Full orthogonality between Dde and Fmoc: The direct synthesis of PNA-peptide conjugates

Article abstract of DOI:10.1021/ol049905y

New deprotection conditions for the Dde amine protecting group that are fully orthogonal to Fmoc are described and successfully applied to the dual synthesis of PNA-peptide conjugates.

Full text of DOI:10.1021/ol049905y

ORGANIC
LETTERS
2004
Vol. 6, No. 7
1127-1129
Full Orthogonality between Dde and
Fmoc: The Direct Synthesis of
PNA−Peptide Conjugates
Juan Jose D´ıaz-Mocho´n, Laurent Bialy, and Mark Bradley*
School of Chemistry, UniVersity of Southampton, Highfield,
Southampton SO17 1BJ, United Kingdom
mb14@soton.ac.uk
Received January 15, 2004
ABSTRACT
New deprotection conditions for the Dde amine protecting group that are fully orthogonal to Fmoc are described and successfully applied to
the dual synthesis of PNA−peptide conjugates.
Over the past decade, a DNA mimic known as Peptide
Nucleic Acid (PNA) has been developed, which hybridizes
to complementary DNA or RNA sequences with higher
affinity than its oligonucleotide counterpart.1,2 This, together
with its stability and ease of synthesis and manipulation, has
turned PNA into a major tool in the biomolecular and
biotechnology fields.3 PNA is seen as a promising tool for
gene therapy in antisense or antigene applications.4 However,
cellular uptake has been one of the main pitfalls and has
given rise to a number of PNA conjugates in attempts to try
to improve cellular permeability.5
In the context of our research program, a highly flexible
strategy for the synthesis of branched PNA-peptide conju-
gates was required. To use commercially available Fmoc-
protected amino acid monomers carrying acid-labile pro-
tecting groups, PNA monomers with an N-terminal protecting
group completely orthogonal to Fmoc were required. The
use of the palladium-labile allyloxycarbonyl (Aloc) group
gave unsatisfactory results with poor repetitive yields,
severely restricting the length of PNA that could be made.
Our attention was attracted to the 1-(4,4-dimethyl-2,6-
dioxacyclohexylidene)ethyl (Dde) group.6 Dde has been used
as a protecting group for primary amines in solid-phase
chemistry and classically is cleaved under nucleophilic
conditions using 2% v/v hydrazine in DMF.6 Dde is stable
under Fmoc and Boc deprotection conditions; unfortunately,
Fmoc is readily deprotected with 2% v/v hydrazine in DMF,
therefore limiting the potential of a dual Dde/Fmoc protecting
group strategy.
The aim of this study was to develop novel Dde depro-
tection conditions that were completely orthogonal to Fmoc
(5) Li, X.; Zhang, L.; Lu, J.; Chen, Y.; Min, J.; Zhang, L. Bioconjugate
Chem. 2003, 14, 153. Cutrona, G.; Carpento, E. M.; Ulivi, M.; Roncella,
S.; Landt, O.; Ferrarini, M.; Boffa, L. C. Nat. Biotechnol. 2000, 18, 300.
Branden, L. J.; Mohamed, A. J.; Smith, C. I. Nat. Biotechnol. 1999, 17,
784. Stock, R. P.; Olvera, A.; Sanchez, R.; Saralegui, A.; Scarfi, S.; Sanchez-
Lopez, R.; Ramos, M. A.; Boffa, L. C.; Benatti, U.; Alagon, A. Nat. Biotech.
2001, 19, 231. Zhou, P.; Wang, M.; Du, L.; Fisher, G. W.; Waggoner, A.;
Ly, D. H. J. Am. Chem. Soc. 2003, 125, 6878.
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991,
245, 1497.
(2) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. Ang. Chem., Int.
Ed. 1998, 37, 2796. Antsypovitch, S. I.; Russ. Chem. ReV. 2002, 71, 71.
(3) Nielsen, P. E. Curr. Opin. Biotechnol. 2001, 12, 16. Lovrinovic, M.;
Seidel, R.; Wacker, R.; Schroeder, H.; Seitz, O.; Engelhard, M.; Goody, R.
S.; Niemeyer, C. M. Chem. Commun. 2003, 822. Rosenbaum, D. M.; Liu,
D. R.; J. Am. Chem. Soc. 2003, 125, 13924.
(6) Bycroft, B. W.; Chan, W. C.; Chanabra, S. R.; Hone, N. D. Chem.
Commun. 1993, 778.
(4) Pooga, M.; Langel, U¨ . Curr. Cancer Drug Targets 2001, 1, 231.
10.1021/ol049905y CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/04/2004

and apply these to the synthesis of branched PNA-peptide
conjugates. We reasoned that this should be possible given
the different mechanisms of deprotection for both protecting
groups. While Fmoc is cleaved by basic elimination, Dde is
cleaved by nucleophiles (trans enamination).6,7 A set of new
deprotection conditions and reagents were evaluated using
Fmoc-Lys(Dde)-Rink-PS resin (1) as a simple model com-
pound. Following deprotection, amino groups were coupled
with Fmoc-Gly-OH as an analytical tag. After cleavage from
the resin with TFA/CH2Cl2/TIS (90/5/5), the crude product
was analyzed by RP-HPLC. As expected, the use of
hydrazine led to both Fmoc and Dde deprotection (after 1
min of hydrazine treatment, products 2-5 were detected,
Scheme 1). Alternative bivalent nucleophiles with reduced
verified. For this purpose, novel PNA monomers 7-10 with
Dde as an N-terminal protecting group and 4-methoxytrityl
(Mmt) as an acid-labile nucleobase protecting group (for
thymine, no protecting group was needed) were synthesized
in good overall yields starting from the known amine 6
(Scheme 2).9,10
Scheme 2.11 Synthesis of Dde-Protected PNA Monomersa
Scheme 1a
a Reagents: (i) DdeOH, DIPEA, CH2Cl2/MeOH, 15 h, 71%; (ii)
CMmtCH2COOH, AMmtCH2COOH, or GMmtCH2COOH, PyBrOP,
NEt(iPr)2, DMF, 15 h (7-9); (iii) T-CH2COOH, DCC, HOBt,
NMM, DMF, 15 h (10); (iv) 1 M Cs2CO3 MeOH/H2O 1/1, 90 min,
overall yields from 6: 25% (7), 75% (8), 45% (9), 37% (10). bNB
) nucleobases.
a Reagents: (i) deprotection mixture; (ii) Fmoc-Gly-OH, HOBt/
DIC, DMF; (iii) TFA/CH2Cl2/TIS (90/5/5).
basicity were investigated. Unfortunately, most of them (1,2-
ethanedithiol, 2-mercaptoethanol, guanidine, 4-hydrazinoben-
zoic acid, diaminomaleonitrile, o-phenylenediamine) did not
deprotect Dde.
Attention was then turned toward screening mixtures of
NH2OH‚HCl along with different bases (N,N-diisopropyl-
ethylamine, pyridine, imidazole) using a range of solvents
(CH2Cl2, N,N-dimethylformamide (DMF), N-methyl-2-pyr-
rolidone (NMP)).
The best results in terms of reactivity and selectivity were
achieved using a mixture of NH2OH‚HCl/imidazole in NMP/
CH2Cl2.8 After 3 h, the Dde group was cleaved cleanly while
the Fmoc group was completely stable, yielding 4 as the only
compound with a quantitative yield. The applicability of these
deprotection conditions to other types of resins (PEGA and
TentaGel (TG)) was then explored. The selectivity was
excellent, while the deprotection proved to be markedly faster
on PEGA resin (1 h in NMP/DMF) than on TentaGel (3 h
in NMP/CH2Cl2).
The synthesis of the conjugates was carried out on PEGA,
PS, and TG resins on the Rink linker using the new Dde
deprotection conditions and traditional Fmoc chemistry in a
stepwise manner. The new conditions were also compatible
with acid-labile (Mmt) and palladium-labile groups (Aloc).12
Of particular interest was the perfect stability of the PNA
nucleobases toward our deprotection conditions, as hydroxy-
lamine has been used to extensively modify nucleobases in
DNA chemistry.13
Although all the resins gave good results, PEGA displayed
the fastest cleavage kinetics and therefore was particularly
(9) Heimer, E. P.; Gallo-Torres, H. E.; Felix, A. M.; Ahmad, M.;
Lambros, T. J.; Scheidl, F.; Meienhofer, J. Int. J. Pept. Protein Res. 1984,
23, 203.
(10) Breipohl, G.; Knolle, J.; Langner, D.; O’Malley, G.; Uhlmann, E.
Bioorg. Med. Chem. Lett. 1996, 6, 665.
t
(11) Side-chains of Fmoc-amino acid monomers were Boc- and Bu-
protected. Abbreviations: DIC (N,N′-diisopropylcarbodiimide), HOBt (1-
hydroxybenzotriazole), NEM (N-ethylmorpholine), NMM (N-methylmor-
pholine), NMP (N-methyl-2-pyrrolidone), PyBOP ((benzotriazol-1-yloxy)-
tripyrrolidinophosphonium hexafluorophosphate), PyBrOP (bromotripyr-
rolidinophosphonium hexafluorophosphate), TFA (trifluoroacetic acid), TIS
(triisopropylsilane).
(12) Hydrazine partially reduces the double-bonds of Aloc groups:
Rohwedder, B.; Mutti, Y.; Dumy, P.; Mutter, M. Tetrahedron Lett. 1998,
39, 1175
(13) Blackburn, G. M.; Jarvis, S.; Ryder, M. C.; Solan, V. J. Chem. Soc.,
Perkin Trans. 1 1975, 370. Rubin, C. M.; Schmid, C. W. Nucleic Acids
Res. 1980, 8, 4613. Shugar, D.; Huber, C. P.; Birnbaum, G. I. Biochim.
Biophys. Acta 1976, 447, 274.
The applicability of these new deprotection conditions to
the synthesis of a branched PNA-peptide conjugate was then
(7) Rubinov, D. B.; Rubinova, I. L.; Akhrem, A. A. Chem. ReV. 1999,
99, 1046.
(8) Deprotection mixture was prepared as follows: 1.25 g (1.80 mmol)
of NH2OH‚HCl and 0.918 g (1.35 mmol) of imidazole were suspended in
5 mL of NMP, and the mixture was sonicated until complete dissolution.
This solution can be stored for at least 2 weeks at -20 °C. Just before
reaction, 5 volumes of this solution were diluted with 1 volume of CH2Cl2
(polystyrene and tentagel resin) or DMF (PEGA resin).
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Org. Lett., Vol. 6, No. 7, 2004

Scheme 3.11 PNA Conjugate Synthesisa
a Reagents: (i) NH2OH‚HCl/imidazole;8 (ii) Dde-PNA-OH (5.5
equiv), PyBop (5 equiv), NEM (11 equiv) in DMF (0.1 M), 3 h;
(iii) 20% piperidine in DMF; (iv) Fmoc-aa-OH (5.5 equiv), PyBop
(5 equiv), DIPEA (11 equiv), HOBt (5.5 equiv) in DMF (0.1 M),
3 h; (v) Repeat steps i-iv. (vi) TFA/TIS/CH2Cl2 (90/5/5).
Figure 1. (a) HPLC trace and acetonitrile gradient (0% for 5 min;
from 0 to 25% over 40 min and then to 90% over 1 min, 90% for
4 min. (b) MALDI-TOF MS of crude conjugate 15.
fields of organic chemistry. Furthermore we have demon-
strated that Dde can be employed as an N-terminal groupfor
PNA synthesis. To the best of our knowledge, this is also
the first report of the synthesis of PNA on PEGA resin.
suited for the synthesis of longer sequences of PNA, as
demonstrated by the synthesis of longer conjugates 14 and
15 (12-mers). The quality of the method was confirmed by
the high purity of the crude compounds, which were analyzed
by HPLC and MALDI-TOF (Figure 1). Conjugates 11-15
were obtained with purities equating to over 99% per
chemical step (Scheme 3). The overall yields of isolated
compounds varied between 50 and 67%.
In conclusion, we have shown that hydroxylamine is the
reagent of choice for mild and orthogonal deprotection of
the Dde group. This was demonstrated by a highly flexible
synthesis of PNA-peptide conjugates. Our new conditions
are milder than hydrazine and compatible with a larger
number of functional groups and solid supports and should
therefore greatly extend the scope of the Dde group to other
Acknowledgment. This research was supported by
BBRSC (EBS). L.B. is grateful to the DAAD for a
scholarship. J.J.D. is grateful to the University of Granada
for a scholarship and the BBRSC.
Supporting Information Available: HPLC and MS (ES
or MALDI-TOF) of crude compounds 11-14. This material
is available free of charge via the Internet at http://pubs.acs.org.
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