of
cytosine was necessary in order to prevent chain exten-
sion from this position in the later peptide-coupling steps.
A Cbz group was selected for this purpose since it had
also been found to render the intermediates sufficiently
soluble for chemical manipulation.24 Thus, the exocyclic
amino group of cytosine 7 was protected prior to alkyla-
tion by treatment with benzyl chloroformate to give N4-
Cbz-cytosine 824 (Scheme 2). The subsequent alkylation
of 8 with 3 was accomplished by firstly generating the
anion, using sodium hydride in anhydrous DMF, followed
by addition of a solution of 3 in anhydrous DMF (Scheme
2). After an aqueous workup and purification by flash
chromatography, 9 was afforded in a 61% yield. Lastly,
the methyl ester was hydrolyzed to give the Cbz-protected
cytosine monomer 10 in an 86% yield (Scheme 2).
Preliminary studies to determine whether racemiza-
tion occurs during the preparation of these amino acids
have been undertaken. The cytosine monomer 10 was
coupled to L-alanine tert-butyl ester via in situ formation
of the N-hydroxysuccinimide activated ester (as described
below for coupling the base-containing amino acids to
glycine ethyl ester) to give the corresponding dipeptide.
This would have yielded a mixture of diastereoisomers
if racemization had occurred anywhere in the synthesis.
However, both NMR and TLC analysis showed only one
detectable product, and hence these results suggest that
little or no racemization has been caused during the
introduction of the nucleotide base.
As opposed to adenine, guanine cannot be alkylated
to give only N-9 substituted products. However, since
(25) Browne, D. T.; Eisenger, J .; Leonard, N. J . J . Am. Chem. Soc.
1968, 90, 7302-7323.
(26) J oshi, R. V.; Zemlicka, J . Tetrahedron 1993, 49, 2353-2360.
(27) Chenon, M.-T.; Pugmire, R. J .; Grant, D. M.; Panzica, R. P.;
Townsend, L. B. J . Am. Chem. Soc. 1975, 97, 4627-4636.
(28) Watkins, B. E.; Kiely, J . S.; Rapoport, H. J . Am. Chem. Soc.
1982, 104, 5702-5708.
(24) Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.;
Hansen, H. F.; Vulpius, T.; Petersen, K. H.; Berg, R. H.; Nielsen, P.
E.; Buchardt, O. J . Org. Chem. 1994, 59, 5767-5773.
(29) Watkins, B. E.; Rapoport, H. J . Org. Chem. 1982, 47, 4471-
4477.

5444 J . Org. Chem., Vol. 62, No. 16, 1997
Howarth and Wakelin
1
was given in a 90% yield. The H and 13C NMR spectra
obtained for 16 proved to be consistent with published
data for N-9 alkylated 2-aminopurines,31,32 thus support-
ing that substitution had occurred at the required N-9
position.
Sch em e 3. Syn th esis of P u r in e Mon om er s
The next step in the synthetic pathway involves
replacement of the 6-chloro group with an oxygen func-
tionality, which would yield the corresponding guanine
carbonyl moiety at a later stage. The simplest and most
obvious approach is to exchange the chloro group for an
alkoxy group, which would liberate guanine on subse-
quent deprotection. However, the use of sodium alkoxide
is not desirable in our case, as it could lead to racemiza-
tion of the amino acid. Therefore, based on work by
Reese et al.,33,34 we chose to replace the chloro group with
a 2-nitrophenoxy group instead. This was accomplished
using the procedure developed by Sproat et al.;35
a
solution of 16 in 1,2-dichloroethane was treated with a
solution of 2-nitrophenol, DABCO, and triethylamine in
1,2-dichloroethane at rt. Following workup and purifica-
tion by flash chromatography, 17 was afforded in quan-
titative yield.
Protection of the N2-amino group of 17 was then
investigated, since there is a risk that it could interfere
in the later peptide-coupling steps. Initially, protection
with a Cbz group was tried, using benzyl chloroformate
and “Rapoport’s reagent”, but it proved to be unsuccessful
with decomposition of 17 occurring under the reaction
conditions. Therefore, we protected the amine with an
acetyl group. This was readily achieved by reacting 17
with acetyl chloride in anhydrous pyridine. After workup
and purification by flash chromatography, 18 was ob-
tained in a 94% yield.
Subsequently, the 2-nitrophenoxy group in 18 was
removed according to the method described by Reese et
al.34 We chose to cleave this group at this stage, prior to
performing the solution phase oligomerization described
below, since it was anticipated that this group would be
unlikely to withstand either the basic conditions used for
ester hydrolysis or the acidic conditions associated with
Boc removal. Thus, a solution of 18 and 2-nitrobenzal-
doxime in anhydrous acetonitrile was treated with a
solution of 1,1,3,3-tetramethylguanidine in anhydrous
acetonitrile. After purification using flash chromatog-
raphy, 19 was given in a 78% yield. Finally, the methyl
ester was cleaved by hydrolysis to afford the N2-
acetylguanine monomer 20 in a 44% yield.
Solu t ion P h a se Oligom er iza t ion . Using solution
phase techniques, the nucleotide base analogues of N-
Boc-L-homoserine (i.e. thymine monomer 6, N4-Cbz-
cytosine monomer 10, N6-Cbz-adenine monomer 14, and
N2-acetylguanine monomer 20) have been coupled to
glycine ethyl ester, and the resulting dipeptides have
been dimerized to give the corresponding tetrapeptides
as outlined in Scheme 4. Thus, firstly these compounds
were converted in situ into their respective N-hydroxy-
succinimide activated esters using N-hydroxysuccinimide
and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hy-
2-amino-6-chloropurine (15) gives almost exclusively N-9
alkylation,30 it is generally employed instead as the
starting material for the synthesis of guanine derivatives.
Consequently, the guanine monomer 20 was prepared
from 15 as shown in Scheme 3. Firstly, alkylation of 15
with 3 was achieved by stirring a mixture of 15, 3, and
anhydrous potassium carbonate in anhydrous DMF at
rt. After workup and purification by flash chromatog-
raphy, the desired 2-amino-6-chloropurine monomer 16
(31) Green, G. R.; Grinter, T. J .; Kincey, P. M.; J arvest, R. L.
Tetrahedron 1990, 46, 6903-6914.
(32) Kjellberg, J .; J ohansson, N. G. Tetrahedron 1986, 42, 6541-
6544.
(33) J ones, S. S.; Reese, C. B.; Sibanda, S.; Ubasawa, A. Tetrahedron
Lett. 1981, 22, 4755-4758.
(34) Reese, C. B.; Stone, P. A. J . Chem. Soc., Perkin Trans. 1 1984,
1263-1271.
(30) Nollet, A. J . H.; Huting, C. M.; Pandit, U. K. Tetrahedron 1969,
25, 5971-5981.
(35) Sproat, B. S.; Beijer, B.; Iribarren, A. Nucleic Acid Res. 1990,
18, 41-49.

A Novel Peptide Nucleic Acid Analogue of DNA
J . Org. Chem., Vol. 62, No. 16, 1997 5445
Sch em e 4. Solu tion P h a se Oligom er iza tion
F igu r e 2. MALDI-TOF mass spectrum of crude H-RCLys-
RTGlyRCGlyRTLysRCGlyRCGlyRTLysRTGlyRTGlyRTLys-
NH2.20 The matrix used was 3,5-dimethoxy-4-hydroxycinnamic
acid (sinapinic acid).
precursors and resulting peptides are poorly soluble in
organic media, and secondly the unprotected 6-oxo func-
tion can cause problems when using carbodiimide re-
agents.36 Thus, it may be better to use a suitably
protected O6-guanine monomer for solution phase oligo-
merzation. However, as our current main objective is to
prepare longer oligomers of R-PNA for biochemical evalu-
ation using the solid phase strategy described below, we
have yet to explore this further. For the solid phase
approach, which uses a uronium salt as the coupling
reagent, the guanine monomer 20 is unlikely to need
further protection.
Solid P h a se Oligom er iza tion . The preparation of
longer R-PNA oligomers employing solid phase peptide
synthesis techniques has been explored using the 20-mer
H-RCLysRTGlyRCGlyRTLysRCGlyRCGlyRTLysRTGly-
RTGlyRTLys-NH220 as a model. Lysine was incorporated
at the C-terminus and at three other positions within the
oligomer in order to suppress self aggregation and to
increase solubility in aqueous media. This R-PNA oli-
gomer was assembled manually in a stepwise fashion
using a similar protocol to that described by Christensen
et al.37 for the solid phase synthesis of PNA. A N1-Boc-
N5-(2-chloro-Cbz)-L-lysine derivatized 4-methylbenzhy-
drylamine (Boc-Lys(2-Cl-Cbz)-MBHA) resin was used.
Amino acids 6, 10, N-Boc-glycine (Boc-Gly-OH), and N1-
Boc-N5-(2-chloro-Cbz)-L-lysine (Boc-Lys(2-Cl-Cbz)-OH) were
coupled using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate (HBTU) as the
activating reagent in the presence of a slight excess of
N,N-diisopropylethylamine (DIEA). In all cases only
single couplings were required. Once all the amino acids
had been coupled, the R-PNA was cleaved from the solid
support using trifluoromethanesulfonic acid (TFMSA)
under identical conditions to those reported by Chris-
tensen et al.37
drochloride (EDC) in anhydrous DMF. Subsequently,
glycine ethyl ester hydrochloride and triethylamine were
added. After workup and purification by flash chroma-
tography, dipeptides 21, 22, 23, and 24 were obtained in
87%, 86%, 99%, and 33% yields respectively.
Dimerization of dipeptides 21-24 was then investi-
gated. Before two dipeptides could be dimerized, one
dipeptide required deprotection of the carboxylic acid
group while the other needed deprotection of the amino
function. The ethyl esters were removed by hydrolysis,
under similar conditions to those which had been used
to hydrolyze the methyl esters of 5, 9, 13, and 19. The
corresponding carboxylic acid derivatives were afforded
in yields ranging from 74% to 91%. The N-Boc groups
were cleaved using a 50% solution of TFA in DCM. The
trifluoroacetic acid salts of the free amino derivatives
were isolated from the reaction mixture by precipitation
with diethyl ether, in yields ranging from 64% to 88%.
Dimerization of these compounds was performed using
N-hydroxybenzotriazole (HOBt) and DCC in anhydrous
DMF. After purification by flash chromatography, tet-
rapeptides 25, 26, 27, and 28 were given in 61%, 59%,
24% and 10% yields. These yields have not been opti-
mized. Only one diastereoisomer has been observed for
each of these tetrapeptides thereby reinforcing our previ-
ous finding that little or no racemization has occurred
during the synthesis of the base-containing amino acids
and in the subsequent peptide-coupling steps.
The identity and purity of the crude material was
determined by MALDI-TOF mass spectrometry (Figure
2). The principal mass peak at 2901.9 Da corresponds
to the protonated form of the desired R-PNA oligomer (i.e.
[M + H]+) and the one at 2926.0 Da (Figure 2: peak z]
(36) (a) Ho, N. W. Y.; Gilham, P. T. Biochemistry 1967, 6, 3632-
3639. (b) Astell, C. R.; Smith, M. Biochemistry 1972, 11, 4114-4120.
(37) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.;
Hansen, H. F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E.;
Coull, J .; Berg, R. J . Pept. Sci. 1995, 3, 175-183.
The low yields obtained for the guanine peptides are
probably a result of two factors. Firstly, both the guanine

5446 J . Org. Chem., Vol. 62, No. 16, 1997
Howarth and Wakelin
Exp er im en ta l Section
Gen er a l P r oced u r es. All anhydrous organic solvents were
either purchased from Aldrich Chemical Company or dried
according to the procedures described by Perrin et al.38 N-Boc-
L-homoserine was purchased from Sigma Chemical Company.
Thin-layer chromatography (TLC) was performed on precoated
plates (Merck TLC aluminum sheets silica 60F254, Art. No.
5554). Products were visualized either by UV light or by
spraying the plate with a 7% phosphomolybdic acid solution
in methanol (w/v) followed by heating. Flash chromatography
refers to the method of Still et al.39 and was carried out using
Silica Gel 60 (Merck particle size 0.040-0.063 mm). Analytical
HPLC was performed at 55 °C on a Deltapak C18-reverse phase
column (0.49 × 15 cm, 10 µm) using a Waters 490 HPLC
system. Buffer A was 0.1% TFA in water, and buffer B was
0.1% TFA in acetonitrile. A linear gradient of 0-50% buffer
B over 30 min at a flow rate of 1 mL/min was used. 1H and
13C NMR spectra were measured on J EOL GX270 and Varian
UNITY 500 NMR spectrometers. 1H and 13C chemical shifts
were recorded in ppm relative to tetramethylsilane (TMS) or
deuterated DMSO. J values are given in hertz. Mixtures of
two rotamers were observed for some of the products contain-
ing amide bonds in the ratio of 2:1 unless otherwise indicated.
As a consequence, several of the NMR signals for these
products were doubled in the rotamer ratio as indicated by
ma for major and mi for minor. Microanalysis was performed
by University College Dublin Chemical Service unit. Mass
spectra were either recorded by University College Dublin
Mass Spectrometry Service or University College London Mass
Spectrometry Service. MALDI-TOF spectra were recorded
curtesy of The Panum Institute, Copenhagen N, Denmark. The
matrix used was 3,5-dimethoxy-4-hydroxycinnamic acid (si-
napinic acid). Prior to each measurement, the spectrometer
was calibrated using a mixture of four Nielsen type PNAs of
appropriate known molecular weights. Experimental errors
of the MALDI-TOF mass peaks are estimated to be (0.1%.
Dicycloh exyla m m on iu m Sa lt of N-(ter t-Bu toxyca r bo-
n yl)-L-h om oser in e. N-Boc-L-homoserine (1.00 g, 4.56 mmol)
was dissolved in ethanol (10 mL), and dicyclohexylamine was
added dropwise, with stirring, to the organic solution until it
became basic. Subsequently, the solvent was removed in
vacuo, and the resulting white precipitate was resuspended
in diethyl ether. The white solid was collected by suction
filtration and dried in a vacuum desiccator over phosphorus
pentoxide (1.55 g, 85%).
F igu r e 3. Analytical reverse-phase HPLC of crude H-RCLys-
RTGlyRCGlyRTLysRCGlyRCGlyRTLysRTGlyRTGlyRTLys-
NH220 on a Deltapak C18-reverse phase column at 55 °C. Buffer
A was 0.1% TFA in water, and buffer B was 0.1% TFA in
acetonitrile. A linear gradient of 0-50% buffer B over 30 min
at a flow rate of 1 mL/min was used. The eluents were
monitored at 260 nm.
to the sodium salt of the oligomer (i.e. [M + Na]+). The
mass spectrum also shows evidence for some capped
failure sequences and one deletion fragment [Figure 2:
peaks v, w, x and y]. Peak v at 2355 Da can be assigned
to the capped failure sequence Ac-RCGlyRTLysRCGly-
RCGlyRTLysRTGlyRTGlyRTLys-NH2,20 peak w at 2621
Da to the sequence Ac-RTGlyRCGlyRTLysRCGlyRCGly-
20
RTLysRTGlyRTGlyRTLys-NH2 and peak x at 2749 Da
to the capped failure sequence Ac-LysRTGlyRCGlyRTLys-
RCGlyRCGlyRTLysRTGlyRTGlyRTLys-NH2.20 Thus, prod-
uct v appears to lack the N-terminal RCLysRTGly,
product w the RCLys and product x the RC. Peak y at
2847 Da has a molecular weight that corresponds to a
full length base sequence minus a glycine residue and
may therefore be a glycine deletion fragment. No other
impurities are discernible in the mass spectrum from
which we estimate the purity of the desired oligomer to
be 80%. The homogeneity of the crude R-PNA oligomer
was also assessed using analytical reverse-phase HPLC
which, at 260 nm, showed only a single peak at 14.97
min (Figure 3).
N-(ter t-Bu t oxyca r b on yl)-L-h om oser in e Met h yl E st er
(2). Methyl iodide (638 µL, 1.45 g, 10.25 mmol) was added
dropwise to a stirred suspension of the dicyclohexylammonium
salt of N-Boc-L-homoserine (3.44 g, 8.60 mmol) in anhydrous
DMF (60 mL) at rt. The reaction mixture was then left to
stir overnight. Subsequently, the reaction was evaporated to
dryness in vacuo and the residue coevaporated with toluene
(3 × 30 mL). Water (55 mL) was added to the residue and
the resulting aqueous solution extracted with ethyl acetate (6
× 68 mL). The combined organic extracts were washed with
water (3 × 80 mL) and brine (80 mL) before being dried over
MgSO4. Filtration followed by solvent evaporation gave a
crude viscous yellow oil which was purified by flash chroma-
tography using diethyl ether as the eluting solvent. Compound
2 was obtained as a colorless viscous oil (1.30 g, 65%): Rf 0.28
Con clu sion
We have synthesized all four nucleotide base-contain-
ing amino acids from N-Boc-L-homoserine via the key
intermediate (S)-2-(N-Boc-amino)-4-bromobutyric acid
methyl ester by nucleophilic substitution of the bromine
with the desired nucleotide base. Apart from the thy-
mine derivative, all other base-containing amino acids
are prepared with their exocyclic functionalities protected
in order to prevent interference in the later peptide-
coupling steps and to render intermediates sufficiently
soluble for chemical manipulation. Using solution phase
techniques, the base-containing amino acids have been
coupled to glycine ethyl ester, and the resulting dipep-
tides have been further dimerized to give tetrapeptides,
which are analogous to dinucleotides, thereby illustrating
the feasibility of preparing short oligo-R-PNAs by these
methods. We have also demonstrated that it is possible
to synthesize longer oligomers with a high degree of
integrity and purity using standard solid phase peptide
synthesis protocols.
1
[diethyl ether]; H NMR (CDCl3) δ 1.45 (s, 9H), 1.65 (m, 1H),
2.13 (m, 1H), 3.28 (br s, exchangeable, 1H), 3.70 (br m, 2H),
3.77 (s, 3H), 4.49 (m, 1H), 5.42 (br d, 1H); 13C NMR (CDCl3) δ
28.68, 36.54, 50.96, 52.97, 58.68, 80.92, 156.85, 173.77; MS[EI]
m/z (%) 234 [(M + H)+] (<1), 174 (5), 160 (3), 118 (38), 102
(11).
(S)-2-[N-(ter t-Bu t oxyca r bon yl)a m in o]-4-br om ob u t yr -
ic Acid Meth yl Ester (3). A solution of triphenylphosphine
(3.78 g, 14.40 mmol, 2.8 equiv) in anhydrous DCM (10.64 mL)
was added dropwise to a stirred suspension of NBS (2.75 g,
(38) Perrin, D. D.; Armarego, W. L. F., Eds.; Purification of Labora-
tory Chemicals, 3rd ed.; Pergamon Press plc: Oxford, 1988.
(39) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923-
2925.

A Novel Peptide Nucleic Acid Analogue of DNA
J . Org. Chem., Vol. 62, No. 16, 1997 5447
15.42 mmol, 3 equiv) in anhydrous DCM (24 mL) at rt. The
resulting reddish brown solution was left to stir for a further
5 min. Anhydrous pyridine (499 µL, 6.17 mmol, 1.2 equiv)
followed by a solution of 2 (1.20 g, 5.14 mmol, 1 equiv) in
anhydrous DCM (10.64 mL) were then added, and the reaction
was left to stir at rt overnight. Subsequently, the reaction
mixture was evaporated to dryness in vacuo and the residue
coevaporated with toluene (3 × 20 mL). The dark brown oily
residue was triturated with diethyl ether (10 mL) and the
ethereal extract applied to a flash column in which the eluting
solvent was 60:40 diethyl ether:hexane. The residue was then
triturated twice with the above eluting solvent (2 × 10 mL)
before running the column. Compound 3 was obtained as a
colorless viscous oil which solidified on standing (1.14 g,
75%): Rf 0.54 [60:40, diethyl ether:hexane]; 1H NMR (CDCl3)
δ 1.45 (s, 9H), 2.22 (m, 1H), 2.40 (br m, 1H), 3.44 (t, 2H, J )
7.03), 3.77 (s, 3H), 4.43 (br m, 1H), 5.18 (br d, 1H); MS[EI]
m/z (%) 296 [M+] (10), 236 (11), 196 (8), 180 (17), 136 (78).
Anal. Calcd for C10H18NO4Br: C, 40.56; H, 6.12; N, 4.73; Br,
26.98. Found: C, 40.60; H, 6.16; N, 4.60; Br, 26.56.
eluting solvent. Compound 12 was obtained as a pale yellow
solid (0.50 g, 79%): Rf 0.20 [90:10, ethyl acetate:methanol];
1H NMR (d6-DMSO) δ 1.38 (s, 9H), 2.07 (m, 1H), 2.27 (m, 1H),
3.55 (s, 3H,), 3.87 (m, 1H), 4.19 (t, 2H), 7.19 (s, exchangeable,
2H), 7.43 (d, 1H), 8.02 (s, 1H), 8.12 (s, 1H); 13C NMR
(d6-DMSO) δ 32.49, 34.86, 44.31, 55.25, 56.46, 82.86, 123.08,
145.12, 153.81, 156.76, 159.84, 160.30, 176.74; MS[EI] m/z (%)
350 [M+] (14), 294 (10), 277 (21), 250 (20), 235 (14), 189 (17),
162 (24). Anal. Calcd for C15H22N6O4‚1/2CH3OH: C, 50.82; H,
6.56; N, 22.95; C/N, 2.21. Found: C, 50.87; H, 6.33; N, 23.02;
C/N, 2.21.
(S)-2-[N-(ter t-Bu toxyca r bon yl)a m in o]-4-[N6-(ben zylox-
yca r bon yl)a d en in -9-yl]bu tyr ic Acid Meth yl Ester (13). A
1 M solution of triethyloxonium tetrafluoroborate in DCM (3.66
mL, 3.66 mmol, 6 equiv) was added dropwise to a stirred
solution of N-(benzyloxycarbonyl)imidazole (0.74 g, 3.66 mmol,
6 equiv) in anhydrous DCM (0.64 mL) at 0 °C under nitrogen.
The reaction was then left to stir for 1 h at this temperature
and for a further 2 h at rt. Subsequently, 12 (0.21 g, 0.61
mmol, 1 equiv) was added to the reaction mixture at rt and
the reaction left to stir overnight. The reaction was quenched
by addition of a saturated solution of NaHCO3 (6 mL) and the
organic layer separated. The aqueous layer was then further
extracted with DCM (6 mL), and the combined organic extracts
were dried over MgSO4. Filtration followed by solvent evapo-
ration gave a crude viscous oil which was purified by flash
chromatography using 96:4 ethyl acetate:methanol as the
eluting solvent. Compound 13 was obtained as a white foam
(0.24 g, 82%): Rf 0.27 [96:4; ethyl acetate:methanol]; 1H NMR
(d6-DMSO) δ 1.38 (s, 9H), 2.13 (m, 1H), 2.33 (m, 1H), 3.54 (s,
3H), 3.91 (m, 1H), 4.31 (t, 2H), 5.20 (s, 2H), 7.32-7.48 (series
of m, 6H), 8.37 (s, 1H), 8.60 (s, 1H), 10.67 (s, 1H); 13C NMR
(d6-DMSO) δ 32.46, 34.64, 44.64, 55.24, 56.28, 70.53, 82.84,
127.62, 132.16, 132.30, 140.70, 148.59, 153.74, 155.79, 156.44,
159.78, 176.62; MS[EI] m/z (%) 484 [M+] (<1), 376 (3), 320 (30),
217 (34), 175 (57). Anal. Calcd for C23H28N6O6‚1/2CH3OH: C,
56.40; H, 6.00; N, 16.80; C/N, 3.36. Found: C, 56.71; H, 5.94;
N, 16.65; C/N, 3.41.
(S)-2-[N-(ter t-Bu toxycar bon yl)am in o]-4-[2-am in o-6-ch lo-
r op u r in -9-yl]bu tyr ic Acid Meth yl Ester (16). A mixture
of 2-amino-6-chloropurine (0.24 g, 1.42 mmol, 1 equiv) and
anhydrous K2CO3 (0.20 g, 1.42 mmol, 1 equiv) were suspended
in anhydrous DMF (4 mL). Compound 3 (0.42 g, 1.42 mmol,
1 equiv) was then added with stirring at rt and the reaction
left overnight. Subsequently, the reaction was evaporated to
dryness in vacuo and the residue redissolved in chloroform
(20 mL). The organic solution was washed with water (3 ×
10 mL) and dried over MgSO4. Filtration followed by solvent
evaporation gave a crude viscous oil which was purified by
flash chromatography using ethyl acetate as the eluting
solvent. Compound 16 was afforded as a white foam (0.49 g,
90%): Rf 0.37 (ethyl acetate); 1H NMR (CDCl3) δ 1.46 (s, 9H),
2.27 (m, 1H), 2.41 (m, 1H), 3.66 (s, 3H), 4.20 (m, 2H), 4.40 (br
s, 1H), 5.25 (s, exchangeable, 2H), 5.56 (br d, 1H), 7.85 (s, 1H);
13C NMR (CDCl3) δ 28.28, 32.47, 40.32, 51.17, 52.69, 80.51,
125.28, 142.63, 151.39, 153.73, 155.54, 159.02, 172.04; MS[EI]
m/z (%) 384 [M+] (7), 311 (17), 284 (20), 223 (17), 196 (21).
Anal. Calcd for C15H21N6O4Cl‚3/4CH3OH: C, 46.27; H, 5.62;
N, 20.55; Cl, 8.68; C/N, 2.25. Found: C, 46.38; H, 5.86; N,
20.56; Cl, 8.78; C/N, 2.26.
(S)-2-[N-(ter t-Bu toxyca r bon yl)a m in o]-4-[2-a m in o-6-(2-
n itr op h en oxy)p u r in -9-yl]bu tyr ic Acid Meth yl Ester (17).
A solution of 2-nitrophenol (0.33 g, 2.34 mmol, 3 equiv),
DABCO (0.09 g, 0.78 mmol, 1 equiv), and triethylamine (330
µL, 2.34 mmol, 3 equiv) in anhydrous 1,2-dichloroethane (750
µL) was added to a stirred slurry of 16 (0.30 g, 0.78 mmol, 1
equiv) in anhydrous 1,2-dichloroethane (3 mL) at rt. The
reaction was left to stir overnight. Subsequently, the reaction
was diluted with DCM (6 mL) and the organic solution washed
with a saturated solution of NaHCO3 (9 mL). The aqueous
layer was re-extracted with DCM (6 mL), and the combined
organic extracts were dried over MgSO4. Filtration followed
by solvent evaporation gave a crude product which was
purified by flash chromatography using ethyl acetate as the
eluting solvent. Compound 17 was obtained as a yellow foam
in quantitative yield: Rf 0.29 (ethyl acetate); 1H NMR (CDCl3)
(S )-2-[N -(t er t -Bu t oxyca r b on yl)a m in o]-4-(t h ym in -1-
yl)bu tyr ic Acid Meth yl Ester (5). A stirred mixture of 3
(1.20 g, 4.06 mmol, 1 equiv), thymine (1.79 g, 14.21 mmol, 3.5
equiv), and anhydrous K2CO3 (1.68 g, 12.18 mmol, 3 equiv) in
anhydrous DMSO (14 mL) was heated at 80 °C for 22.5 h
under nitrogen. Subsequently, water (100 mL) was added to
the reaction and the resulting aqueous solution extracted with
chloroform (6 × 60 mL). The combined organic extracts were
washed with water (3 × 60 mL) and brine (60 mL) and dried
over MgSO4. Filtration followed by solvent evaporation gave
a crude viscous yellow oil which was purified by flash chro-
matography using ethyl acetate as the eluting solvent. Com-
pound 5 was obtained as a foamy white solid (0.98 g, 71%):
1
Rf 0.37 (ethyl acetate); H NMR (CDCl3) δ 1.45 (s, 9H), 1.92
(s, 3H), 2.02-2.24 (series of m, 2H), 3.68 (s, 1H), 3.73 (s, 3H),
4.06-4.40 (series of m, 2H), 5.40 (br m, 1H), 7.07 (s, 1H); 13C
NMR (CDCl3) δ 12.24, 28.20, 31.51, 45.32, 50.96, 52.61, 80.34,
110.74, 140.76, 151.01, 155.57, 164.54, 172.00; MS[EI] m/z (%)
341 [M+] (1), 282 (23), 226 (14), 181 (16), 153 (47), 140 (19).
Anal. Calcd for C15H23N3O6: C, 52.79; H, 6.74; N, 12.32.
Found: C, 52.49; H, 6.92; N, 12.03.
(S)-2-[N-(ter t-Bu toxyca r bon yl)a m in o]4-[N4-(ben zylox-
yca r bon yl)cytosin -1-yl]bu tyr ic Acid Meth yl Ester (9).
Sodium hydride (60% disp, 0.079 g, 1.98 mmol, 1.1 equiv) was
added to a vigorously stirred suspension of N4-Cbz-cytosine
824 (0.49 g, 1.98 mmol, 1.1 equiv) in anhydrous DMF (5 mL)
at rt. After hydrogen production had ceased, a solution of 3
(0.53 g, 1.80 mmol, 1 equiv) in anhydrous DMF (5 mL) was
added, and the reaction mixture was left to stir overnight.
Subsequently, water (25 mL) was added to the reaction and
the aqueous solution extracted with chloroform (6 × 25 mL).
The combined organic extracts were dried over MgSO4. Filtra-
tion followed by solvent evaporation afforded a crude product
which was purified by flash chromatography using ethyl
acetate as the eluting solvent. Compound 9 was obtained as
1
a white foam (0.50 g, 61%): Rf 0.24 (ethyl acetate); H NMR
(CDCl3) δ 1.43 (s, 9H), 2.10 (br m, 1H), 2.32 (br m, 1H), 3.70
(s, 3H), 3.83 (br m, 1H), 4.11 (br m, 1H), 4.31 (br m, 1H), 5.21
(s, 2H), 5.56 (br s, 1H), 7.20 (br m, 1H), 7.38 (s, 5H), 7.77 (d,
1H, J ) 7.31); 13C NMR (CDCl3) δ 28.26, 31.75, 47.78, 51.00,
52.68, 67.96, 80.36, 94.94, 127.75, 128.34, 128.70, 134.97,
149.32, 155.72, 162.38, 172.31; MS[EI] m/z (%) 460 [M+] (3),
429 (30), 404 (29), 387 (17), 343 (20). Anal. Calcd for C22H28
-
N4O7‚1/2H2O: C, 56.29; H, 6.18; N, 11.94; C/N, 4.71. Found:
C, 56.05; H, 6.16; N, 11.68; C/N, 4.80.
(S)-2-[N-(ter t-Bu t oxyca r b on yl)a m in o]-4-[a d en in -9-yl-
]bu tyr ic Acid Meth yl Ester (12). Sodium hydride (60% disp,
0.079 g, 1.98 mmol, 1.1 equiv) was added to a vigorously stirred
suspension of adenine (0.24 g, 1.80 mmol, 1.1 equiv) in
anhydrous DMF (4.43 mL) at rt and the reaction left to stir
for 2 h. A solution of 3 (0.53 g, 1.80 mmol, 1 equiv) in
anhydrous DMF (5.32 mL) was then added and the reaction
mixture left to stir overnight. Subsequently, the reaction was
concentrated in vacuo and the residue coevaporated with
toluene (3 × 10 mL). The crude product was purified by flash
chromatography using 90:10 ethyl acetate:methanol as the

5448 J . Org. Chem., Vol. 62, No. 16, 1997
Howarth and Wakelin
δ 1.47 (s, 9H), 2.26-2.48 (series of m, 2H), 3.65 (s, 3H), 4.20
(m, 2H), 4.40 (br s, 1H), 4.82 (br s, exchangeable, 2H), 5.80
(br d, 1H), 7.40-7.46 (m, 2H), 7.66-7.76 (series of m, 3H),
8.09-8.12 (m, 1H); 13C NMR (CDCl3) δ 28.25, 32.35, 40.00,
51.19, 52.58, 80.40, 114.80, 125.34, 125.51, 126.10, 134.56,
140.90, 142.47, 145.46, 155.22, 155.80, 158.65, 159.10, 172.03;
MS[EI] m/z (%) 487 [M+] (21), 387 (10), 328 (9), 286 (100), 226
(22).
(S)-2-[N-(ter t-Bu toxyca r bon yl)a m in o]-4-[N4-(ben zylox-
yca r bon yl)cytosin -1-yl]bu tyr ic Acid [Boc-rC (Cbz)-OH20
]
(10). The above procedure was followed using 9 (0.80 g, 1.74
mmol). After acidification, 10 precipitated from solution as a
white solid and was collected by suction filtration. It was
washed thoroughly with water and once with diethyl ether
before being dried in a vacuum desiccator over phosphorus
pentoxide (0.67 g, 86%): 1H NMR (d6-DMSO) δ 1.36 (s, 9H),
1.73-2.15 (br d, 2H), 3.80 (br s, 3H), 5.16 (s, 2H), 6.95 (d, 1H,
J ) 7.31), 7.23 (d, 1H, J ) 7.88), 7.37 (m, 5H), 7.94 (d, 1H, J
) 7.31); MS[EI] m/z (%) 446 [M+] (1), 354 (1), 320 (4), 229 (6),
214 (4). Anal. Calcd for C21H26N4O7‚1/4H2O: C, 55.94; H, 5.88;
N, 12.43; C/N, 4.50. Found: C, 55.89; H, 5.76; N, 12.22; C/N,
4.57.
(S)-2-[N-(ter t-Bu toxyca r bon yl)a m in o]-4-[2-(a cetyla m i-
n o)-6-(2-n itr op h en oxy)p u r in -9-yl]bu tyr ic Acid Meth yl
Ester (18). Acetyl chloride (290 µL, 4.08 mmol, 5 equiv) was
carefully added dropwise to a stirred solution of 17 (0.40 g,
0.82 mmol, 1 equiv) in anhydrous pyridine (4 mL) at 0 °C. The
reaction was allowed to warm slowly to rt before being left to
stir overnight. Subsequently, the reaction was poured onto
ice (15 mL) and the resulting aqueous solution extracted with
ethyl acetate (6 × 12 mL). The combined organic extracts were
dried over MgSO4. Filtration followed by solvent evaporation
gave a crude viscous orange oil which was purified by flash
chromatography using 95:5 ethyl acetate:methanol as the
eluting solvent. Compound 18 was obtained as an orange foam
(0.41 g, 94%): Rf 0.30 (95:5, ethyl acetate:methanol); 1H NMR
(CDCl3) δ 1.47 (s, 9H), 2.22 (s, 3H), 2.29-2.47 (series of m,
2H), 3.67 (s, 3H), 4.32 (br m, 3H), 5.63 (br s, 1H), 7.41-7.52
(series of m, 2H), 7.71-7.78 (series of m, 1H), 7.82 (br s, 1H),
8.01 (br s, 1H), 8.15-8.19 (series of m, 1H); 13C NMR (CDCl3)
δ 24.71, 28.23, 32.50, 40.60, 51.11, 52.64, 80.42, 117.49, 125.45,
125.80, 126.70, 134.88, 142.17, 143.23, 145.44, 151.50, 154.46,
159.14, 171.88; MS[EI] m/z (%) 529 [M+] (21), 456 (19), 370
(16), 352 (33), 328 (73), 315 (100).
(S)-2-[N-(ter t-Bu toxyca r bon yl)a m in o]-4-[N6-(ben zylox-
yca r bon yl)a d en in -9-yl]bu tyr ic Acid [Boc-rA (Cbz)-OH20
]
(14). The above procedure was followed using 13 (0.20 g; 0.41
mmol). After acidification, the aqueous layer was extracted
with ethyl acetate (6 × 4 mL). The combined organic extracts
were washed with brine (5 mL) before being dried over MgSO4.
Filtration followed by solvent evaporation and coevaporation
with toluene (3 x 10 mL) gave 14 as a white foam, which was
dried further in a vacuum desiccator over phosphorous pen-
toxide (0.14 g, 72%). It was evident from the microanalysis
that the product contained some salt but the C/N ratio
corresponded well with the calculated value: 1H NMR (d6-
DMSO) δ 1.40 (s, 9H), 2.09-2.50 (br d, 2H), 3.82 (br m, 1H),
4.32 (br m, 2H), 5.22 (s, 2H), 7.28-7.48 (series of m, 6H), 8.38
(s, 1H), 8.62 (s, 1H), 10.70 (br s, exchangeable, 1H); MS[EI]
m/z (%) 470 [M+] (<1), 447 (<1), 411 (<1), 392 (<1), 387 (<1),
370 (<1). Anal. Calcd for C22H26N6O6: C, 56.16; H, 5.57; N,
17.86; C/N, 3.14. Found: C, 55.89; H, 5.27; N, 16.55; C/N, 3.38.
(S)-2-[N-(ter t-Bu toxyca r bon yl)a m in o]-4-[N2-a cetylgu a -
n in -9-yl]bu tyr ic Acid Meth yl Ester (19). A solution of
1,1,3,3-tetramethylguanidine (306 µL, 2.44 mmol, 9 equiv) in
anhydrous acetonitrile (2.71 mL) was added to a stirred
solution of 18 (0.14 g, 0.27 mmol, 1 equiv) and 2-nitrobenzal-
doxime (0.45 g, 2.71 mmol, 10 equiv) in anhydrous acetonitrile
(2.71 mL) at rt under nitrogen. The reaction was left to stir
overnight. Subsequently, the reaction mixture was evaporated
to dryness in vacuo and the resulting residue purified using
flash chromatography with 90:10 DCM:methanol as the elut-
ing solvent. Compound 18 was afforded as a beige powder
(S)-2-[N-(ter t-Bu toxyca r bon yl)a m in o]-4-(N2-a cetylgu a -
n in -9-yl)bu tyr ic Acid [Boc-rG (Ac)-OH20] (20). The above
procedure was followed using 19 (0.23 g, 0.58 mmol). After
acidification, 20 precipitated from solution as a beige solid and
was collected by suction filtration. It was washed thoroughly
with water and once with diethyl ether before being dried in
a
vacuum desiccator over phosphorus pentoxide (0.10 g,
44%): 1H NMR (d6-DMSO) δ 1.39 (s, 9H), 1.89-2.36 (br d, 1H),
2.17 (s, 3H), 3.82 (br m, 1H), 4.12 (br m, 2H), 7.30 (d, 1H, J )
8.72), 7.90 (s, 1H), 11.75 (s, 1H), 12.03 (s, 1H); MS[FAB] m/z
(%) 395 [(M+1)+] (100). Anal. Calcd for C16H22N6O6‚11/4H2O:
C, 46.10; H, 5.88; N, 20.17; C/N, 2.29. Found: C, 46.46; H,
5.77; N, 19.96; C/N, 2.33.
1
(0.087 g, 78%): Rf 0.33 (90:10, DCM:methanol); H NMR (d6-
DMSO) δ 1.37 (s, 9H), 2.05 (br series of m, 1H), 2.17 (s, 3H),
2.26 (br series of m, 1H), 3.59 (s, 3H), 3.92 (br series of m,
1H), 4.11 (br series of m, 2H), 7.43 (d, 1H), 7.88 (br m, 1H),
11.71 (br m, 1H), 12.02 (s, 1H); 13C NMR (d6-DMSO) δ 29.00,
33.36, 35.79, 45.09, 56.22, 57.25, 83.84, 125.38, 144.91, 152.91,
153.60, 160.13, 160.74, 177.33, 178.74; MS[EI] m/z (%) 408
[M+] (16), 335 (12), 308 (15), 249 (9), 207 (74). Anal. Calcd
for C17H24N6O6‚3/4CH3OH: C, 49.30; H, 6.29; N, 19.43; C/N,
2.54. Found: C, 49.57; H, 6.16; N, 19.03; C/N, 2.60.
Solu tion P h a se Syn th esis of Dip ep tid es 21-24. Sta n -
d a r d P r otocol. EDC (1 equiv) was added to a stirred solution
of 6, 10, 14, or 20 (1 equiv) and N-hydroxysuccinimide (1 equiv)
in anhydrous DMF (10 mL/g) at rt. The reaction was left to
stir for 1 h whereupon a further quantity of EDC (0.5 equiv)
was added. The reaction was then left for another 0.5 h before
glycine ethyl ester hydrochloride (5 equiv) and triethylamine
(7 equiv) were added. The reaction was left to stir at rt
overnight. Subsequently, water (27 mL/mmol) was added to
the reaction mixture and the resulting aqueous solution
extracted with ethyl acetate (6 × 40 mL/mmol). The combined
organic extracts were dried over MgSO4. Filtration followed
by solvent evaporation afforded a crude product.
Ba se-Con ta in in g Am in o Acid s 6, 10, 14, a n d 20. Sta n -
d a r d P r otocol. A 0.67 M(aq) solution of sodium hydroxide
(2.5 equiv) was added to a stirred solution of 5, 9, 13, or 19 (1
equiv) in dioxane (1.75 mL/mmol) at rt. The reaction was left
for 0.5 h before being diluted with water (6.65 mL/mmol). The
aqueous solution was washed with DCM (3 × 6.65 mL/mmol)
and the pH adjusted to 3.0 with a 2 M (aq) solution of citric
acid.
Dip ep tid e 21. The above procedure was followed using 6
(0.86 g, 2.63 mmol). After workup, the crude product obtained
was purified using flash chromatography with 96:4 ethyl
acetate:methanol as the eluting solvent. The dipeptide 21 was
given as a glassy white solid (0.94 g, 87%): Rf 0.29 (96:4, ethyl
acetate:methanol]; 1H NMR (d6-DMSO) δ 1.16 (t, 3H, J ) 7.03),
1.38 (s, 9H), 1.60-2.07 (series of m, 2H), 1.72 (ma) and 1.76
(mi) (s, 3H), 3.60-4.00 (series of m, 5H), 4.05 (mi) and 4.06
(ma) (q, 2H, J ) 7.03), 6.90 (mi) and 7.10 (ma) (d, 1H), 7.30
(mi) and 7.35 (ma) (s, 1H), 8.27 (mi) and 8.32 (ma) (br t, 1H);
13C NMR (d6-DMSO) δ 11.93 (ma) and 12.37 (mi), 13.92, 28.08,
30.04 (mi) and 30.73 (ma), 40.59 (mi) and 40.64 (ma), 44.32,
51.39 (ma) and 52.40 (mi), 60.33, 78.06 (mi) and 78.17 (ma),
107.09 (mi) and 108.23 (ma), 136.19 (mi) and 141.35 (ma),
150.70 (ma) and 150.80 (mi), 155.14, 163.60 (mi) and 164.24
(ma), 169.54 (mi) and 169.63 (ma), 172.05; MS[EI] m/z (%) 413
(S )-2-[N -(t er t -Bu t oxyca r b on yl)a m in o]-4-(t h ym in -1-
yl)bu tyr ic Acid [Boc-rT-OH20] (6). The above procedure
was followed using 5 (0.98 g, 2.87 mmol). After acidification,
the aqueous layer was extracted with ethyl acetate (6 × 20
mL), and the combined organic extracts were dried over
MgSO4. Filtration followed by solvent evaporation and co-
evaporation with toluene (3 × 20 mL) gave 6 as a glassy white
solid which was dried further in a vacuum desiccator over
phosphorus pentoxide (0.86 g, 92%): 1H NMR (d6-DMSO) δ
1.39 (s, 9H), 1.74 (ma) and 1.77 (mi) (s, 3H), 1.74-2.12 (br m,
2H), 3.68 (br m, 1H), 3.83 (br m, 2H), 7.18-7.24 (series of m,
1H), 7.31 (mi) and 7.42 (ma) (s, 1H), 10.90 (mi) and 11.25 (ma)
(s, exchangeable, 1H); MS[CI] m/z (%) 328 [(M + H)+] (2), 303
(9), 289 (8), 284 (39), 272 (8), 245 (11), 228 (30). Anal. Calcd
for C14H21N3O6‚CH3OH: C, 50.14; H, 6.96; N, 11.70; C/N, 4.29.
Found: C, 49.83; H, 6.61; N, 11.56; C/N, 4.31.
[M+] (6), 226 (15), 182 (16), 127 (13). Anal. Calcd for C18H28
-

A Novel Peptide Nucleic Acid Analogue of DNA
J . Org. Chem., Vol. 62, No. 16, 1997 5449
N4O7‚3/4CH3OH: C, 51.61; H, 7.11; N, 12.84; C/N, 4.02.
Found: C, 51.59; H, 6.80; N, 12.78; C/N, 4.04.
was extracted with ethyl acetate (7 × 10 mL/mmol), and the
combined organic extracts were washed with brine (10 mL/
Dip ep tid e 22. The above procedure was followed using 10
(0.67 g, 1.49 mmol). After workup, the crude product obtained
was purified using flash chromatography with 96:4 ethyl
acetate:methanol as the eluting solvent. The dipeptide 22 was
given as a cream powder (0.68 g, 86%): Rf 0.29 [96:4, ethyl
acetate:methanol]; 1H NMR (d6-DMSO) δ 1.16 (t, 3H, J ) 7.03),
1.37 (s, 9H), 1.73-2.10 (br series of m, 2H), 3.70-4.00 (series
of m, 5H), 4.06 (q, 2H, J ) 7.03), 5.17 (s, 2H), 6.96 (br d, 1H,
J ) 7.03), 7.09 (d, 1H, J ) 8.16), 7.36-7.40 (m, 5H), 7.92 (d,
1H, J ) 7.60), 8.34 (br t, 1H); 13C NMR (d6-DMSO) δ 14.03,
28.17, 30.74, 40.73, 46.87, 51.66, 60.44, 66.44, 78.32, 94.01,
127.94, 128.16, 128.49, 136.00, 149.86, 153.20, 154.94, 155.24,
162.75, 169.73, 172.07; MS[EI] m/z (%) 487 [(M - Et + H)+]
(1), 280 (6), 267 (7), 221 (7), 138 (43), 125 (94). Anal. Calcd
for C25H33N5O8: C, 56.50; H, 6.21; N, 13.18. Found: C, 56.27;
H, 6.23; N, 12.96.
mmol) before being dried over MgSO4
. Filtration followed by
solvent evaporation gave the free carboxylic acid dipeptide
which was dried in a vacuum desiccator over phosphorus
pentoxide.
Th ym in e Der iva tive. The above procedure was followed
for dipeptide 21 (0.12 g, 0.29 mmol). The desired compound
was obtained as a pale yellow powder (0.10 g, 91%).
Cytosin e Der iva tive. The above procedure was followed
for dipeptide 22 (0.06 g, 0.10 mmol). The desired compound
was given as a white powder (0.05 g, 90%).
Ad en in e Der iva tive. The above procedure was followed
for dipeptide 23 (0.10 g, 0.19 mmol). The desired compound
was afforded as a white powder (0.08 g, 81%).
Gu a n in e Der iva tive. The above procedure was followed
for dipeptide 24 (0.11 g, 0.23 mmol). The desired compound
was obtained as a glassy white solid (0.076 g, 74%).
(iii) F or m a tion of th e Tetr a p ep tid es. The appropriate
free amino dipetide (1 equiv), free carboxylic acid dipeptide (1
equiv), and HOBt (1.2 equiv) were dissolved in the relevant
amount of anhydrous DMF so that the reaction was 0.2 M in
both the amino and acid components. Subsequently, triethyl-
amine (2 equiv) was added to the stirred mixture and the
reaction cooled in an ice-salt bath to ca. -10 °C. DCC (1.1
equiv) was added and the reaction left to stir at this temper-
ature for 1 h. The reaction was then allowed to warm slowly
to rt over the next hour before being left to stir overnight. The
precipitate, which formed during the course of the reaction,
was removed by filtration and the filtrate concentrated in
vacuo. The crude residue was finally coevaporated with
toluene (3 × 10 mL/100 mg).
Dip ep tid e 23. The above procedure was followed using 14
(0.14 g, 0.29 mmol). After workup, the crude product obtained
was purified using flash chromatography with 92:8 ethyl
acetate:methanol as the eluting solvent. The dipeptide 23 was
given as a cream powder (0.16 g, 99%): Rf 0.27 [92:8, ethyl
acetate:methanol]; 1H NMR (d6-DMSO) δ 1.14 (t, 3H, J ) 7.03),
1.37 (s, 9H), 1.94-2.36 (series of m, 2H), 3.66-4.40 (series of
m, 5H), 4.05 (q, 2H, J ) 7.03), 5.20 (s, 2H), 7.16 (d, 1H, J )
8.16), 7.34-7.46 (m, 5H), 8.32 (br s, 2H), 8.59 (s, 1H), 10.66
(br s, 1H); 13C NMR (d6-DMSO) δ 18.05, 32.21, 35.65, 44.25,
44.73, 55.50, 64.56, 70.20, 82.35, 127.32, 131.87, 132.02,
132.44, 140.42, 148.25, 153.40, 155.43, 156.03, 159.30, 165.65,
173.73, 176.01; MS[FAB] m/z (%) 556 [(M + 1)+] (100).
Dip ep tid e 24. The above procedure was followed using 20
(0.11 g, 0.27 mmol). After workup, the crude product obtained
was purified by flash chromatography using 90:10 DCM:
methanol as the eluting solvent. The dipeptide 24 was given
as a cream powder (0.042 g, 33%); Rf 0.31 (90:10, DCM:
Tetr a p ep tid e 25. The above procedure was used to couple
the free carboxylic acid derivative of dipeptide 21 (0.05 g, 0.13
mmol) and the TFA salt of the free amino acid analogue of
dipeptide 21 (0.055 g, 0.13 mmol). After workup the crude
residue was purified using flash chromatography with 90:10
DCM:methanol as the eluting solvent. The tetrapeptide 25
was obtained as a pale yellow waxy solid (0.053 g, 61%): Rf
0.23 (90:10, DCM:methanol); 1H NMR (d6-DMSO) δ 1.145 (mi)
and 1.15 (ma) (t, 3H, J ) 7.07), 1.37 (s, 9H), 1.66-2.11 (series
of m, 4H), 1.71 (s, 3H), 1.74 (s, 3H), 3.54-4.32 (series of m,
10H), 4.05 (br q, 2H, J ) 7.07), 7.25 (br s, 1H), 7.30 (br m,
1H), 7.37 (br s, 1H), 8.00-8.50 (br series of m, 3H), 10.89 (mi)
and 11.21 (ma) (br s, 2H); MS[FAB] m/z (%) 679 [(M + 1)+]
(1).
1
methanol); H NMR (d6-DMSO) δ 1.15 (t, 3H, J ) 7.03), 1.37
(s, 9H), 1.89-2.34 (series of m, 2H), 2.16 (s, 3H), 3.64-4.00
(series of m, 5H), 4.06 (mi) and 4.07 (ma) (q, 2H, J ) 7.03),
7.16 (d, 1H, J ) 8.16), 7.86 (s, 1H), 8.37 (br s, 1H), 11.72 (br
t, 1H), 12.02 (br s, 1H); 13C NMR (d6-DMSO) δ 18.00, 27.73,
32.13, 35.70 (mi) and 39.76 (ma), 44.57 (ma) and 45.41 (mi),
44.71, 55.44, 64.37 (mi) and 64.46 (ma), 82.42, 124.15, 143.64,
151.59, 152.63, 158.93 (ma) and 159.25 (mi), 161.79, 173.70
(ma) and 173.91 (mi), 175.32 (ma) and 175.87 (mi), 177.47;
MS[FAB] m/z (%) 480 [(M + 1)+] (100).
Solu tion P h a se Syn th esis of Tetr a p ep tid es 25-28. (i)
Rem ova l of th e Boc P r otectin g Gr ou p s. TFA (0.5 mL/100
mg) was added dropwise to a stirred solution of 21, 22, 23, or
24 in anhydrous DCM (0.5 mL/100 mg) at rt. The reaction
was left to stir for 4 h. The reaction was then saturated with
diethyl ether until a precipitate formed; this was the TFA salt
of the free amino dipeptide. The ethereal solution was
decanted off from the precipitate, and the precipitate was
triturated twice with further quantities of diethyl ether before
being dried in a vacuum desiccator over phosphorus pentoxide.
TF A Sa lt of th e Th ym in e Der iva tive. The above proce-
dure was applied to dipeptide 21 (0.11 g, 0.26 mmol). The
desired compound was given as a white solid (0.093 g, 85%).
TF A Sa lt of th e Cytosin e Der iva tive. The above proce-
dure was applied to dipeptide 22 (0.20 g, 0.37 mmol). The
desired compound was afforded as a cream solid (0.14 g, 69%).
TF A Sa lt of th e Ad en in e Der iva tive. The above proce-
dure was applied to dipeptide 23 (0.10 g, 0.19 mmol). The
desired compound was obtained as a cream solid (0.093g, 88%).
TF A Sa lt of th e Gu a n in e Der iva tive. The above proce-
dure was applied to dipeptide 24 (0.12 g, 0.26 mmol) except
that the reaction was left for 5.5 h before workup. The desired
compound was afforded as a cream solid (0.082 g, 64%).
(ii) Hyd r olysis of th e Eth yl Ester s. A 0.67 M (aq)
solution of sodium hydroxide (2.5 equiv) was added to a stirred
solution of 21, 22, 23, or 24 (1 equiv) in dioxane (1.75 mL/
mmol) at rt. The reaction was left for 0.5 h before being diluted
with water (6.65 mL/mmol). The aqueous solution was washed
with DCM (3 × 6.65 mL/mmol) and the pH adjusted to 3.0
with a 2 M (aq) solution of citric acid. The aqueous solution
Tetr a p ep tid e 26. The above procedure was used to couple
the free carboxylic acid derivative of dipeptide 22 (0.13 g, 0.25
mmol) and the TFA salt of the free amino acid analogue of
dipeptide 22 (0.14 g, 0.25mmol). After workup the crude
residue was purified using flash chromatography with 90:10,
DCM:methanol as the eluting solvent. The tetrapeptide 26
was obtained as a pale yellow powder (0.14 g, 59%): Rf 0.36
1
(90:10, DCM:methanol); H NMR (d6-DMSO) δ 1.15 (t, 3H, J
) 7.03), 1.36 (s, 9H), 1.69-2.26 (series of m, 4H), 3.64-4.32
(series of m, 10H), 4.06 (q, 2H, J ) 7.03), 5.15 (s, 4H), 6.92 (d,
1H, J ) 7.31), 6.97 (d, 1H, J ) 7.03), 7.17 (br m, 1H), 7.37 (m,
10H), 7.88 (d, 1H, J ) 7.59), 7.97 (d, 1H, J ) 7.31), 8.12-8.54
(br series of m, 3H), 10.73 (br s, 2H); MS[FAB] m/z (%) 917
[(M + 1)+] (39).
Tetr a p ep tid e 27. The above procedure was used to couple
the free carboxylic acid derivative of dipeptide 23 (0.086 g, 0.16
mmol) and the TFA salt of the free amino acid analogue of
dipeptide 23 (0.093 g, 0.16 mmol). After workup the crude
residue was purified using flash chromatography with 85:15
DCM:methanol as the eluting solvent. The tetrapeptide 27
was obtained as a cream powder (0.038 g, 24%): Rf 0.47 (85:
1
15, DCM:methanol); H NMR (d6-DMSO) δ 1.14 (t, 3H, J )
7.08), 1.35 (s, 9H), 2.06 (series of m, 2H), 2.36 (series of m,
2H), 3.70-4.38 (series of m, 10H), 4.05 (q, 2H, J ) 7.08), 5.21
(s, 4H), 7.25 (br d, 1H, J ) 7.82), 7.30-7.46 (series of m, 10H),
8.20-8.49 (br series of m, 3H), 8.38 (br s, 2H), 8.60 (br s, 2H),
10.66 (br s, 2H); MS[FAB] m/z (%) 965 [(M + 1)+] (4).
Tetr a p ep tid e 28. The above general procedure was used
to couple the free carboxylic acid derivative of dipeptide 24
(0.062 g, 0.14 mmol) and the TFA salt of the free amino acid

5450 J . Org. Chem., Vol. 62, No. 16, 1997
Howarth and Wakelin
analogue of dipeptide 24 (0.068 g, 0.14 mmol). After workup
the crude residue was purified using flash chromatography
with 75:25, DCM:methanol as the eluting solvent. The tet-
rapeptide 28 was obtained as a beige powder (0.011 g, 10%):
Rf 0.68 (75:25, DCM:methanol); 1H NMR (d6-DMSO) δ 1.15
(t, 3H, J ) 7.03), 1.37 (s, 9H), 1.89-2.34 (series of m, 4H),
2.16 (s, 6H), 3.64-4.32 (series of m, 10H), 4.06 (q, 2H, J )
7.03), 7.16 (d, 1H, J ) 8.16), 7.86 (s, 2H), 8.12-8.50 (br series
of m, 3H), 11.72 (br t , 2H), 12.02 (br s, 2H); MS[FAB] m/z (%)
814 [(M + 2)+] (19).
Solid P h a se Oligom er iza tion . Step w ise Assem bly of
H -rCLysrTGlyrCGlyrTLysrCGlyrCGlyrTLysrTGly-
rTGlyrTLys-NH2.20 The protected R-PNA was assembled
manually on a Boc-Lys (2-Cl-Cbz) derivatized MBHA resin in
a stepwise manner. The synthesis was initiated on 50 mg (dry
weight) Boc-Lys (2-Cl-Cbz)-MBHA resin, which was preswollen
overnight in DMF. The procedure is as follows: (1) Boc
deprotection with TFA/m-cresol (95:5, v/v), 2 × 4 min. (2)
Washing with DMF/DCM (1:1, v/v), 3 × 20 s; washing with
pyridine, 2 × 20 s. (3) Coupling using a 0.1 M solution of Boc-
amino acid (i.e. 6, 10, Boc-Lys (2-Cl-Cbz)-OH or Boc-Gly-OH)
in DMF/pyridine (3:1) containing HBTU (0.95 equiv) and DIEA
(1.1 equiv). The mixture was preactivated for 1 min prior to
coupling, and coupling was allowed to proceed for 20 min at
rt. (4) Washing with DMF, 2 × 20 s. (5) Capping of unreacted
amino groups using Ac2O/DMF/collidine (1:8:1, v/v/v), 1 × 5
min. (6) Washing with DMF, 3 × 20 s; washing with 5%
piperidine in DMF, 1 × 4 min; washing with DMF/DCM (1:1,
v/v), 3 × 20 s. Steps 1-6 were repeated until the desired
sequence was obtained. All the couplings were monitored
using Kaiser tests40 and all were negative. After the final
coupling, the resin was dried under vacuum, and the R-PNA
oligomer was concomitantly deprotected and cleaved from the
resin using the TFMSA procedure described by Christensen
et al.:37 yield 10.5 mg (crude: purity > 80% by MS analysis);
MALDI-TOF MS: calcd average mass 2904.08, found 2901.9
[M + H]+, 2926.0 [M + Na]+. Analytical reverse phase HPLC
recorded a single peak at 14.97 min (λmax ) 260 nm).
Ack n ow led gm en t. The authors gratefully acknowl-
edge Professor Peter E. Nielsen and Mr. Leif Chris-
tensen of The Panum Institute, Copenhagen N., Den-
mark, for their assistance with the solid phase
oligomerization and for many useful discussions. This
work was supported by St. Luke’s Institute of Cancer
Research. NMH is a Health Research Board postdoc-
toral fellow.
J O970111P
(40) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 84, 595.