J . Org. Chem. 2000, 65, 7697-7699
7697
We also required an acid-labile group to protect the
exocyclic amines in purine-containing R-helical peptide
nucleic acids (RPNAs).7 Boc protection was envisaged as
a highly attractive strategy since this protecting group
is orthogonal to our Fmoc-based SPPS protocol and, in
contrast to the known acid labile monomethoxytrityl
(Mmt) protecting group, can sustain mildly acidic condi-
tions. Thus, Boc-protected RPNAs can be cleaved from
the resin via mild acidolysis, and further synthetic
chemistry involving conjugation, fragment condensation,
etc., can be carried out. With the appropriate choice of
resin/linker, on-resin deprotection and modification of a
specific amine group would also be possible. One could,
for example, deprotect an orthogonally protected Lys and
then attach a fluorophore to the RPNA. We now report
practical syntheses of Boc-protected adenine, 6-chloro-
2-aminopurine (6Cl2AP), and guanine for incorporation
into our RPNA monomer synthesis. Gram quantities of
these Boc-protected purines can now be synthesized from
inexpensive starting materials without the need for
elaborate purification steps.
Our initial attempts to make the Boc-protected adenine
by treating adenine with Boc2O and a catalytic amount
of DMAP were not very successful. The use of polar
solvents such as DMSO and DMF (to solubilize adenine)
gave mono-, bis-, and tris-Boc protected adenines, along
with a major amount of free adenine. Significantly, the
ratio of these products remained constant over time, and
warming the reaction mixture led to a more complicated
reaction mixture along with the development of highly
colored species. While evaluating different reaction con-
ditions, it was observed that use of excess (4.5 equiv)
Boc2O, a catalytic amount of DMAP, and THF as solvent
gave a single productsthe tris-Boc-protected adenine 3
(Scheme 1). Purification was easily effected by simple
filtration through silica gel to give 3 in 90% yield. Tris-
Boc adenine 3 can be converted to bis-Boc adenine 5
almost quantitatively by treatment with aq NaHCO3, and
the latter can be converted to the desired mono-Boc
derivative 7 in very good yield by treatment with NaOH
for 3 days at room temperature.
Syn th esis of ter t-Bu toxyca r bon yl
(Boc)-P r otected P u r in es
Subhakar Dey and Philip Garner*
Department of Chemistry, Case Western Reserve University,
Cleveland, Ohio 44106-7078
ppg@po.cwru.edu
Received J une 29, 2000
Interest in the chemical synthesis of nucleic acids and
their analogues as well as nucleoside antibiotics has been
the driving force behind research on purines and pyri-
midines. One issue involves how to mask or protect amine
functionality that may be present in the nucleobases. In
a typical solid-phase oligonucleotide synthesis, for ex-
ample, the exocyclic amine groups of adenine, cytosine,
and guanine are blocked with acyl protecting groups. At
the end of synthesis, global deprotection under basic
conditions gives the fully deblocked nucleic acids. With
the advent of Nielsen’s peptide nucleic acids (PNAs)1 and
related amide-linked oligonucleotide surrogates,2 there
is a need for protecting groups that can be removed under
acidic or neutral conditions and are compatible with
Fmoc-mediated solid-phase synthesis protocols. Such
protecting groups would also be useful for the synthesis
of DNA-peptide conjugates.3 Unfortunately, the applica-
tion of existing protecting group strategies to free
nucleobasessespecilly the purinessoften leaves much to
be desired. Although there are well-documented examples
of nucleobases with acid-labile protecting groups,4 the
most common acid-labile protecting group for amines, the
tert-butoxycarbonyl group (Boc), has, to our knowledge,
not been successfully extended to the parent purine
nucleobases.5 The Boc protecting group has the additional
virtue in that it can also be removed under neutral
conditions.6
(1) For reviews that focus on Nielsen’s contributions, see: (a)
Dueholm, K. L.; Nielsen, P. E. New J . Chem. 1997, 21, 19. (b) Eriksson,
M.; Nielsen, P. E. Quart. Rev. Biophys. 1996, 29, 369. (c) Uhlmann,
E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int. Ed. 1998,
37, 2796.
(2) For a recent review that includes a section on “modified” PNAs,
see: Falkiewicz, B.; Acta Biochim. Pol. 1999, 46, 503. (Also see
literature cited in ref 7a.).
(3) Dreef-Tromp, C. M.; van der Maarel, J . C. M.; van den Elst, H.;
van der Marel, G. A.; van Boom, J . H. Nucleic Acids Res. 1992, 20,
4015.
(4) Breiphol, G.; Knolle, J .; Langner, D.; O’Malley, G.; Uhlmann, E.
Bioorg. Med. Chem. Lett. 1996, 6, 665.
(5) The Boc group has been used to protect purine-containing
nucleosides, but the starting material has generally been the nucleoside
and not the free bases. (a) Ubukata, M.; Isono, K. Tetrahedron Lett.
1986, 27, 3907. (b) Schirmeister, H.; Pfleiderer, W. Helv. Chim. Acta
1994, 77, 10. (c) Ubukata, M.; Osada, H.; Magae, J .; Isono, K. Agric.
Biol. Chem. 1988, 52, 1117. (d) Nagatsugi, F.; Uemura, K.; Nakashima,
S.; Maeda, M.; Sasaki, S. Tetrahedron Lett. 1995, 36, 421. A bis-Boc
derivative of 2-amino-9-benzyloxy-6-methoxypurine has been prepared.
(e) Harnden, M. R.; Wyatt, P. G. Ibid. 1990, 31, 2185. Nielsen’s adenine
PNA monomer has been Boc-protected starting from N9-alkylated
adenine. (f) Fare`se, A.; Patino, N. Condom, R.; Dalleu, S.; Guedj, R.
Ibid. 1996, 37, 1413.
(6) (a) Ceric ammonium nitrate (CAN): Hwu, J . R.; J ain, M. L.;
Tsay, S.-C.; Hakimelahi, H. Tetrahedron Lett. 1996, 37, 2035. (b)
Microwave irradiation: Siro, J . G.; Mart´ın, J .; Garc´ıa, J . L.; Remuin˜an,
M. J .; Vaquero, J . J . Synlett 1998, 147. (c) NaI/acetone: Ham, J .;
Maeda, Choi, K.; Ko, J .; Lee, H., J ung, M. Protein Pept. Lett. 1998, 5,
257.
Care has to be taken during the conversion of bis-Boc
adenine 5 to mono-Boc adenine 7. The best result was
obtained by stopping the reaction after 72 h by acidifica-
tion and isolation of the product. Longer reaction times
led to the formation of significant quantities of adenine.
Once isolated, mono-Boc adenine 7 is very stable and can
be stored on the benchtop for months without any
degradation. Tris-Boc adenine 3 can be converted directly
to 7 by treatment with NaOH without even isolating 5.
Similar chemistry was observed with 6-chloro-2-ami-
nopurine (6Cl2AP) 2 with the exception that the mono-
Boc species is quite stable to NaOH. The regiochemistry
of the tris-Boc species 3 and 4 (N7 vs N9) was verified by
UV spectroscopy since it is known that the λmax for the
(7) (a) Garner, P.; Dey, S.; Huang, Y.; Zhang, X. Org. Lett. 1999, 1,
403. (b) Garner, P.; Dey, S.; Huang, Y. J . Am. Chem. Soc. 2000, 122,
2405.
(8) Ohrt, J . M.; Srikrishnan, T.; Parthasarathy, R.; Dutta, S. P.;
Chheda, G. B. J . Am. Chem. Soc. 1978, 100, 5232.
(9) Coull, J . M.; Hodge, R. P. Guanine Synthons for Peptide Nucleic
Acid Synthesis and Method for Production. International Patent
Application No. PCT/US94/14742, December 22, 1994.
10.1021/jo000983i CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/10/2000