J. Beythien, P. D. White / Tetrahedron Letters 46 (2005) 101–104
103
By making the chromophore part of the linker, the need
for additional synthetic steps for chromophore introduc-
tion or the use of expensive labelled amino acid deriva-
tives are avoided. In the context of high-throughput
synthesis, the assurance that the chromophore is present
at the outset is particularly important, as checking for
complete chromophore incorporation is not practical
when dealing with large numbers of reactions. This pro-
cedure provides a support of defined and reproducible
substitution (typically ꢀ0.5mmol/g).
The potential of resin 2 for the synthesis of EDANS-
labelled peptides was evaluated using peptides 3 and 4
as examples (Scheme 2). In the case of peptide 3, the
C-terminal residue was attached by double coupling
Fmoc-Arg(Pbf)-OH that was activated with HATU in
the presence of DIEA. A double coupling was used to
ensure complete condensation, as activated arginine
derivatives are prone to intramolecular lactamisation
to unreactive c-lactams. For peptide 4, a single coupling
of Fmoc-Leu-OH under identical conditions was used.
Satisfactory substitutions have also been obtained using
Fmoc-amino acid pre-formed Pfp esters. Under the
above coupling conditions, the secondary anilino func-
tionality of EDANS5 was found to be unreactive and
was therefore not protected. The use of the more aggres-
sive coupling reagent such as PyBrOP, however, did lead
to the formation of anilide by-products and should
therefore be avoided.
Figure 3. HPLC profile of crude peptide 4. (HPLC conditions:
Nucleosil 33-5 C18 column; gradient: 30–50% B in 30min, 1ml/min;
A: 0.1% TFA aq; B: 0.1% TFA in MeCN.
peptidyl resin with 95% TFA cocktail for 3h. For pep-
tide 4, the use of the more acidic cleavage cocktail
10% TMSBr in TFA afforded the desired peptide in
moderate yield (63%) and excellent purity (Fig. 3) in
only 1h following reprecipitation from AcOH/MeCN
with ether/MeCN.9 The identities of both peptides were
confirmed by LC–ESMS.
Additions of subsequent Fmoc-protected amino acids
were carried out using PyBOP/DIEA. 20% piperidine
in DMF and 4% DBU in DMF were used for Fmoc
removal during the synthesis of peptides 3 and 4, respec-
tively. Typically, the use of DBU is preferred when
coupling methods employed are capable of activating
the EDANS sulfonate group, as the use of piperidine
in such circumstances could lead to the formation of
piperidinylsulfonamides. In both cases the N-terminal
Dabcyl group was introduced using Dabcyl-OSu. Pep-
tide 3 was obtained in good purity (Fig. 2) and good
yield (76%) following treatment of the corresponding
In summary, our recently developed EDANS linker 1
provides a simple and effective method for the prepara-
tion of EDANS-labelled peptide by solid phase synthe-
sis. The linker attached to aminomethyl polystyrene is
now commercially available as EDANS NovaTag resin
from Novabiochem, Switzerland.
References and notes
1. Maly, D. J.; Huang, L.; Elman, J. A. Chembiochem 2002,
3, 16–37.
2. Matayoshi, E. D.; Wang, G. T.; Krafft, G. A.; Erickson, J.
Science 1990, 247, 954–958.
3. Wang, G. T.; Matayoshi, E. D.; Huffaker, H. J.; Krafft, G.
A. Tetrahedron Lett. 1990, 31, 6493–6496.
4. Garcia-Echeverria, C.; Rich, D. H. FEBS Lett. 1992, 297,
100–102.
5. Maggiora, L. L.; Smith, C. W.; Zhang, Z.-Y. J. Med.
Chem. 1992, 35, 3727–3730.
6. Drijfhout, J. W.; Nagel, J.; Beekman, B.; Te Koppele, J.
M.; Bloemhoff, W. In Peptides: Chemistry, Structure &
Biology: Proc. 14th American Peptide Symposium; Kau-
maya, P. T. P., Hodges, R. S., Eds.; Mayflower Scientific
Ltd: Birmingham, 1996; pp 129–131.
Figure 2. HPLC profile of crude peptide 3. (HPLC conditions: Vydac
peptide/protein C18 column; gradient: 20–100%B in 20min, 1ml/min;
A: 0.1% TFA aq; B: MeCN/water/TFA (90:10:0.1).
7. Jensen, K. J.; Alsina, J.; Songster, M. F.; Vagner, J.;
Albericio, F.; Barany, G. J. Am. Chem. Soc. 1998, 120,
5441–5452.