Solid-phase synthesis: A linker for side-chain anchoring of arginine

Article abstract of DOI:10.1016/S0040-4039(03)01203-6

A new linker based on a chroman system is described for the side-chain anchoring of Arg and other guanidine-containing molecules. The system is compatible with the Fmoc/tBu solid-phase strategy, because the release of the final product is achieved by treatment with TFA in the presence of scavengers.

Full text of DOI:10.1016/S0040-4039(03)01203-6

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TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 5319–5321
Solid-phase synthesis: a linker for side-chain anchoring
of arginine
Oscar Garc´ıa,a Ernesto Nicola´sa,* and Fernando Albericioa,b,
*
aDepartment of Organic Chemistry, University of Barcelona, E-08028 Barcelona, Spain
bBarcelona Biomedical Research Institute, Barcelona Science Park, University of Barcelona, Josep Samitier 1,
E-08028 Barcelona, Spain
Received 9 April 2003; accepted 10 May 2003
Abstract—A new linker based on a chroman system is described for the side-chain anchoring of Arg and other guanidine-contain-
ing molecules. The system is compatible with the Fmoc/tBu solid-phase strategy, because the release of the final product is
achieved by treatment with TFA in the presence of scavengers. © 2003 Published by Elsevier Science Ltd.
1. Introduction
guanidine, the guanidine group is an important motif in
a broad range of therapeutic programmes. The protec-
tion of the guanidine group can be accomplished using
arylsulfonyl- or bis(alkoxycarbonyl)-based groups.4,5
The latter system involves a double protection and so
only arysulfonyl systems represent a real alternative to
be converted into a handle. However, only two p-
alkoxybenzenesulfonyl linkers have been described in
the literature.
A key aspect in all solid-phase programmes is to specify
the mode of attachment of the first building block to
the solid support.1 This is usually accomplished
through the use of bifunctional spacer molecules known
as handles or linkers.2 Such handles become attached
permanently to a functionalised resin at one end, often
through a stable amide bond, and are linked temporar-
ily to the growing molecule.3 At the end of the solid-
phase synthetic process, cleavage of the temporary
building blockꢀhandle bond results in release of the
molecule from the solid support. In this regard, handles
can be considered as temporary protecting groups.
Hundreds of handles have been described in the litera-
ture and the choice is such that they are compatible
with the majority of organic functional groups.1–3
Treatment with acid is considered a very convenient
cleavage method, but there are handles that are suscep-
tible to cleavage by other reagents/chemical mecha-
nisms such as electrophiles, nucleophiles, photolysis,
metals, oxidative and reductive conditions, and
cycloadditions/cycloreversions.1c Despite this myriad of
handles, there is a niche to be filled in terms of systems
for anchoring guanidine groups. Besides being present
in natural building blocks such as arginine or
Linker 1 was used to prepare a guanidinium-based
‘tweezer’ receptor6 and 2 was used to anchor Arg
through the side-chain to prepare small peptides.7 Both
of these examples required the use of strong acids such
as CF3SO3H or HF and are not therefore the best
choice for synthetic schemes that require mild condi-
tions, such as the Fmoc/tBu8 strategy used in peptide
chemistry. Finally, Bernhardt et al.9 have reported the
anchoring with low yields of Arg residue via its side-
chain to a Barlos resin, but acidolytical cleavage from
the resin is problematic and led to complex mixture of
products. So, as a consequence, they anchored Orn
residue and after cleavage guanidination was
performed.
Keywords: benzofuran; benzopyran; combinatorial chemistry; handle;
linker; protecting group; solid phase.
* Corresponding authors: Tel.: +34-93-402-9057; fax: +34-93-339-
7878 (E.N.); Tel.: +34-93-403-7088; fax: +34-93-403-7126 (F.A.);
e-mail: nicolas@qo.ub.es; albericio@pcb.ub.es
0040-4039/03/$ - see front matter © 2003 Published by Elsevier Science Ltd.
doi:10.1016/S0040-4039(03)01203-6

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O. Garc´ıa et al. / Tetrahedron Letters 44 (2003) 5319–5321
The work described here concerns a new linker strategy
(3) based on a chroman structure.
method first proposed by Yamaguchi et al.13 for similar
molecules.14 Practically quantitative yields were
obtained in the catalytic reduction of the double bond
and subsequent hydrolysis of the ethyl ester. The han-
dle (3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-
2-yl)acetic acid (8) was obtained with an overall yield of
47% after four steps. Incorporation of 8 to Val (internal
reference amino acid)15 containing p-methylbenzhydryl-
amine was carried out with DIPCDI/HOBt in DCM.
A key step in this strategy is the chlorosulfonation of
the resin 9. Chlorosulfonation of polystyrene resins is
usually carried out with ClSO3H in CHCl3 at reflux.16
However, these strong conditions are not necessary in
this case because the aromatic ring of the linker in 9 is
activated by the electron-donating substituents. Fur-
thermore, forcing conditions will also lead to chlorosul-
fonation of the aromatic rings of polystyrene itself.
After several trials, the chlorosulfonation was carried
out with 4 equiv. of freshly distilled ClSO3H in anhy-
drous CHCl3 under a nitrogen atmosphere for 30 min
at −10°C, then 30 min at 0°C, and 90 min at 25°C. The
resin was washed with cold H2O for 5 min, followed by
dioxane/H2O, dioxane, and DCM.
2. Results and discussion
Among the thirteen proteinogenic amino acids whose
side-chain requires protection, Arg is the most problem-
atic case. A system that satifies all the characteristics of
an ideal protecting group has not been found to date
for this residue.4 For peptides containing a single Arg
residue,
2,2,7,7,8-pentamethylchroman-6-sulfonyl
(Pmc)10 and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-
sulfonyl (Pbf)11 are the protecting groups of choice. By
considering the Pmc group as a model, the new linker
was prepared from 2,3,4-trimethylphenol (4) according
the synthetic pathway outlined in Scheme 1.
Reaction of 4 with TiCl4 (2.2 equiv.) followed by
addition of dichloromethyl methyl ether led after 4 h to
a mixture of 2-hydroxy-3,4,6-trimethylbenzaldehyde (5)
and 4-hydroxy-2,3,6-trimethylbenzaldehyde (93%). Sep-
aration of the two isomers was achieved easily by
crystallisation from ethanol/water to give 5 with 71%
overall yield (4-hydroxy-2,3,6-trimethylbenzaldehyde
derivative was obtained in 15% overall yield; ratio
5:1).12 Formation of the benzopyran structure (6, 75%)
was achieved by reaction of 5 with diethyl isopropyli-
denemalonate following a modified version of the
Incorporation of Fmoc-Arg-OAllyl (5 equiv.)17 was car-
ried out in CHCl3 in the presence of DIEA (10 equiv.)
for 8 h at 25°C.5 The resin was washed with DMF and
DCM. Acid hydrolysis and amino acid analysis (AAA)
of a sample of resin-bound peptide showed that the
incorporation of Fmoc-Arg-OAllyl took place to give a
79% yield. Cleavage of the Fmoc-Arg-OAllyl [97%
purity; MALDI-TOF MS (DHB): m/z: [M+H]+ 437. 78
(calcd 437.53); [M+K]+ 475.78 (calcd 476.62)] from the
resin was carried out with TFA/H2O(10:1) for 3 h at
Scheme 1.

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O. Garc´ıa et al. / Tetrahedron Letters 44 (2003) 5319–5321
5321
25°C. The crude compound was analyzed by HPLC
and had a purity of 97%.
M.; Felix, A.; Moroder, L.; Toniolo, C., Eds.; Georg
Thieme: Stuttgart, 2001; pp. 315–333.
5. Fmoc-Arg(Trt)-OH has also been described (Caciagli, V.,
Verdini, A. S. In Peptide Chemistry 1987; Shiba, T.;
Sakakibara, S., Eds.; Protein Research Foundation:
Osaka, Japan, 1998; pp. 283), but its poor solubility in
the solvents generally employed in SPPS has precluded its
use in peptide synthesis. On the other hand, the 5H-
dibenzo[a,d]cycloheptene-based groups (Noda, M.; Kiffe,
M. J. Peptide Res. 1997, 50, 329–335) appear to be more
promising for use in SPPS.
Unreacted chlorosulfonyl groups could be capped with
either Et2NH/DMF (1:19) or piperidine/DMF (2:8),
which can also be used to remove the Fmoc group.
After removal of the Fmoc, incorporation of Fmoc-
Phe-OH was carried with DIPCDI/HOBt in DCM. The
protected dipeptide was obtained with excellent purity,
as shown by HPLC after treatment with TFA in the
manner described above [97% purity; MALDI-TOF
MS (DHB): m/z: [M+H]+ 585.14 (calcd 584.53)].
6. Bonnat, M.; Bradley, M.; Kilburn, J. D. Tetrahedron
Lett. 1996, 30, 5409–5412.
7. Zhong, H. M.; Greco, M. N.; Maryanoff, B. E. J. Org.
Chem. 1997, 62, 9326–9330.
3. Conclusions
8. Abbreviations used for amino acids and the designations
of peptides follow the rules of the IUPAC-IUB Commis-
sion of Biochemical Nomenclature in J. Biol. Chem. 1972,
247, 977–983. The following additional abbreviations are
used: Boc, tert-butyloxycarbonyl; t-Bu, tert-butyl; Barlos
resin, Cl-TrtCl-resin, 2-chlorotrityl chloride-resin; DCM,
dichloromethane; DIEA, N,N-diisopropylethylamine;
DIPCDI, N,N%-diisopropylcarbodiimide; DMF, N,N-
dimethylformamide; Et2NH, diethylamine; Fmoc, 9-
A new handle, (3,4-dihydro-2,5,7,8-tetramethyl-2H-1-
benzopyran-2-yl)acetic acid, is easily synthesised and
can be used, after attachment to an amino resin and
subsequent chlorosulfonation, for anchoring arginine
derivatives (through their side-chain) as well as other
guanidine-containing molecules. Compounds are
released from the solid support by treatment with TFA
in the presence of scavengers. This strategy, which is
compatible with the Fmoc/tBu approach for peptide
synthesis, is currently being used in our laboratory for
the solid-phase preparation of C-terminal Arg p-
nitroanilide18 and cyclic peptides through side-chain
anchoring of Arg.19
fluorenylmethoxycarbonyl;
triazole; TFA, trifluoroacetic acid. Amino acid symbols
denote the -configuration.
HOBt,
1-hydroxybenzo-
L
9. Bernhardt, A.; Drewello, M.; Schutkowski, M. J. Peptide
Res. 1997, 50, 143–152.
10. Ramage, R.; Green, J.; Blake, A. J. Tetrahedron 1991, 47,
6353–6370.
11. Carpino, L. A.; Shroff, H.; Triolo, S. A.; Mansour, E. M.
E.; Wenschuh, H.; Albericio, F. Tetrahedron Lett. 1993,
34, 7829–7832.
Acknowledgements
12. Garc´ıa, O.; Nicola´s, E.; Albericio, F. Tetrahedron Lett.,
submitted for publication.
We are grateful to the University of Barcelona for a
predoctoral fellowship (O.G.). This work was partially
supported by CICYT (BQU2000-0235), Generalitat de
Catalunya [Grup Consolidat and Centre de Refere`ncia
en Biotecnologia].
13. (a) Yamaguchi, S.; Saitoh, T.; Kamiumezawa, M.;
Enomoto, H.; Kawase, Y. J. Heterocyclic Chem. 1992,
29, 755–758; (b) Yamaguchi, S.; Takahashi, K.; Kawase,
Y. J. Heterocyclic Chem. 1992, 29, 759–761.
14. The use of large excesses of diethyl isopropyldene-
malonate (16 equiv.) led to clear improvements in the
yield.
References
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