A novel solid-phase linker strategy for the side-chain anchoring of arginine: an expeditious route to arginine 7-amido-4-methylcoumarins

Article abstract of DOI:10.1016/j.tetlet.2006.03.019

A novel linker strategy for the efficient side-chain anchoring of arginine is described. The utility of this approach was demonstrated by the facile synthesis of arginine-specific fluorogenic peptide substrates by standard Fmoc solid phase peptide synthesis methods.

Full text of DOI:10.1016/j.tetlet.2006.03.019

Tetrahedron Letters 47 (2006) 3009–3012
A novel solid-phase linker strategy for the side-chain anchoring
of arginine: an expeditious route to arginine
7-amido-4-methylcoumarins
a
a
a
b,
*
´ ´
Joerg Beythien, Sophie Barthelemy, Peter Schneeberger and Peter D. White
aNovabiochem, Merck Biosciences AG, Weidenmattweg 4, CH-4448, La¨ufelfingen, Switzerland
bNovabiochem, Merck Biosciences Ltd, Padge Road, Nottingham NG9 2JR, UK
Received 31 January 2006; revised 21 February 2006; accepted 2 March 2006
Available online 20 March 2006
Abstract—A novel linker strategy for the efficient side-chain anchoring of arginine is described. The utility of this approach was
demonstrated by the facile synthesis of arginine-specific fluorogenic peptide substrates by standard Fmoc solid phase peptide syn-
thesis methods.
Ó 2006 Elsevier Ltd. All rights reserved.
In solid-phase peptide synthesis (SPPS), the standard
practice of linking the first amino acid residue to the
solid support through its a-carboxyl group does not
readily facilitate synthesis of C-terminally modified pep-
tides. Strategies have therefore been developed where
anchoring is either through the a-amino group1 or more
commonly through a side-chain functionality, thereby
leaving the a-carboxyl group available for modification.
Most trifunctional amino acids can be simply linked via
their side chains to standard synthesis supports, such as
Wang2 or 2-chlorotrityl resin.3 However, these methods
are not satisfactory for arginine since the initial attach-
ment and final cleavage yields are poor and the protec-
tion afforded by the resin handle is insufficient to
prevent side reactions during SPPS.3 To overcome these
problems, a number of handles based on standard
arenesulfonyl arginine-protecting groups have been
developed.4–7 However, none has found general applica-
tion, as the synthesis of such linkers is either complex, in
one case requiring an on-resin chlorosulfonation step,7
or attachment and detachment of the arginine to the lin-
ker is difficult and proceeds in poor yields.
to Rink amide resin, and the resulting thiourea con-
verted into a guanidino group by S-methylation and
ammonolysis immediately prior to cleavage. To avoid
the need for post-assembly guanidinylation, we devel-
oped a two-step strategy in which the guanidine is pre-
pared maximally protected on the solid-phase prior to
commencement of Fmoc SPPS. This approach also
eliminates the risk of ornithine formation9 and prema-
ture chain loss from the resin (Fig. 1) which can
potentially occur in Martinez’s approach where the
guanidine is not protected. In the first step, an appropri-
ate Fmoc-protected ornithine bearing the desired
C-terminal modification is simply immobilised on a solid
support by reductive amination to a backbone-amide-
type linker.1 For our purposes, we selected the 3-formyl-
indol-1-ylacetyl linker10 supported on aminomethyl
polystyrene as we believed this would confer the desired
acid-lability to the final construct. The second step
involved carbodiimide-mediated guanidinylation of the
resulting immobilized secondary amine with bis-Boc-
thiourea.11–14 (Treatment of this resin with electrophiles
such as isocyanates, thiocyanates, or chloroformates
may also be used to generate ureas, thioureas or carba-
mates, respectively.15)
Our strategy aimed to avoid these difficulties by forming
the guanidine moiety on the solid phase, employing an
approach similar to that described by Martinez et al.8
Here, an ornithine-derived isothiocyanate was attached
To demonstrate the utility of the approach, we used it to
prepare a resin for the synthesis of peptides containing
C-terminal arginyl-7-amino-4-methylcoumarin (Fig. 2).
Such peptides are widely used as fluorogenic peptide sub-
strates for the detection of trypsin-like serine proteases,
*
Corresponding author. Tel.: +44 115 9430840; fax: +44 115
9430951; e-mail: peter.white@merckbiosciences.co.uk
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.03.019

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J. Beythien et al. / Tetrahedron Letters 47 (2006) 3009–3012
R1
R1
H
HN
Fmoc
A
N
NR
HN
NHR
O
N
R1
H
N
RN
O
NH
H
NH
Fmoc
N
+
H
piperidine
O
NH2
N
H
N
H
O
O
N
H
O
Figure 1. Formation of orthinine residues via deguanidinylation of arginine residues.
H
N
H
Boc-N
NH-Boc
N
N
N
H
N
H
O
O
N
N
N
O
H
ii
i
O
AMC
AMC
3
2
Fmoc-NH
Fmoc-NH
O
O
Figure 2. Synthesis of resin 3. (i) Fmoc-Orn-AMC 1, TMOF, DCM, DIEA, NaBH(OAc)3; (ii) N,N0-bis-Boc-thiourea, DIC, DIEA, DCM.
a class of enzymes which includes important therapeutic
targets such as thrombin, factor IXa and factor Xa.
Cleavage by the enzyme of the amide bond between the
arginine and 7-amino-4-methylcoumarin (AMC) releases
free fluorophore, which can be detected at 441 nm upon
excitation with light at 342 nm. The synthesis of argi-
nine-AMC derivatives is particularly problematic owing
to the poor nucleophilicity of AMC and the propensity
for arginine to undergo c-lactam formation. It is for this
reason that the synthesis of such compounds is usually
carried out in solution. Solid-phase strategies such as
those described above employing side-chain anchoring
of arginine have also been used; however, these methods
are subject to the limitations previously mentioned.
Another approach utilizes an AMC derivative (ACC,
7-amino-4-carbamoylmethylcoumarin) which contains
a carboxylic acid group to provide the means of attach-
ment to amino-functionalised support.16 However, the
synthesis of ACC is complex and loading of arginine
onto the polymer-bound aromatic amino group is extre-
mely difficult (50% after two exposures).17 Therefore, a
simple and practical method for making such peptides
would be of enormous value to researchers studying
trypsin-like proteases.
conjunction with DIC,12 to afford the supported bis-
Boc-protected arginine AMC derivative 3. (The use of
Mukaiyama’s reagent19 instead of DIC gave poorer
results.) Treatment of a small sample of 3 with TFA/
DCM (1:1) and TLC analysis of the cleaved product
indicated complete guanidinylation. The loading of 3
was determined by the quantitative Fmoc test20 to be
0.26 mmol/g.
Resin 3 was evaluated in the synthesis of peptides 4 and
5 (Fig. 3).21 In both examples, coupling of the C-termi-
nal residue was performed twice using Fmoc-protected
amino acids activated with PyBOPÒ in the presence of
DIEA. This double coupling procedure was used to
ensure complete condensation, as it was anticipated that
the arginine amino group would be hindered by the
two Boc groups protecting the side-chain. Additions of
subsequent Fmoc-protected amino acids were carried
out using a single coupling with PyBOPÒ/DIEA. A solu-
tion of 3% DBU in DMF was used for Fmoc removal as
in our experience its use in the synthesis of fluorescently
labelled peptides frequently gives better results than
piperidine.
In all cases, cleavage of products from the solid support
was achieved by treating the peptidyl resins with TFA/
DCM (1:1) for 2 h. Peptides 4 and 5 were obtained in
excellent purities (Fig. 4) and in good yields (4, 73%;
5, 62%). Detailed LC-MS of the products indicated that
there were no orthinine-containing by-products in the
crude peptides.
Fmoc-Orn-AMC 1 was prepared from Fmoc-Orn(Boc)-
OH by published methods.8 Reductive amination of 3-
formylindol-1-ylacetylaminomethyl polystyrene with 1
was effected with sodium triacetoxyborohydride in
TMOF/DCM/DIEA.18 The resin-bound secondary
amine 2 was guanidinylated with bis-Boc-thiourea in

J. Beythien et al. / Tetrahedron Letters 47 (2006) 3009–3012
3011
H
H
Boc-N
NH-Boc
N
Boc-N
NH-Boc
N
N
N
O
N
O
N
3
3
AMC
AMC
Fmoc-NH
Fmoc-NH
O
O
i, ii
i, ii
i, iii
i, iv
H
H
Boc-N
NH-Boc
N
Boc-N
NH-Boc
N
N
N
O
N
O
N
AMC
AMC
Bz-Phe-Val-NH
Ac-Val-Gly-Leu-NH
O
O
v
v
Bz-Phe-Val-Arg-AMC 4
Ac-Val-Gly-Leu-Arg-AMC 5
Figure 3. (i) 3% DBU in DMF; (ii) Fmoc-Aaa-OH, PyBOPÒ, HOBt, DIEA, DMF; (iii) Bz2O, DIEA, THF; (iv) Ac2O, DIEA, THF; (v) TFA/DCM.
Figure 4. HPLC profile of crude peptide (a) 4 and (b) 5. (HPLC conditions: Vydac peptide/protein C18 column; gradient: 20–100% B in A over
20 min, 1 mL/min; A: 0.1 TFA aq; B: MeCN/water/TFA (90:10:0.1).
2. (a) Albericio, F.; Van Abel, R.; Barany, G. Int. J. Pept.
Protein Res. 1990, 35, 284–286; (b) Yan, L. Z.; Edwards,
P.; Flora, D.; Mayer, J. P. Tetrahedron Lett. 2004, 45,
923–925.
Investigations are currently underway to extend the
approach to the synthesis of p-nitroanilides and 7-amido-
4-trifluoromethylcoumarins.
3. Bernhardt, A.; Drewello, M.; Schutkowski, M. J. Pept.
Res. 1997, 50, 143–152.
4. Bonnet, M.; Bradley, M.; Kilburn, J. D. Tetrahedron Lett.
1996, 37, 5409–5412.
In summary, our recently developed side-chain anchor-
ing strategy is an efficient method for preparing peptides
modified on the C-terminal carboxyl group of arginine.
5. Zhong, H. M.; Greco, M. N.; Maryanoff, B. E. J. Org.
Chem. 1997, 62, 9326–9330.
6. Edwards, P. D.; Mauger, R. C.; Cottrell, K. M.; Morris,
F. X.; Pine, K. K.; Sylvester, M. A.; Scott, C. W.; Furlong,
S. T. Bioorg. Med. Chem. Lett. 2000, 10, 2291–2294.
References and notes
1. Jensen, K. J.; Alsina, J.; Songster, M. F.; Vagner, J.;
Albericio, F.; Barany, G. J. Am. Chem. Soc. 1998, 120,
5441–5452.
´
7. Garcia, O.; Nicolas, E.; Albericio, F. Tetrahedron Lett.
2003, 44, 5319–5321.

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J. Beythien et al. / Tetrahedron Letters 47 (2006) 3009–3012
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8. Hamze, A.; Martinez, J.; Hernandez, J.-F. J. Org. Chem.
20. Gude, M.; Ryf, J.; White, P. D. Lett. Pept. Sci. 2003, 9,
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2004, 69, 8394–8402.
9. (a) Photaki, I.; Yiotakis, A. J. Chem. Soc., Perkin Trans.
1 1976, 259; (b) Atherton, E.; Cammish, L. E.; Goodard,
P.; Richards, J. D.; Sheppard, R. C. In Peptides 1984:
Proc. 18th European Peptide Symposium; Ragnarsson, U.,
Ed.; Almqvist and Wiksell: Stockholm, 1984; pp 153–
156.
10. Estep, K. G.; Neipp, C. E.; Stramiello, L. M. S.; Adam,
M. D.; Allen, M. P.; Robinson, S.; Roskamp, E. J. J. Org.
Chem. 1998, 63, 5300–5301.
11. Atwal, K. S.; Ahmed, S. Z.; O’Reilly, B. C. Tetrahedron
Lett. 1989, 30, 7313–7316.
12. Shey, J.-Y.; Sun, C.-M. Synlett 1998, 1423–1425.
13. Martinez-Perez, J. A.; Pickel, M. A.; Caroff, E.; Waggon,
W. D. Synlett 1999, 1875–1878.
14. Poss, M.; Iwanowicz, E.; Reid, J. A.; Lin, J.; Gu, Z.
Tetrahedron Lett. 1992, 33, 5933–5936.
15. Yan, B.; Nguyen, N.; Liu, L.; Holland, G.; Raju, B. J.
Comb. Chem. 2000, 2, 66–74.
16. Harris, J. L.; Backes, B. J.; Leonetti, F.; Mahrus, S.;
Ellman, J. A.; Craik, C. S. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 7754–7759.
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Harris, J. L.; Craik, C. S.; Ellman, J. A. J. Org. Chem.
2002, 67, 910–915.
21. Resin 3 (0.2 g; loading 0.26 mmol/g) pre-swollen with
DMF was treated twice with 3% DBU in DMF (5 min,
15 min). After washing the resin with DMF, it was
treated with Fmoc-Val-OH (70 mg, 0.2 mmol), HOBt
(10 mg, 0.065 mmol), PyBOPÒ (100 mg, 0.21 mmol) and
DIEA (100 ll, 0.6 mmol) in a minimum volume of DMF.
The mixture was left to stand with gentle agitation for 1 h,
after which time the resin was washed with DMF and the
coupling reaction repeated with fresh reagents for 30 min.
The resin was removed by filtration, washed with DMF
and then treated twice with 3% DBU in DMF as described
above. The resin was washed with DMF, and a mixture
of Fmoc-Phe-OH (80 mg, 0.2 mmol), HOBt (10 mg,
0.065 mmol), PyBOPÒ (100 mg, 0.21 mmol) and DIEA
(100 ll, 0.6 mmol) in DMF was added to the resin and it
was gently agitated for 30 min, after which time the TNBS
test was negative. Following removal of the final Fmoc
group with DBU in DMF, the resin was end-capped by
treatment with benzoic anhydride (100 mg, 0.44 mmol)
and DIEA (100 ll) in THF. The resin is removed by
filtration, washed with DMF, isopropanol, H2O, THF,
isopropanol, MeOH, ether, hexane and dried overnight.
The peptide was cleaved from the resin by treatment with
TFA/DCM (1:1) for 2 h. The resin was removed by
filtration and the peptide precipitated from the filtrate by
the addition of ether. The precipitate was washed with
ether and dried under vacuum to afford the desired peptide
4 (30 mg, 73%); m/z (+ES) 682.53 (MH+), calcd for
C37H44N7O6 682.33.
18. Doerner, B.; White, P. In Peptides 1998: Proc. 25th
European Peptide Symposium; Bajusz, S., Hudecz, F.,
´
´
Eds.; Akademiai Kiado: Budapest, 1999; pp 90–91.
19. Yong, Y.-F.; Kowalski, J. A.; Lipton, M. A. J. Org. Chem.
1997, 62, 1540–1542.