A convenient route to N-[2-(Fmoc)aminoethyl]glycine esters and PNA oligomerization using a bis-N-Boc nucleobase protecting group strategy

Article abstract of DOI:10.1021/jo800195j

(Chemical Equation Presented) A simple and practical synthesis of the benzyl, allyl, and 4-nitrobenzyl esters of N-[2-(Fmoc)aminoethyl]glycine is described starting from the known N-(2-aminoethyl)glycine. These esters are stored as stable hydrochloride salts and were used in the synthesis of peptide nucleic acid monomers possessing bis-N-Boc-protected nucleobase moieties on the exocyclic amino groups of ethyl cytosin-1-ylacetate, ethyl adenin-9-ylacetate and ethyl (O⁶-benzylguanin-9-yl)acetate. Upon ester hydrolysis, the corresponding nucleobase acetic acids were coupled to N-[2-(Fmoc)aminoethyl] glycine benzyl ester or to N-[2-(Fmoc)aminoethyl]glycine allyl ester in order to retain the O⁶ benzyl ether protecting group of guanine. The Fmoc/bis-N-Boc-protected monomers were successfully used in the Fmoc-mediated solid-phase peptide synthesis of mixed sequence 10-mer PNA oligomers and are shown to be a viable alternative to the currently most widely used Fmoc/Bhoc-protected peptide nucleic acid monomers.

Full text of DOI:10.1021/jo800195j

A Convenient Route to N-[2-(Fmoc)aminoethyl]glycine Esters and
PNA Oligomerization Using a Bis-N-Boc Nucleobase Protecting
Group Strategy
Filip Wojciechowski and Robert H. E. Hudson*
Department of Chemistry, The UniVersity of Western Ontario, London, Ontario, Canada N6A 5B7
robert.hudson@uwo.ca
ReceiVed January 24, 2008
A simple and practical synthesis of the benzyl, allyl, and 4-nitrobenzyl esters of N-[2-(Fmoc)amino-
ethyl]glycine is described starting from the known N-(2-aminoethyl)glycine. These esters are stored as
stable hydrochloride salts and were used in the synthesis of peptide nucleic acid monomers possessing
bis-N-Boc-protected nucleobase moieties on the exocyclic amino groups of ethyl cytosin-1-ylacetate,
ethyl adenin-9-ylacetate and ethyl (O6-benzylguanin-9-yl)acetate. Upon ester hydrolysis, the corresponding
nucleobase acetic acids were coupled to N-[2-(Fmoc)aminoethyl]glycine benzyl ester or to N-[2-
(Fmoc)aminoethyl]glycine allyl ester in order to retain the O6 benzyl ether protecting group of guanine.
The Fmoc/bis-N-Boc-protected monomers were successfully used in the Fmoc-mediated solid-phase peptide
synthesis of mixed sequence 10-mer PNA oligomers and are shown to be a viable alternative to the
currently most widely used Fmoc/Bhoc-protected peptide nucleic acid monomers.
Introduction
non-natural nucleobases.7 All of these endeavors depend on
accessible methods of PNA monomer synthesis and especially
so for the synthesis of PNA bearing additional functional groups.
As part of our current research, we are interested in the
synthesis of nucleobase modified PNA by modifying the non-
Watson/Crick base-pairing face of pyrimidines with charged,
hydrophilic, luminescent, and potential helix-stabilizing sub-
stituents.8 The synthesis of such modified nucleobase mono-
mers relies on the assembly of a protected nucleobase acetic
acid derivative with a suitably protected N-(2-aminoethyl)
glycine backbone. Appending charged or hydrophilic groups
onto the nucleobase is sometimes difficult to carry out due
to the required orthogonal protection. Synthetic routes toward
N-[2-(Boc)aminoethyl]glycine esters have been extensively
investigated since the introduction of Boc/Cbz oligomeriza-
tion of PNA, giving an assortment of synthetic methods
Peptide nucleic acid (PNA) is an achiral and uncharged
nucleic acid analogue constructed from a polyamide backbone
composed of N-(2-aminoethyl)glycine to which the nucleobases
are attached by a methylene carbonyl group.1 PNA oligomers
hybridize to cDNA and cRNA, obeying Watson-Crick base-
pairing rules, with very high affinity and sequence specificity
and are resistant to nucleases and proteases. Because of these
remarkable properties, PNA has received much attention. PNA
oligomers are intensively pursued and applied as probes in
clinical diagnostics2,3 and in potential therapeutic applications
as antisense/antigene agents.4 Due to the modular nature of PNA,
many backbone-modified PNA analogues have been syn-
thesized.5,6 PNA has also stimulated the synthesis of numerous
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991,
254, 1497–1500.
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(3) Leijon, M.; Mousavi-Jazi, M.; Kubista, M. Mol. Aspects. Med. 2006, 27,
160–175.
(6) Porcheddu, A.; Giacomelli, G. Curr. Med. Chem. 2005, 12, 2561–2599.
(7) Wojciechowski, F.; Hudson, R. H. E. Curr. Top. Med. Chem. 2007, 7,
667–679.
(4) Nielsen, P. E. Curr. Med. Chem. 2001, 8, 545–550.
(5) Ganesh, K. N.; Nielsen, P. E. Curr. Org. Chem. 2000, 4, 931–943.
(8) Hudson, R. H. E.; Viirre, R. D.; Liu, Y. H.; Wojciechowski, F.;
Dambenieks, A. K. Pure Appl. Chem. 2004, 76, 1591–1598.
10.1021/jo800195j CCC: $40.75
Published on Web 04/16/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 3807–3816 3807

Wojciechowski and Hudson
toward such molecules.9–11 Conversely, synthetic routes
toward N-[2-(Fmoc)aminoethyl]glycine esters are less well
documented since the first report on Fmoc-mediated synthesis
of PNA by Thomson et al. in 1995.12 Currently there are
three Fmoc backbones in use: methyl ester, allyl ester and
tert-butyl ester of N-[2-(Fmoc)aminoethyl]glycine. The methyl
ester has not been extensively used since partial removal of
the Fmoc group is a hazard during its hydrolysis. Use of the
methyl ester then requires tedious optimization of the
hydrolysis conditions13,14 or the subsequent addition of excess
Fmoc-OSu to reintroduce the Fmoc group.9 The orthogonal
allyl ester has previously been synthesized by esterification
of N-[2-(Fmoc)aminoethyl]glycine15 or N-(2-aminoethyl)g-
lycine dihydrochloride,13 but both routes are inefficient giving
the product in an overall low yield. Likewise, the most widely
used tert-butyl ester is synthesized in low yield (54%) by
reacting N-(2-aminoethyl)glycine tert-butyl ester with Fmoc-
OSu.12 Furthermore, the most convenient removal of the tert-
butyl ester is by acidolysis and therefore limited to nucleo-
bases and other compounds that contain no acid labile groups.
Since several common amino protecting groups (Boc, Mmt,
Bhoc) are acid labile, the removal of the tert-butyl ester is
accomplished with unwanted removal of the amino protecting
groups or in low yield as illustrated in the recent literature.16,17
Therefore, a convenient synthetic route to N-[2-(Fmoc)ami-
noethyl]glycine esters would advance the synthesis of
molecules that were previously difficult to synthesize and
allow for modified nucleobases or reporter groups to be more
easily introduced into PNA by Fmoc-mediated oligomerization.
the Fmoc/Cbz-,12 Fmoc/Mmt-,21 and Fmoc/Bhoc-protected
monomers were used for the synthesis of PNA-peptide
conjugates.
Solid-phase peptide synthesis employing Fmoc chemistry
usually involves the use of commercially available amino acids,
such as lysine or ornithine, which have their side-chain amino
groups Boc-protected. PNA is often appended with lysine at
the N- or C-terminus in order to improve aqueous solubility
and reduce self-aggregation. Although the side-chain amino
group of lysine is protected with the Boc group, a strategy for
the oligomerization of PNA using Boc protection of the
nucleobases has not yet been reported. Only a brief description
of Fmoc/Boc monomer synthesis has appeared in the literature,
but it does neither include detailed experimental procedures nor
demonstrate the oligomerization of the monomers.22Although
the Boc group has been used intermittently in nucleobase and
nucleoside chemistry and for many years, there are only a few
examples that are directly related to the PNA-based work
reported herein. A bis-N-Boc-protected adenine PNA monomer
was first utilized by Condom and co-workers for the solution-
phase synthesis of a PNA dimer; however, this report contained
only a brief description of experimental conditions which were
disclosed later.23a,b Much later, the same group communicated
the use of a bis-N-Boc-protected cytosine PNA acetic acid
derivative for the solution-phase synthesis of a PNA dimer, but
without disclosing the experimental details.23c More recent work
from Condom’s group has used Cbz-protected cytosine.23d Some
relevant work on the use of the Boc-protecting group for purines
was reported by Garner and co-workers, presumably in support
of their research into R-helical peptide nucleic acids (R-PNA),
wherein bis-N-Boc adenine and mono-N-Boc guanine were
prepared. These nucleobase derivatives were not alkylated at
N9 or used further for the synthesis of PNA-like monomers.24
Finally, Hultin and Sikchi quite recently reported an elegantly
simple solventless approach to bis-N-Boc protected nucleosides
which also indicated the feasibility of our approach reported
herein.25
The solid-phase peptide synthesis of PNA was first
accomplished by Boc/Cbz-protected building blocks where
the Boc group is used for the protection of the amino group
of the backbone and the Cbz for protection of the exocyclic
amino groups of the nucleobases cytosine and adenine, while
guanine was protected with the O6-benzyl group.18 In part
due to the harsh conditions required to remove the Cbz
groups, most commonly HF or TFMSA, other protecting
group strategies were investigated to enable the synthesis of
PNA-peptide or PNA-oligonucleotide conjugates which
demanded milder deprotection conditions. The Mmt/acyl19
and Fmoc/acyl20 monomers were prepared and evaluated for
the preparation of PNA-oligonucleotide conjugates, while
To date, there exists no attempt at the synthesis of Fmoc/
bis-N-Boc PNA monomers for the oligomerization of PNA. We
have decided to pursue the bis-N-Boc protection of the adenine,
guanine, and cytosine for the following reasons: (i) Fmoc/Bhoc
monomers are expensive and have limited commercial sources,26
(ii) introduction of the Bhoc group onto the nucleobases requires
expensive reagents such as carbonyldiimidazole and triphosgene,
(iii) the Bhoc group is unstable toward hydrogenation or mild
acidolysis rendering the Fmoc monomer synthesis difficult, (iv)
both the bis-N-Boc protecting group and Boc group may be
removed under neutral conditions or in a single acidolysis step
which is compatible with a convenient single deprotection/
cleavage postsynthetic step, (vi) bis-N-Boc-protected monomers
(9) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int.
Ed. 1998, 37, 2823.
(10) Kofoed, T.; Hansen, H. F.; Orum, H.; Koch, T. J. Peptide Sci. 2001, 7,
402–412.
(11) Viirre, R. D.; Hudson, R. H. E. J. Org. Chem. 2003, 68, 1630–1632.
(12) Thomson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.; Hassman, F.;
Luzzio, M. J.; Pipe, A. J.; Reed, K. L.; Ricca, D. J.; Wiethe, R. W.; Noble,
S. A. Tetrahedron 1995, 22, 6179–6194.
(13) Hudson, R. H. E.; Goncharenko, M.; Wallman, A. P.; Wojciechowski,
F. Synlett 2005, 9, 1442–1446.
(21) Breipohl, G.; Knolle, J.; Langner, D.; Omalley, G.; Uhlmann, E. Bioorg.
Med. Chem. Lett. 1996, 6, 665–670.
(14) Hudson, R. H. E.; Liu, Y. H.; Wojciechowski, F. Can. J. Chem. 2007,
85, 302–312.
(22) Sugiyama, T.; Kittaka, A.; Takemoto, Y.; Takayama, H.; Kuroda, R.
Nucleic. Acids. Res. Suppl. 2002, 2, 145–146.
(15) Seitz, O.; Ko¨hler, O. Chem. Eur. J. 2001, 18, 3911–3925.
(16) Ausin, C.; Ortega, J. A.; Robles, J.; Grandas, A.; Pedroso, E. Org. Lett.
2002, 4, 4073–4075.
(23) (a) Farese, A.; Patino, N.; Condom, R.; Dalleu, S.; Duedj, R. Tetrahedron
Lett. 1996, 37, 1413–1416. (b) Farese, A.; Pairot, S.; Patino, N.; Ravily, V.;
Condom, R.; Aumelas, A.; Guedj, R. Nucleosides Nucleotides 1997, 16, 1893–
1906. (c) Leroux, M.-L.; Di Giorgio, C.; Patino, N.; Condom, R. Tetrahedron
Lett. 2002, 43, 1641–1644. (d) For example: Upert, G.; Mehiri, M.; Goddard,
M.-L.; Di Giorgio, A.; Benhida, R.; Condom, R.; Patino, N. Tetrahedron Lett.
2005, 46, 4081–4085.
(17) Timar, Z.; Kovacs, L.; Kovacs, G.; Schmel, Z. J. Chem. Soc., Perkin
Trans. 1 2000, 1, 19–26.
(18) 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.
(24) Dey, S.; Garner, P.; Dey, S.; Garner, P. J. Org. Chem. 2000, 65, 7697–
7699.
(19) Breipohl, G.; Will, D. W.; Peyman, A.; Uhlmann, E. Tetrahedron 1997,
53, 14671–14686.
(25) Sikchi, S.; Hultin, P. G. J. Org. Chem. 2006, 71, 5888–5891.
(26) Available from ASM Research Chemicals. www.asm-research-chemi-
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J. Chem. Soc., Perkin Trans. 1 2002, 1266–1270.
3808 J. Org. Chem. Vol. 73, No. 10, 2008

ConVenient Route to N-[2-(Fmoc)aminoethyl]glycine Esters
are highly lipophilic and do not possess any amide functionality
which is expected to improve monomer solubility and reduce
self-aggregation of these residues during oligomerization.
Synthesis of PNA oligomers by alternative terminal amino
protecting groups has also received attention. Protective group
strategies that have recently appeared include: Dde/Mmt,27
NVOC/acyl,28 and azide/Bhoc.29 Of late, the synthesis of PNA
oligomers using novel self-activated PNA monomers has been
reported. Although this strategy allows the synthesis of PNA
oligomers in excellent purity, it requires 20 equiv of the Bhoc
protected cytosine, guanine, and adenine monomers for each
coupling and extended coupling times.30 This is a severe
drawback since the incorporation of custom-made nucleobases
or reporter groups would waste a significant amount of the
monomer.
toluenesulfonic acid and the removal of water is achieved by
azeotropic distillation using benzene or toluene,32 and this is
the approach that we have adopted.
Our route to N-[2-(Fmoc)aminoethyl]glycine esters began
with the alkylation of ethylenediamine by chloroacetic acid to
give N-(2-aminoethyl)glycine,31 which was isolated by precipi-
tation from a DMSO/ethanol/ether mixture.33 Treatment of N-(2-
aminoethyl)glycine with p-toluenesulfonic acid under reflux
gave the p-toluenesulfonate salt of N-(2-aminoethyl)glycine after
solvent removal under reduced pressure and precipitation with
ether. Compound 1 may be easily synthesized on a large scale
(ca. 75 g); in contrast to N-(2-aminoethyl)glycine, it is not
hygroscopic and stable to storage for extended periods. This
material allows for the synthesis of N-[2-(Fmoc)aminoethyl]g-
lycine esters with benzyl alcohol, 4-nitrobenzyl alcohol, and
allyl alcohol, compounds 2, 3, and 4, respectively, in excellent
yield and without the need for an exogenous acid. As reported
by Thompson et al., the introduction of the Fmoc group can
proceed with low yield (53%) onto tert-butyl N-(2-aminoeth-
yl)glycinate.12 Likewise, our previous attempts at introducing
the Fmoc group onto allyl N-(2-aminoethyl)glycinate dihydro-
chloride gave a low yield (63%).13 This low yield may be due
to the low solubility of aminoethylglycine ester dihydrochloride,
lactam formation, or extending the reaction time to longer than
12 h may cause Fmoc removal in the presence of Hu¨nig’s base.
We reasoned that introduction of the Fmoc group onto the
primary amine of 2 in the presence of an appropriate base with
an excess of Fmoc-OSu under concentrated conditions would
drive the reaction to completion at the primary amino group
and any di-Fmoc product could be removed in the workup.
Instead of neutralizing compound 2 followed by dropwise
addition of Fmoc-OSu, which could lead to lactam formation,
we decided to take advantage of the appreciable solubility of 2
in organic solvents and pursue in situ neutralization of 2 in the
presence of 1.1 equivalents of Fmoc-OSu. Initially, introduction
of the Fmoc group onto 2 with Fmoc-OSu was performed in a
dioxane/water mixture with the dropwise addition of 4.0 equiv
of sodium bicarbonate to give the desired product in a modest
yield of 66%. The yield improved significantly by using 3.3
equiv of Hu¨nig’s base (N,N-diisopropylethylamine) instead of
sodium bicarbonate and performing the reaction in THF. The
reaction was arbitrarily decided to be complete within 1.5 h
giving compound 5 in 85% yield. Workup involved filtering
through a short plug of silica to remove any polar impurities
and isolating 5 as the hydrochloride salt by the dropwise addition
of HCl in ether. The last step may be substituted for column
chromatography to remove any unreacted Fmoc-OSu. Likewise,
6 is isolated in good yield, thereby improving on previous
procedures for the synthesis of the allyl backbone, which
required the synthesis of tert-butyl N-[2-(Fmoc)aminoethyl]-
glycinate followed by acidolysis and re-esterification.15 The
4-nitrobenzyl ester, as contained in 7, may be removed by
hydrogenation or mildly acidic reducing conditions (Zn/acetic
acid).34 This protecting group could find use in the rare case
that a compound or non-natural nucleobase contains protecting
groups susceptible to hydrogenation, acidolysis, and deallylation.
Results and Discussion
Synthesis of Fmoc-protected PNA monomers containing
natural and non-natural nucleobases may be divided into the
synthesis of the protected nucleobase-substituted acetic acids
and N-[2-(Fmoc)aminoethyl]glycine esters. First, we desired to
devise a practical route to 2-aminoethylglycine derivatives that
would ultimately possess orthogonal ω-amino-, carboxyl-, and
nucleobase-protecting groups. For convenience, this scheme was
to result in PNA monomers compatible with standard Fmoc-
based oligomerization chemistry. Thus, the Fmoc group and
acid-labile nucleobase protecting groups were already specified,
and we focused on carboxyl protecting groups that could be
removed under neutral or other mild conditions.
Our initial attempt at the synthesis of benzyl N-[2-(Fmoc)am-
inoethyl]glycinate involved making N-(2-aminoethyl)glycine
benzyl ester, which would be reacted with Fmoc-OSu to form
the desired product. Following the procedure of Thomson et
al. for the synthesis of tert-butyl N-(2-aminoethyl)glycinate,12
we attempted to alkylate an excess ethylenediamine with benzyl
bromoacetate. Unfortunately, upon aqueous workup, no benzyl
N-(2-aminoethyl)glycinate was isolated. Presumably, 1 equiv
of ethylenediamine reacts with benzyl bromoacetate to form the
desired product, which in the presence of excess ethylenediamine
can further undergo cyclization resulting in lactam formation.
Alternatively, the disubstituted water-soluble N-(2-aminoethyl)-
2-(2-aminoethylamino)acetamide could form in the presence of
excess ethylenediamine. As an alternative approach, the esteri-
fication of N-(2-aminoethyl)glycine31 was pursued. Previously,
we have described the esterification of N-(2-aminoethyl)glycine
in an excess of allyl alcohol saturated with HCl(g) to give the
allyl ester in 98% yield.13 Unfortunately, esterification with
benzyl alcohol saturated with HCl(g) failed to give the desired
benzyl ester and only N-(2-aminoethyl)glycine dihydrochloride
was isolated. It is suspected that the benzyl ester failed to form
due to the poor solubility of N-(2-aminoethyl)glycine dihydro-
chloride in benzyl alcohol coupled with the inability to remove
water from the reaction mixture. Classically, in peptide chem-
istry, this problem was remedied by replacing HCl with p-
1
Compounds 1-4 were characterized by H and 13C NMR but
(27) Bialy, L.; Diaz-Mochon, J. J.; Specker, E.; Keinicke, L.; Bradley, M.
Tetrahedron 2005, 8295–8305.
resisted characterization by mass spectrometry. Protecting the
(28) Liu, Z.-C.; Shin, D.-S.; Lee, K.-T.; Jun, B.-H.; Kim, Y.-K.; Lee, Y.-S.
Tetrahedron 2005, 61, 7967–7973.
(29) Dabaene, F.; Winssinger, N. Org. Lett. 2003, 5, 4445–4447.
(30) Lee, H.; Jeon, J. H.; Lim, J. C.; Choi, H.; Yoon, Y.; Kim, S. K. Org.
Lett. 2007, 9, 4503–4506.
(32) Bodanszky, M. Principles of Peptide Synthesis, Ed.; Springer-Verlag:
Berlin, 1993, pp 75-78.
(33) Byk, G.; Gilon, C. J. Org. Chem. 1992, 57, 5687–5692.
(34) Green, T. W.; Wuts, P. G. M. ProtectiVe Groups in Organic Synthesis;
Wiley-Interscience, New York, 1999; pp 518-525.
(31) Heimer, E. P.; Gallo-Torres, H. E.; Felix, A. M.; Ahmad, M.; Lambros,
T. J.; Scheidl, F. Int. J. Pep. Protein Res. 1984, 23, 203–211.
J. Org. Chem. Vol. 73, No. 10, 2008 3809

Wojciechowski and Hudson
SCHEME 1. Synthesis of Fmoc Backbones
SCHEME 2. Synthesis of Bis-N-Boc-Protected Cytosine
SCHEME 3. Synthesis of Bis-N-Boc-Protected Adenine
amino group of 2, 3, and 4 gave the desired Fmoc backbones
5, 6, and 7, respectively, which were amenable to mass
spectroscopic characterization, Scheme 1.
We now routinely use these backbones, particularly N-[2-
(Fmoc)aminoethyl]glycine benzyl ester, in the synthesis of novel
nucleobases containing appended amino groups protected by
Boc groups. Catalytic hydrogenation proceeds readily and in
very good yield without any detectable loss of the Fmoc group.
The Boc group which is used commonly as an amino protecting
group in peptide and heterocyclic chemistry is stable to
hydrogenation and is usually removed with acid (TFA or HCl)
giving t-BuOH or isobutylene and CO2 as the byproduct.34
Furthermore, the bis-N-Boc group may be selectively converted
to the mono-N-Boc derivative, and if so desired, the remaining
Boc group may be removed without the use of acid under neutral
conditions.34,35
While we decided to pursue the synthesis of bis-N-Boc-
protected cytosine, adenine, and guanine for reasons already
outlined, our main objective in this paper was to investigate
whether the introduction of this lipophilic protecting group
would occur readily on the exocyclic amino groups of the
nucleobases and if the bis-N-Boc protected nucleobases are
compatible with Fmoc oligomerization, thereby providing a
useful alternative to Bhoc protection.
Alkylation of cytosine and adenine with ethyl bromoacetate
was performed prior to bis-N-Boc protection of the exocyclic
amino group due to the increased organic solubility of the
alkylated nuclobases and to lower the amount of (Boc)2O
needed for protection. The alkyation of cytosine was
performed in the same fashion as described for the synthesis
of methyl cytosin-1-ylacetate,36 using NaH and ethyl bro-
moacetate to yield ethyl cytosin-1-ylacetate 8. Compound 8
was reacted with a catalytic amount of DMAP, 2.2 equiv of
(Boc)2O, and triethylamine under reflux to give 9 in excellent
yield (89%). The ethyl ester was hydrolyzed using 8 equiv
of NaOH in a dioxane:water mixture to give 10 in near-
quantitative yield (Scheme 2).
in DMF or THF to give the bis-N-Boc-protected compounds in
57% and 62% yield, respectively. Under these conditions, we
obtained 12 in 66% yield. In order to reduce the amount of
(Boc)2O and DMAP, ethyl adenin-9-ylacetate 1119 was refluxed
with a catalytic amount of DMAP, 2.2 equiv of (Boc)2O, and
triethylamine to give 12 in 68% yield. Compound 12 was
hydrolyzed to give 13 in 90% yield (Scheme 3).
With the uncomplicated synthesis of compounds 10 and 13
established, we turned our attention to the Boc-protection of
guanine, which was expected to be the most challenging
nucleobase to derivatize. Direct alkylation of guanine, which
itself has very poor organic solubility, is known to give a
complex reaction mixture including both N9- and N7-alkylated
regioisomers.12 To circumvent the low solubility and poor
regioselectivity, other derivatives such as 2-amino-6-chloropu-
rine, N2-acetyl-O6-diphenylcarbamoylguanine,38 and N2-isobu-
tyrylguanine have been used.28 Synthesis of 14 was performed
as described in the literature by reacting N2-acetyl-O6-diphe-
nylcarbamoylguanine with ethyl bromoacetate in the presence
of Hu¨nig’s base.21 Compound 14 contains the dipenylcarbamoyl
protecting group (Dpc) which can be removed by either TFA21
or NH3(aq)/MeOH.38 Removing the acetyl group in 14 and
reacting the exocyclic amino group with (Boc)2O would give
our desired bis-N-Boc protected guanine. Unfortunately, our
attempts to remove the acetyl group by treatment with EtOH/
NEt3 at reflux failed, and only starting material was recovered.
We therefore decided to remove the Dpc group by use of TFA
and subsequent reflux of the intermediate N2-acetyl-9-ethylcar-
The introduction of bis-N-Boc onto N9-alkylated adenine has
been previously reported in the liquid-phase synthesis of a PNA
dimer23a or in the synthesis of lipophilic PNA containing a 1,3-
diyne group.37 In both cases the bis-N-Boc protecting group
was introduced using 3 equiv of (Boc)2O and 3 equiv DMAP
(35) Stafford, J. A.; Brackeen, M. F.; Karanewsky, D. S.; Valvano, N. L.
Tetrahedron Lett. 1993, 34, 7873–7876.
(36) Schwergold, C.; Depecker, G.; Di Giorgio, C.; Patino, N.; Jossinet, F.;
Ehresmann, B.; Terreux, R.; Cabrol-Bass, D.; Condom, R. Tetrahedron 2002,
58, 5675–5687.
(37) Howarth, N. M.; Lindsell, E.; Murray, E.; Preston, P. N. Tetrahedron
2005, 61, 8875–8887.
(38) Zou, R.; Robins, M. J. Can. J. Chem. 1987, 65, 1436–1437.
3810 J. Org. Chem. Vol. 73, No. 10, 2008

ConVenient Route to N-[2-(Fmoc)aminoethyl]glycine Esters
SCHEME 4. Synthesis of Compound 16
SCHEME 6. Bis-N-Boc Protection of 20
SCHEME 5. Synthesis of Compound 19
hydrogenation or acidolysis with TFA. Although reaction of
ethyl [2-amino-6-(benzyloxy)purin-9-yl]acetate 20 with benzy-
loxycarbonyl chloride or “Rapoport’s reagent” was unsuccessful,
treatment of 20 with the capping reagent used in the oligomer-
ization (acetic anhydride/pyridine/CH2Cl2 1:2:2) for 1.5 h led
to 23% of the acylated product.18 The protection of the exocyclic
amino group has been also investigated by Koch and co-workers
who treated [2-amino-6-(benzyloxy)purin-9-yl]acetic acid with
isobutyryl chloride to give the product in 30%.10 The protection
was improved by avoiding the use of the carboxylic acid
derivative and treating compound 20 with isobutyryl chloride
in pyridine to give ethyl [N2-(isobutyryl)-O6-(benzyl)guanin-9-
yl]acetate in 80% yield. Recently the high yielding synthesis
of O6-benzyl guanine has been described.41 We have decided
not to start with this reagent because its alkylation with ethyl
bomoacetate will most likely proceed with poor regioselectivity
for N9/N7. Therefore, 20 was synthesized using 2-amino-6-
chloropurine as described in the literature.10 Compound 20 was
bis-N-Boc-protected in 65% yield using 2.2 equiv of (Boc)2O
and DMAP to give 21, which upon hydrolysis gave 22 in 95%
(Scheme 6).
boxymethylguanine in EtOH/NEt3 gave 15 in 65% yield. This
illustrates that under identical conditions, removal of the acetyl
group proceeds easily in the lactam structure of guanine but
not in the lactim form. Next, 15 was reacted with an excess of
(Boc)2O in an attempt to form N2,N2-(bis-Boc)-9-ethylcar-
boxymethylguanine; unfortunately, only compound 16 was
isolated (Scheme 4).
Although compound 16 was considered a good candidate as
a lipophilic protected guanine, it was isolated in a disappointing
yield of 36%. All attempts to improve the yield for the synthesis
of 16 were unsuccessful. To overcome the failure of introducing
the tris-Boc groups, compound 1736 was reacted with (Boc)2O
in the presence of DMAP to give 18 in 51% yield. Refluxing
the reaction did not improve the yield possibly due to the
consumption of DMAP by its reaction with 6-Cl group of 17
giving the 6-dimethylaminopyridinium derivative.39 It is known
that 6-chloropurines react with 3-hydroxypropionitrile to give
the guanine derivative.40 Unfortunately, converting 18 to N2,N2-
(bis-Boc)-9-carboxymethylguanine resulted in displacement of
one of the Boc groups leading to compound 19 (Scheme 5),
which has poor organic solubility.
Exhausting the routes to bis-N-Boc-protected guanine by way
of compounds 14 and 17, we decided to work with guanine in
the lactim form in order to focus on Boc protection at the
exocyclic amino group. We reasoned that guanine protected at
the 6-position with a benzyl group would be a feasible approach.
The first report of PNA oligomerization used this protecting
group strategy,18 the benzyl ether may be removed by either
The attachment of compounds 10 and 13 onto N-[2-
(Fmoc)aminoethyl]glycine benzyl ester and compound 22 onto
allyl N-[2-(Fmoc)aminoethyl]glycinate was accomplished by
neutralizing the hydrochloride salts to give the free bases 5 and
6 prior to coupling. Coupling of 5 or 6 to the backbone was
accomplished using EDC, DCC/NHS, or EDC/HOBt. Removal
of the benzyl ester of 23 was accomplished using Pd/C and
1,4-cyclohexadiene42,43 to give monomer 26 in 89%. Removal
of the benzyl ester from 24 using Pd/C and H2 gave the desired
monomer 27 in 94% (Scheme 7).
Compound 22 was coupled to the allyl protected backbone
6 in order to retain the O6 benzyl ether as a lipophilic group.
Removal of the allyl group in 25 was achieved using a
catalytic amount of Pd(PPh3)4 and N-methylmorpholine as
the scavenger to give 28 in 84% (Scheme 8). Alternatively,
in order to avoid the use of the allyl ester, 22 may be coupled
to 5 and hydrogenation of both the benzyl ester and O6 benzyl
ether would give the bis-N-Boc-protected guanine monomer.
In order to investigate whether the monomers were compatible
with Fmoc-mediated oligomerization and if the removal of the
protecting groups occurred readily, the newly synthesized
(39) Schirrmacher, R.; Wangler, B.; Schirrmacher, E.; August, T.; Rosch,
F. Synthesis 2002, 4, 538–542.
(41) Barth, C.; Seitz, O.; Kunz, H. Z. Naturforsch. 2004, 59b, 802–806.
(42) Watkins, B. E.; Kiely, J. S.; Rapoport, H. J. Am. Chem. Soc. 1982,
104, 5702–5708.
(40) Ashwell, M.; Bleasdale, C.; Golding, B. T.; O‘Neill, I. K J. Chem. Soc.,
Chem. Commun. 1990, 95, 5–957.
(43) Johnson, D.C., II; Widlanski, T. S. Org. Lett. 2004, 6, 4643–4646.
J. Org. Chem. Vol. 73, No. 10, 2008 3811

Wojciechowski and Hudson
TABLE 1. ESI-TOF MS Analysis of Oligomers
SCHEME 7. Monomer Synthesis: Coupling of 10 and 13
with 5
entry
sequencea (N f C)
expected (Da) found (Da)
1
2
3
H-GTA GAT CAC T-Lys-NH2
H-GTA GAT CAC T-Lys-NH2
H-GTA GAT CAC T-Lys-NH2
2854.8
2854.8
2854.8
2854.75
2854.88
2854.01
a PNA sequences possess
a free N-terminal amino group and
C-terminal amide. Bold type indicates use of bis-N-Boc-protected
monomers.
The monomers performed equivalently to the conventional
Fmoc/Bhoc monomers during the machine-assisted synthesis.
The crude oligomers were analyzed and subsequently purified
by RP-HPLC. The identity of the oligomers was confirmed by
ESI-MS. Analysis of the crude product mixture indicated that
the desired oligomer represented >90% of the material (by
HPLC area, Figure S1, Supporting Information), which leads
to an average cycle (deprotection and coupling) yield of >99%
per monomer.
Conclusion
We have described a convenient method for the synthesis of
N-[2-(Fmoc)aminoethyl]glycine esters (benzyl, allyl, 4-nitroben-
zyl) starting with inexpensive N-(2-aminoethyl)glycine. The
syntheses of both N-[2-(Fmoc)aminoethyl]glycine benzyl ester
and N-[2-(Fmoc)aminoethyl]glycine 4-nitrobenzyl ester have not
yet been described in the literature, and we have improved the
synthesis of allyl N-[2-(Fmoc)aminoethyl]glycinate. Both es-
terification of 1 and introduction of the Fmoc group onto 2-4
proceeds in very good yield. These backbones will find use in
the synthesis of modified reporter groups or modified nucleo-
bases that were previously difficult to make or altogether not
attempted due to the lack of orthogonal protecting groups present
in the backbone and nucleobase. Introduction of the bis-N-Boc
as a nucleobase protecting group strategy proceeds easily with
alkylated cytosine and in excellent yield. Likewise protection
of N9-alkylated adenine is performed in good yield. The
protection of guanine proved to be most challenging but was
accomplished with the aid of the O6 benzyl ether protecting
group to give a fully protected guanine derivative. Removal of
the benzyl ester in 23 and 24 is achieved with Pd/C and H2 or
Pd/C and 1,4-cyclohexadiene. Monomers 26, 27, and 28 were
incorporated into a PNA oligomer and are compatible with
commercially available Bhoc-protected monomers or may
substitute completely for them with no modification to the
standard automated cycles and conditions.
SCHEME 8.
Monomer Synthesis: Coupling of 22 onto 6
During solid-phase PNA oligomer synthesis, aggregation of
growing polyamide chains is a commonly encountered problem
which leads to inaccessibility of the terminal Fmoc group to
piperidine deprotection. Besides extended deprotection times
that are sometimes required, the on-resin aggregation may result
in the formation of truncated oligomers as impurities that
complicate the purification of the final product or even to
complete failure of the synthesis.45 It is also known that the
Fmoc/Bhoc-protected cytosine and guanine monomers have
limited organic solubility and often require sonication for full
monomers were incorporated into a 10-mer PNA sequence.
Oligomers were synthesized under identical conditions whether
using either Fmoc/Bhoc- or Fmoc/bis-N-Boc-based monomers
and employed standard conditions.44 The consecutive incorpora-
tion of monomers 26, 27, and 28 was performed in order to
judge if the monomers were compatible with commercially
Bhoc-protected monomers (Table 1).
(44) PNA synthesis followed the manufacturer’s guidelines for small-scale
synthesis (5 µmol) employing FastMoc cycles on an Applied Biosystems 433A
peptide synthesizer. This protocol uses 5 equiv of monomer. The preactivation
time for the monomers was 120 s. ABI 433A Peptide Synthesis 3 mL reaction
vessel User’s Manual, 2001, Chapter 4.
(45) Gildea, B. D.; Casey, S.; MacNeill, J.; Perry-O’Keefe, H.; Sørensen,
D.; Coull, J. M. Tetrahedron Lett. 1998, 39, 7255–7258.
Sequence 3 was synthesized using all three bis-N-Boc
protected monomers 26, 27 and 28 to give the crude oligomer.
3812 J. Org. Chem. Vol. 73, No. 10, 2008

ConVenient Route to N-[2-(Fmoc)aminoethyl]glycine Esters
Benzyl N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]glycin-
ate Hydrochloride (5 ·HCl). To 2 (11.1 g, 20.0 mmol) and Fmoc-
OSu (7.5 g, 22.2 mmol) was added THF (50 mL), and then the
resultant suspension was cooled to 0 °C and a solution of Hu¨nig’s
base (11 mL, 63.0 mmol) in THF (15 mL) was added dropwise
over 15 min. The reaction was stirred at 0 °C for 1 h, removed
from the ice bath, and stirred for a further 15 min. The reaction
was poured into a separatory funnel containing EtOAc (200 mL)
and saturated NaHCO3 (50 mL). The organic layer was washed
with saturated NaHCO3 (50 mL), water (50 mL), and brine (2 ×
50 mL), dried over Na2SO4, and concentrated in vacuo. The organic
layer was dissolved in a minimum amount of EtOAc and filtered
through silica gel (5.0 × 10 cm bed) eluting with a gradient of
(EtOAc to EtOAc/acetone 6:4). The solvent was removed in vacuo,
the crude oil was dissolved in ether (50 mL) and cooled to 0 °C,
and HCl(g) in ether (20 mL, 2 M) was added dropwise. The flask
was kept at -20 °C for 2 h, after which the precipitate was collected
by filtration and rinsed with ether (25 mL) to give 5 as a white
solid (7.96 g, 85%): 1H NMR (400 MHz, DMSO-d6) δ 9.40 (br s,
2H), 7.90 (d, 2H, J ) 7.6), 7.69 (d, 2H, J ) 7.4), 7.57 (t, 1H, J )
5.4), 7.42 (m, 5H), 7.33 (m, 3H), 5.25 (s, 2H), 4.33 (d, 2H, J )
6.6), 4.23 (t, 1H, J ) 6.6), 4.07 (s, 2H), 3.33 (m, 2H), 3.03 (t, 2H,
J ) 6.1); 13C NMR (100 MHz, DMSO-d6) δ 166.6, 156.3, 143.8,
140.7, 135.2, 128.5, 128.3, 128.2, 127.7, 127.1, 125.2, 120.1, 66.9,
65.7, 46.7, 46.6, 36.7; HRMS (EI) m/z calcd for C26H26N2O4 [M+]
430.1893, found 430.1895. Compound 5·HCl may be neutralized
and extracted into an organic solvent (CH2Cl2 or EtOAc) by
partitioning against a solution of saturated aqueous NaHCO3. The
organic solvent was dried over anhydrous Na2SO4 and evaporated
to give a quantitative yield of the free base 5.
Allyl N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]glycinate
Hydrochloride (6·HCl). The reaction was performed as described
for 5 using 10.0 mmol of 3. After removal of the solvent, the residue
was redissolved in a minimum amount of CH2Cl2 and filtered through
silica gel (2.5 × 5 cm bed) eluting with a gradient of (CH2Cl2 to
CH2Cl2/acetone 5:5). The solvent was removed, the crude oil was
dissolved in diethyl ether (50 mL) and cooled to 0 °C, and HCl(g) in
ether (10 mL, 2 M) was added dropwise. The flask was kept at -20
°C for 2 h, after which time the precipitate was collected by filtration
and rinsed with diethyl ether to give 6·HCl as a white solid (3.46 g,
83%): 1H NMR (400 MHz, DMSO-d6) δ 9.40 (br s, 2H), 7.90 (d, 2H,
J ) 7.4), 7.70 (d, 2H, J ) 7.4), 7.57 (t, 1H, J ) 5.7), 7.42 (t, 2H, J
) 7.4), 7.34 (t, 2H, J ) 7.4), 5.94 (ddt, 1H, J ) 17.0, J ) 10.8, J )
5.5), 5.38 (dd, 1H, J ) 17.2, J ) 1.4) 5.27 (dd, 1H, J ) 10.6, J )
1.2), 4.7 (d, 2H, J ) 5.5), 4.34 (d, 2H, J ) 6.8), 4.23 (t, 1H, J ) 7.0),
4.06 (s, 2H), 3.35 (m, 2H), 3.02 (t, 2H, J ) 6.0); 13C NMR (100 MHz,
DMSO-d6) δ 166.3, 156.3, 143.8, 140.8, 131.7, 127.7, 127.1, 125.3,
120.1, 118.5, 67.8, 46.7, 46.6, 36.7; HRMS (EI) m/z calcd for
C22H24N2O4 [M+] 380.1736, found 380.1743. Compound 6·HCl may
be extracted into CH2Cl2 or EtOAc from aqueous NaHCO3 to give
the free base 6.
solubilization. The bis-N-Boc protecting group strategy may
alleviate these solubility issues and reduce aggregation while
increasing solvation in the growing oligomer as indicated by
the trouble-free syntheses performed already; however, this
aspect will be evaluated more fully elsewhere.
With an ever increasing interest in PNA applications, new
methods and inexpensive reagents for the synthesis of PNAs
continue to be of great interest in the scientific community in
order to allow the flourishing synthesis and evaluation of
variously modified PNA in medicinal and diagnostic applica-
tions. This report describes useful improvements to standard
PNA synthesis to support such endeavors.
Experimental Section
N-(2-Aminoethyl)glycine·2TsOH (1). A suspension of N-(2-
aminoethyl)glycine (19.0 g, 161 mmol) and p-toluenesulfonic acid
(68.0 g, 358 mmol) in toluene (400 mL) was refluxed for 3 h. The
solvent was removed in vacuo, and ether (300 mL) was added.
After the mixture was kept at -20 °C for 2 h, the precipitate was
collected by filtration and rinsed with diethyl ether (100 mL) to
give 1 as a white solid (74.0 g, 99%): 1H NMR (400 MHz, DMSO-
d6) δ 7.98 (br s, 3 H), 7.53 (d, 4H, J ) 8.0), 7.14 (d, 4H, J ) 8.0),
3.96 (s, 2H), 3.24 (m, 2H) 3.18 (m, 2H) 2.28 (s, 6H); 13C NMR
(100 MHz, DMSO-d6) δ 168.2, 144.8, 138.5, 128.5, 125.6, 47.4,
43.9, 35.3, 21.0.
Benzyl N-(2-Aminoethyl)glycinate ·2TsOH (2). Benzyl alcohol
(60 mL) was added to a suspension of 1 (14.9 g, 32.0 mmol) and
p-toluenesulfonic acid (2.5 g, 13.1 mmol) in toluene (250 mL).
The suspension was refluxed (oil bath, 135 °C) for 6 h, and then
the reaction mixture was concentrated in vacuo to approximately
100 mL. Ether (250 mL) was added to the reaction mixture, and
the resultant suspension was stored at -20 °C for 12 h. The
precipitate was collected by filtration and rinsed with diethyl ether
1
(150 mL) to give 2 as an off-white solid (19.3 g, 96%): H NMR
(400 MHz, D2O) δ 7.61 (d, 4H, J ) 7.4), 7.33 (br s, 5H, J ) 8.0),
7.2 (d, 4H, J ) 7.8), 5.14 (s, 2H), 3.99 (s, 2H), 3.43 (m, 2H) 3.39
(m, 2H) 2.26 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 166.7,
144.6, 138.7, 135.2, 128.6, 128.5, 128.4, 125.7, 67.2, 47.3, 44.1,
35.3, 21.0.
Allyl N-(2-Aminoethyl)glycinate·2TsOH (3). Allyl alcohol
(150 mL) was added to a suspension of 1 (11.7 g, 25.0 mmol) and
p-toluenesulfonic acid (1.0 g, 5.3 mmol) in benzene (150 mL). The
suspension was refluxed (oil bath, 110 °C) for 12 h. The solution
was concentrated to an oil, diethyl ether (200 mL) was added, and
the solution was sonicated for 30 min to initiate precipitation. At
this point, the suspension was stored at -20 °C for 12 h. The
precipitate was collected by filtration and rinsed with diethyl ether
1
(100 mL) to give 3 as an off-white solid (12.0 g, 96%): H NMR
(400 MHz, D2O) δ 7.72 (d, 4H, J ) 7.8), 7.38 (d, 4H, J ) 8.2),
5.97 (ddd, 1H, J ) 16.4, 10.2, 5.9) 5.38 (m, 2H), 4.76 (d, 2H, J )
5.9), 4.13 (s, 2H), 3.54 (m, 2H) 3.47 (m, 2H) 2.40 (s, 6H); 13C
NMR (100 MHz, D2O) δ 166.3, 142.0, 139.7, 130.6, 129.3, 125.3,
119.3, 67.3, 47.5, 44.1, 35.4, 20.5.
4-Nitrobenzyl N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-
glycinate Hydrochloride (7 ·HCl). The reaction was performed
as described for 5 using 5.0 mmol of 4 to give 2.09 g (81%) of 7
1
as a white solid: H NMR (400 MHz, DMSO-d6) δ 9.52 (br s,
2H), 8.25 (d, 2H, J ) 8.6), 7.90 (d, 2H, J ) 7.4), 7.70 (m, 4H),
7.59 (t, 1H, J ) 5.7), 7.42 (t, 3H, J ) 7.4), 7.33 (t, 3H, J ) 7.4),
5.40 (s, 2H), 4.33 (d, 2H, J ) 6.8), 4.22 (t, 1H, J ) 6.8), 4.14 (s,
2H), 3.35 (m, 2H), 3.05 (t, 2H, J ) 5.5); 13C NMR (100 MHz,
DMSO-d6) δ 166.5, 156.4, 147.2, 143.9, 143.0, 140.8, 128.7, 127.7,
127.2, 125.3, 123.6, 120.2, 65.8, 65.6, 46.7, 46.6, 37.0; HRMS (ESI)
m/z calcd for C26H26N3O6 [MH+] 476.1822, found 476.1824.
Compound 7·HCl may be extracted into CH2Cl2 or EtOAc from
aqueous NaHCO3 to give the free base 7.
4-Nitrobenzyl N-(2-aminoethyl)glycinate ·2TsOH (4). To 4-ni-
trobenzyl alcohol (7.0 g, 45.7 mmol), 1 (2.5 g, 5.4 mmol), and
p-toluenesulfonic acid (0.25 g, 1.3 mmol) was added toluene
(50 mL). The suspension was then refluxed (oil bath, 135 °C)
for 12 h. The solution was concentrated in vacuo, the solid
residue was triturated with warm acetone (50 mL) and cooled
to 0 °C for 12 h, and the white solid was collected by filtration
1
to give 4 as a white solid (2.97 g, 91%): H NMR (400 MHz,
Ethyl [N4,N4-Bis(tert-butoxycarbonyl)cytosin-1-yl]acetate (9).
To ethyl cytosin-1-yl acetate (8, 3.5 g, 17.7 mmol), Boc2O (8.6 g,
39.5 mmol), and DMAP (215 mg, 1.76 mmol) was added THF
(100 mL) followed by NEt3 (4.9 mL, 35.0 mmol), and the reaction
mixture was heated at 60 °C for 12 h under N2. The orange solution
D2O) δ 8.19 (d, 2H, J ) 6.8), 7.63 (d, 4H, J ) 8.2), 7.56 (d,
2H, J ) 7.0), 7.3 (d, 4H, J ) 7.8), 5.35 (s, 2H), 4.16 (s, 2H),
3.48 (m, 2H) 3.41 (m, 2H) 2.34 (s, 6H); 13C NMR (100 MHz,
DMSO-d6) δ 166.6, 147.3, 144.6, 142.9, 138.5, 128.8, 128.4,
125.6, 123.6, 65.8, 47.2, 44.1, 35.3, 20.9.
J. Org. Chem. Vol. 73, No. 10, 2008 3813

Wojciechowski and Hudson
was concentrated in vacuo, and the residue was purified by FCC
on silica gel eluting with a gradient of hexanes/EtOAc (7:3 to 2:8).
The desired fractions were concentrated to give a light yellow oil,
which was coevaporated with hexanes (2 × 25 mL) and dried under
vacuum to give 9 as an off-white solid (6.3g, 87%): 1H NMR (400
MHz, CDCl3) δ 7.48 (d, 1H, J ) 7.6), 7.10 (d, 1H, J ) 7.0), 4.55
(s, 2H), 4.23 (q, 2H, J ) 7.0), 1.55 (s, 18H) 1.28 (t, 3H, J ) 7.0);
13C NMR (100 MHz, CDCl3) δ 167.1, 162.6, 154.7, 149.2, 149.0,
96.2, 84.6, 61.5, 50.8, 27.4, 13.8; HRMS (EI) m/z calcd for
C18H27N3O7 [M+] 397.1849, found 397.1860.
[N4,N4-Bis(tert-butoxycarbonyl)cytosin-1-yl]acetic Acid (10).
A suspension of compound 9 (1.5 g, 3.8 mmol) in dioxane/water
(40 mL/12 mL) and water (12 mL) was placed in an ice bath,
followed by the dropwise addition of NaOH (2.5 M, 12 mL) over
1 min. The suspension was removed from the ice bath and stirred
until TLC analysis indicated complete consumption of the starting
material (15 min). The reaction mixture was poured into a separatory
funnel containing KHSO4 (1.0 M, 75 mL) and EtOAc (75 mL).
The aqueous layer was further extracted with EtOAc (4 × 75 mL)
and the combined organic fractions were dried over Na2SO4 and
concentrated in vacuo. The colorless oil was coevaporated with
hexanes (2 × 25 mL), and dried under vacuum to give 10 as a
white solid (1.3 g, 93% yield): 1H NMR (400 MHz, CDCl3) δ 13.2
(br s, 1H) 8.14 (d, 1H, J ) 7.6), 6.83 (d, 1H, J ) 7.2), 4.56 (s,
2H), 1.49 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 166.8, 153.5,
152.2, 150.4, 150.3, 145.2, 128.4, 83.7, 62.4, 44.4, 27.8, 14.1;
HRMS (ESI) m/z calcd for C16H24N3O7 [MH+] 370.1614, found
370.1620.
concentrated in vacuo. The colorless oil was coevaporated with
hexanes (2 × 25 mL) and placed in vacuo to give 2.9 g (90%) of
13 as a off-white solid: 1H NMR (400 MHz, CDCl3) δ 13.46 (br s,
1H) 8.84 (s, 1H), 8.61 (s, 1H), 5.15 (s, 2H), 1.38 (s, 18H); 13C
NMR (100 MHz, CDCl3) δ 168.8, 153.4, 152.4, 150.1, 150.0, 146.7,
127.6, 84.4, 44.6, 27.9; HRMS (EI) m/z calcd for C17H23N5O6 [M+]
393.1648, found 393.1646.
Ethyl Guanin-9-ylacetate (15). A suspension of 14 (3.5 g, 7.38
mmol) in CH2Cl2 (20 mL) was cooled to 0 °C, followed by
dropwise addition of TFA (10 mL) over 10 min. The reaction was
taken off the ice bath and stirred for a further 3 h, after which time
the solvent was removed in vacuo, followed by coevaporation with
CH2Cl2 (3 × 25). To the residue were added EtOH (150 mL) and
NEt3 (4.2 mL, 30 mmol), and the suspension was refluxed for 12 h.
The solvent was reduced to approximately 25 mL and placed at
-20 °C for 12 h. A precipitate formed that was collected by
filtration and washed with ethanol and diethyl ether to give 15 as
1
a white solid (1.34 g, 65%): H NMR (400 MHz, DMSO-d6) δ
10.73 (s, 1H), 6.54 (br s, 1H), 4.86 (s, 2H), 4.14 (q, 2H, J ) 7.0),
1.19 (t, 3H, J ) 7.0); 13C NMR (100 MHz, DMSO-d6) δ 168.0,
156.9, 153.8, 151.5, 138.0, 116.1, 61.3, 43.9, 14.0; HRMS (EI)
m/z calcd for C9H11N5O3 [M+] 237.0862, found 237.0871.
Ethyl [N2,N2-Bis(tert-butoxycarbonyl)-O6-(tert-butoxycarbo-
nyl)purin-9-yl]acetate (16). A suspension of 15 (577 mg, 2.43
mmol), DMAP (980 mg, 8.02 mmol), and Boc2O (2.12 g, 9.71
mmol) in THF (7 mL) was stirred for 36 h under N2. The solvent
was removed in vacuo, the residue was extracted with EtOAc (50
mL), and the organic layer was subsequently washed with water
(25 mL) and brine (25 mL) and then separated and dried over
Na2SO4. After removal of the solvent, the organic residue was
subjected to FCC eluting with a gradient of (hexanes/EtOAc 9:1
to 8:2). The combined fractions were dried to give 16 as a yellow
solid (472 mg, 36%): 1H NMR (400 MHz, CDCl3) δ 7.94 (s, 1H),
4.95 (s, 2H), 4.23 (q, 2H, J ) 7.0), 1.72 (s, 9H), 1.39 (s, 18H),
1.28 (t, 3H, J ) 7.0); 13C NMR (100 MHz, CDCl3) δ 166.8, 161.0,
152.6, 151.3, 150.6, 142.8, 120.4, 83.8, 82.8, 82.1, 44.3, 28.3, 27.8,
14.0; LRMS (ESI) m/z calcd for C24H35N5O9 [M+] 537.2, found
438.1 [MH+ - Boc].
Ethyl [N6,N6-Bis(tert-butoxycarbonyl)adenin-9-yl]acetate (12).
Method A. A suspension of ethyl adenin-9-ylacetate 11 (3.3 g,
15.0 mmol) in THF (100 mL) was cooled to approximately 0 °C,
in an ice bath, followed by the addition of Boc2O (9.8 g, 45.0 mmol)
and DMAP (5.5 g, 45.0 mmol). The reaction mixture was purged
with N2, removed from the ice bath, and stirred for 24 h under N2
after which time TLC analysis indicated complete consumption of
the starting material. The solution was concentrated in vacuo
(approximately 20 mL), and the residue was extracted with CH2Cl2
(200 mL) and consecutively washed with 1 M NaH2PO4 (pH )
4.0, adjusted with H3PO4, 2 × 50 mL), saturated solution of
NaHCO3 (50 mL), and brine (50 mL). The organic layer was
separated, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by FCC on silica gel eluting with a gradient
of (CH2Cl2/hexanes 8:2 to CH2Cl2:acetone 8:2). The fractions
containing the desired product were concentrated to give a light
yellow oil which was coevaporated with hexanes (2 × 25 mL) and
placed under vacuum to give 4.3 g (68% yield) of 12 as an off-
Ethyl [N2,N2-(Bis(tert-butoxycarbonyl)amino)-6-chloropurin-
9-yl]acetate (18). To a suspension of 17 (2.0 g, 7.82 mmol), DMAP
(98.0 mg, 0.8 mmol), and Boc2O (5.15 g, 23.5 mmol) in THF (25
mL) was added Hu¨nig’s base (4.1 mL, 23.5 mmol) dropwise, and
the suspension was stirred for 36 h under N2. The solvent was
removed in vacuo, and the residue was extracted with EtOAc (150
mL), washed with water (50 mL) and brine (50 mL), dried over
Na2SO4, and reduced in vacuo. The residue was subjected to FCC
eluting with (hexanes/EtOAc 8:2). The solvent was removed from
the combined fractions to give 18 as a yellow solid (1.82 g, 51%):
1H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 5.02 (s, 1H), 4.26 (q,
2H, J ) 7.2), 1.42 (s, 18 H), 1.29 (t, 3H, J ) 7.2); 13C NMR (100
MHz, CDCl3) δ 166.3, 152.8, 151.9, 150.9, 150.3, 146.8, 129.5,
83.5, 62.3, 44.6, 27.7, 14.0; HRMS (ESI) m/z calcd for
C19H26ClN5O6Na [M+] 478.1469, found 478.1446.
1
white solid: H NMR (400 MHz, CDCl3) δ 8.80 (s, 1H), 8.12 (s,
1H), 5.01 (s, 2H), 4.21 (q, 2H, J ) 7.2), 1.48 (s, 18H) 1.23 (t, 3H,
J ) 7.2); 13C NMR (100 MHz, CDCl3) δ 167.1, 162.6, 154.7, 149.2,
149.0, 96.2, 84.6, 61.5, 50.8, 27.4, 13.8; HRMS (EI) m/z calcd for
C19H27N5O6 [M+] 421.1961, found 421.1944.
Method B. To a suspension of ethyl adenin-9-ylacetate 11 (1.8
g, 8.15 mmol) in THF (50 mL) was added Boc2O (3.9 g, 17.9 mmol)
and DMAP (100 mg, 0.82 mmol) followed by NEt3 (2.2 mL, 15.7
mmol). The reaction mixture was refluxed for 12 h under N2. The
reaction was concentrated in vacuo, and the residue was purified
by FCC on silica gel eluting with a gradient of (hexanes/EtOAc
7:3 to 5:5), the fractions were concentrated to give 2.27 g (66%)
of 12 as an off-white solid.
[N2-(tert-Butoxycarbonyl)guanin-9-yl]acetic Acid (19). A solu-
tion of 3-hydroxypropionitrile (0.65 mL, 9.5 mmol) in THF (10
mL) was cooled to -78 °C, and NaH (375 mg, 9.5 mmol) was
added under N2. The reaction was allowed to warm to 0 °C and
stirred for 1 h, at which point 18 (1.0 g, 2.33 mmol) was added
in one portion. The reaction was stirred at 0 °C for 3 h and for
a further 8 h at room temperature. The solvent was evaporated
in vacuo, and to the residue was added 10 mL of water and the
pH adjusted to 8.0 by the addition of KHSO4. The aqueous phase
was washed with EtOAc (2 × 25 mL) which was discarded,
and then the aqueous phase was acidified to pH ) 2.0 with
KHSO4. The aqueous phase was extracted with EtOAc (4 × 150
mL), which was subsequently separated and dried over Na2SO4
and reduced in vacuo. The residue was triturated with equal
volumes of diethyl ether/hexanes (50 mL) and collected by
[N6,N6-Bis(tert-butoxycarbonyl)adenin-9-yl]acetic Acid (13).
To 12 (3.47 g, 8.23 mmol) was added dioxane (65 mL) and water
(25 mL), the solution was then placed in an ice bath, followed by
the dropwise addition of NaOH (2.5 M, 25 mL) over 1 min. The
reaction mixture was removed from the ice bath and stirred until
TLC indicated complete consumption of the starting material (10
min). The reaction mixture was poured into a separatory funnel
containing KHSO4 (1.0 M, 150 mL) and EtOAc (150 mL). The
aqueous layer was further extracted with EtOAc (3 × 100 mL),
and the combined organic fractions were dried over Na2SO4 and
1
filtration to give 19 as an off-white solid (370 mg, 52%): H
3814 J. Org. Chem. Vol. 73, No. 10, 2008

ConVenient Route to N-[2-(Fmoc)aminoethyl]glycine Esters
NMR (400 MHz, DMSO-d6) δ 13.26 (s, 1H), 11.39 (s,1H), 11.12
(s, 1H), 7.91 (s, 1H), 4.85 (s, 2H), 1.48 (s, 9H); 13C NMR (100
MHz, DMSO-d6) δ 169.1, 155.2, 153.9, 151.5, 149.5, 147.8,
140.2, 119.1, 82.6, 44.4, 27.8; HRMS (ESI) m/z calcd for
C12H16N5O5 [M+] 310.1151, found 310.1157.
mL). After the organic phase was dried and the solvent removed,
the residue was subjected to FCC using CH2Cl2/acetone as the
eluent. The combined fractions were reduced in vacuo to give 1.0 g
1
(62%) of 24 as a white solid: H NMR (400 MHz, DMSO-d6) δ
8.8 (s, 0.4H, mi.), 8.74 (s, 0.6H, ma.), 8.45 (m, 1H), 7.88 (m, 2H),
7.68 (m, 2H), 7.33 (m, 10H), 5.42-5.11 (m, 3H), 4.53-4.21 (m,
4H), 4.16 (s, 1H), 3.60-3.13 (m, 5H); 13C NMR (100 MHz, CDCl3)
δ 169.3, 156.8, 153.5, 151.9, 150.4, 150.3, 150.1, 146.1, 146.0,
143.8, 143.7, 141.3, 134.9, 128.8, 128.7, 128.6, 128.3, 127.8, 127.0,
125.0, 124.9, 120.0, 83.8, 83.7, 67.4, 49.0, 48.7, 47.3, 27.8; HRMS
(ESI) m/z calcd for C43H48N7O9 [MH+] 806.3514, found 806.3500.
Allyl N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[N2,N2-
bis(tert-butoxycarbonyl)-O6-benzylguanin-9-yl)acetyl]glyci-
nate (25). To an ice-cooled suspension of 22 (700 mg, 1.4 mmol)
and 6 (533 mg, 1.4 mmol) in CH2Cl2 (5 mL) were added EDC
(403 mg, 2.1 mmol) and HOBt hydrate (54 mg, 0.35 mmol), and
the reaction was stirred on ice for 0.5 h and at room temperature
for 12 h. The reaction was diluted with CH2Cl2 (10 mL) and
NaHCO3 (25 mL) and then extracted with CH2Cl2 (100 mL) and
brine (50 mL). The residue was subjected to FCC using a gradient
of EtOAc/hexanes (7:3) to EtOAc/CH2Cl2 (8:2) as the eluent. The
combined fractions were reduced in vacuo to give 25 as a pale
Ethyl [N2,N2-Bis(tert-butoxycarbonyl)-O6-benzylguanin-9-
yl]acetate (21). A suspension of 20 (1.0 g, 3.0 mmol), DMAP (600
mg, 4.9 mmol), and Boc2O (1.5 g, 6.9 mmol) in THF (7 mL) was
stirred for 36 h under N2. The solvent was removed, the residue
was extracted with EtOAc (150 mL) and washed with water (50
mL) and brine (50 mL), and the organic phase was dried over
Na2SO4. The solvent was reduced in vacuo and the residue subjected
to FCC using EtOAc/hexanes (8:2) as the eluent. The combined
fractions were dried to give 21 as a yellow solid (1.03 g, 65%): 1H
NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.51 (m, 2H), 7.35 (m,
3H), 5.64 (s, 2H), 4.97 (s, 2H), 4.24 (q, 2H, J ) 7.0), 1.40 (s, 18
H), 1.28 (t, 3H, J ) 7.0); 13C NMR (100 MHz, CDCl3) δ 166.6,
160.7, 153.1, 151.7, 150.6, 143.6, 135.7, 128.3, 128.1, 128.0, 119.3,
82.83, 68.6, 61.9, 44.2, 27.6, 13.9; HRMS (EI) m/z calcd for
C26H33N5O7 [M+] 527.2380, found 527.2384.
[N2,N2-Bis(tert-butoxycarbonyl)-O6-benzylguanin-9-yl]ace-
tic Acid (22). A solution of 21 (860 mg, 1.6 mmol) in THF/water
(10 mL/12 mL) was placed in an ice bath followed by the dropwise
addition of NaOH (2.5 M, 5 mL) over 1 min. The resultant
suspension was removed from the ice bath and stirred until TLC
indicated complete consumption of the starting material (10 min).
The reaction mixture was transferred into a separatory funnel
containing KHSO4 (2.5 M, 10 mL) and EtOAc (100 mL). The
aqueous layer was further extracted with EtOAc (3 × 50 mL), and
the combined organic fractions were dried over Na2SO4 and
concentrated in vacuo. The yellow oil was coevaporated with
hexanes (2 × 25 mL) and placed in vacuo to give 750 mg (94%)
1
yellow solid (905 mg, 75%): H NMR (400 MHz, DMSO-d6) δ
8.27 (s, 1H), 7.89 (d, 2H, J ) 7.0), 7.67 (t, 2H, J ) 7.2), 7.51-7.29
(m, 10H), 5.88 (m, 1H), 5.60 (s, 2H), 5.40-5.16 (m, 4H),
4.70-4.36 (m, 4H), 4.32-4.30 (m, 2H), 4.12 (s, 1H), 3.54-3.10
(m, 4H), 1.34 (s, 7H, mi.), 1.32 (s, 11H, ma.); 13C NMR (100 MHz,
CDCl3) δ 169.1, 160.8, 153.1, 151.6, 151.0, 144.3, 143.8, 143.7,
135.8, 131.3, 128.4, 128.3, 128.2, 127.7, 127.0, 124.9, 120.0, 119.9,
119.0, 83.2, 68.7, 66.1, 49.0, 48.9, 47.2, 27.8; HRMS (ESI) m/z
calcd for C46H52N7O10 [MH+] 862.3776, found 862.3734.
N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(N4,N4-bis-
(tert-butoxycarbonyl)cytosin-1-yl)acetyl]glycine (26). Compound
23 (536 mg, 0.686 mmol) dissolved in EtOH (20 mL) was purged
with N2, and 10% Pd/C (600 mg) was added, followed by
cyclohexadiene (1.0 mL, 10.6 mmol). The reaction was stirred for
1.5 h and filtered through a short pad of Celite, and the solvent
was removed in vacuo. The residue was coevaporated with hexanes
(4 × 25 mL) to give the title compound in 89% yield as a white
1
of 22 as a yellow solid: H NMR (400 MHz, CDCl3) δ 8.16 (s,
1H), 7.47 (m, 2H), 7.34 (m, 3H), 5.59 (s, 2H), 4.90 (s, 2H), 1.37
(s, 18H); 13C NMR (100 MHz, CDCl3) δ 168.8, 160.7, 152.9, 152.3,
151.0, 144.4, 135.8, 128.6, 128.4, 128.2, 118.0, 83.6, 68.9, 44.6,
27.9; HRMS (ESI) m/z calcd for C24H30N5O7 [MH+] 500.2145,
found 500.2144.
Benzyl N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(N4,N4-
bis(tert-butoxycarbonyl)cytosin-1-yl)acetyl]glycinate (23). To an
ice-cooled suspension of 10 (443 mg, 1.2 mmol) in THF (7 mL)
were added DCC (250 mg, 1.2 mmol) and NHS (135 mg, 1.2
mmol). The reaction was stirred at 0 °C for 15 min and at room
temperature for 1.5 h, followed by the dropwise addition of 5 (473
mg, 1.1 mmol) in THF (5 mL). The reaction was stirred for 36 h,
the formed DCU was filtered, and the product was extracted with
EtOAc (100 mL) and washed with NaHCO3 (2 × 50 mL) and brine
(50 mL). The residue was subjected to FCC using EtOAc/MeOH
as the eluent. The combined fractions were reduced in vacuo to
give (585 mg, 68%) of 23 as a white solid. Compound 23 exists in
solution as a pair of slowly exchanging rotamers; the signals due
1
solid: H NMR (400 MHz, DMSO-d6) (two rotamers) δ 7.93 (m,
1H), 7.83 (d, 2H, J ) 7.4), 7.62 (m, 2H), 7.37-7.25 (m, 5H), 6.75
(m, 1H), 4.89-4.57 (m, 2H), 4.29-4.14 (m, 4H), 3.94 (s, 1H),
3.41-3.22 (m, 5H), 1.44 (s, 18H); 13C NMR (100 MHz, CDCl3) δ
167.8, 167.0, 162.7, 162.5, 156.8, 155.7, 149.2, 143.7, 140.9, 127.5,
127.0, 128.9, 125.2, 119.7, 85.0, 84.8, 66.7, 46.8, 27.5; HRMS (ESI)
m/z calcd for C35H41N5O10Na [M+] 714.2751, found 714.2742.
N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(N6,N6-bis-
(tert-butoxycarbonyl)adenin-9-yl)acetyl]glycine (27). Compound
24 (720 mg, 0.89 mmol) was dissolved in MeOH (50 mL) and
purged with N2, and then Pd/C (200 mg) was added and the solution
was saturated with H2 by slow bubbling over 2 h. The reaction
was filtered through a short pad of Celite, and the solvent was
removed in vacuo. The residue was coevaporated with hexanes (4
1
to the major (ma.) and minor (mi.) rotamers are designated: H
NMR (400 MHz, DMSO-d6) δ 8.0 (m, 1H), 7.89 (d, 2H, J ) 7.6),
7.69 (m, 2H), 7.40-7.29 (m, 10H), 6.84 (m, 1H), 5.21 (s, 0.6 H,
mi.), 5.12 (s, 1.4 H, ma.), 4.88 (s, 1.4 H, ma.), 4.71 (s, 0.6 H, mi.),
4.42-4.23 (m, 4H), 4.14 (s, 1H), 3.48-3.10 (m, 5H), 1.49 (s, 18
H); 13C NMR (100 MHz, CDCl3) δ 169.2, 167.2, 162.6, 156.7,
154.9, 149.4, 143.8, 141.1, 135.2, 128.5, 128.2, 128.0, 127.5, 127.0,
125.0, 119.8, 96.0, 84.7, 67.0, 66.7, 49.6, 48.8, 48.5, 47.0, 27.5;
HRMS (ESI) m/z calcd for C42H48N5O10 [MH+] 782.3401, found
782.3378.
1
× 50 mL) to give the title compound in 94% as a white solid: H
NMR (400 MHz, DMSO-d6) (two rotamers) δ 10.03 (s, 1H),
8.78-8.48 (m, 2H), 8.28 (s,1H), 7.89-7.87 (m, 3H), 7.69-7.29
(m, 11H), 7.18 (br m, 1H), 5.39-5.14 (m, 3H), 4.37-4.22 (m, 4H),
3.99 (s, 1H), 3.54-3.09 (m, 5H), 1.48 (s, 8H) 1.37 (s, 12H); 13C
NMR (100 MHz, CDCl3) δ 156.9, 153.5, 151.9, 150.5, 150.3, 149.7,
143.6, 141.1, 127.6, 127.0, 125.0, 119.8, 84.0, 47.0, 40.7, 27.7.
HRMS (ESI) m/z calcd for C36H41N7O9Na [M+] 738.2863, found
738.2849.
Benzyl N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[(N6,N6-
bis(tert-butoxycarbonyl)adenin9-yl)acetyl]glycinate (24). To an
ice-cooled suspension of 13 (775 mg, 2.0 mmol) and 5 (820 mg,
1.9 mmol) in DMF (7 mL) was added EDC (775 mg, 4.0 mmol),
and the reaction was stirred on ice for 0.5 h and at room temperature
for 12 h. The reaction was poured into NaHCO3 (50 mL) and
extracted into CH2Cl2 (150 mL). The organic phase was washed
sequentially with aqueous NaHCO3 (2 × 50 mL) and brine (50
N-[2-(Fluorenylmethoxycarbonyl)aminoethyl]-N-[N2,N2-bis-
(tert-butoxycarbonyl)-O6-benzylguanin-9-yl)acetyl]glycine (28).
Compound 25 (850 mg, 1.0 mmol) was dissolved in a degassed
solution of chloroform/acetic acid/N-methylmorpholine (25 mL, 1.5
mL, 0.75 mL), the solution was purged with N2, and Pd(PPh3)4
(115 mg, 0.1 mmol) was added. The solution was deoxygenated
with freeze-pump-thaw degassing and stirred under N2 for 12 h.
J. Org. Chem. Vol. 73, No. 10, 2008 3815

Wojciechowski and Hudson
The solvent was removed in vacuo and the residue was extracted
with CH2Cl2 (100 mL) and KHSO4 (1 M, 2 × 25 mL), dried over
Na2SO4, and reduced in vacuo. The residue was subjected to FCC
using CH2Cl2/MeOH as the eluent. The combined fractions were
reduced in vacuo to give 28 as an off-white solid (691 mg, 84%):
1H NMR (400 MHz, DMSO-d6) (two rotamers) δ 8.28 (s, 1H),
7.89 (d, 2H, J ) 7.4), 7.67 (t, 2H, J ) 7.0), 7.49 (d, 2H, J ) 7.0),
7.46-7.29 (m, 8H), 5.61 (s, 2H), 5.32 (s, 1H), 5.14 (s, 1H),
4.37-4.20 (m, 4H), 4.01 (s, 1H), 3.54-3.09 (m, 5H), 1.32 (s, 18H);
13C NMR (100 MHz, CDCl3) δ 171.1, 166.1, 160.3, 156.6, 156.5,
152.8, 151.6, 150.6, 144.6, 143.6, 143.4, 140.9, 135.5, 131.8, 128.2,
127.9, 127.4, 126.8, 124.9, 124.8, 119.7, 118.2, 83.1, 68.5, 66.4,
48.8, 27.6; HRMS (ESI) m/z: calcd for C43H48N7O10Na [MH+]
845.3360, found 845.3359.
Acknowledgment. We thank the Natural Sciences and En-
gineering Research Council of Canada (NSERC) for funding
this work through their Discovery Grants program. Ms. Paula
Pittock of the UWO Biological Mass Spectrometry Laboratory
is thanked for expert collection of mass spectral data for the
polyamides.
Supporting Information Available: General experimental
1
conditions and copies of H and 13C NMR spectra for com-
pounds 1-7, 9, 10, 12, 13, 15, 16, 18, 19, and 21-28 and an
HPLC spectrum for sequence 3. This material is available free
of charge via the Internet at http://pubs.acs.org.
JO800195J
3816 J. Org. Chem. Vol. 73, No. 10, 2008