A nonacid degradable linker for solid-phase synthesis

Article abstract of DOI:10.1021/ol701879s

Synthesis and applications of two new nonacid degradable linkers as an alternative to the Wang linker for solid-phase synthesis are described. Resin from linker 2 looks superior to linker 1 in terms of yields for both anchoring of the first building block and cleavage and in terms of higher purity of the final product. Use of linker 2 avoids side reactions associated with the use of Wang resin due to an undesired cleavage during final acid treatment.

Full text of DOI:10.1021/ol701879s

ORGANIC
LETTERS
2007
Vol. 9, No. 21
4319-4322
A Nonacid Degradable Linker for
Solid-Phase Synthesis
Aina Colombo,† Natalia De la Figuera,† Joan Carles Ferna`ndez,†
|
Dolors Ferna´ndez-Forner,‡ Fernando Albericio,*,†,§, and Pilar Forns*,†
Almirall-Barcelona Science Park Unit, Josep Samitier 1-5, E-08028 Barcelona, Spain,
Research Center, Almirall & Prodesfarma, Cardener 68-74, E-08024 Barcelona,
Spain, Institute for Research in Biomedicine, Barcelona Science Park, UniVersity of
Barcelona, Josep Samitier 1-5, E-08028 Barcelona, Spain, and Department of Organic
Chemistry, UniVersity of Barcelona, 08028-Barcelona, Spain
albericio@pcb.ub.es; pforns@pcb.ub.es
Received August 2, 2007
ABSTRACT
Synthesis and applications of two new nonacid degradable linkers as an alternative to the Wang linker for solid-phase synthesis are described.
Resin from linker 2 looks superior to linker 1 in terms of yields for both anchoring of the first building block and cleavage and in terms of
higher purity of the final product. Use of linker 2 avoids side reactions associated with the use of Wang resin due to an undesired cleavage
during final acid treatment.
Solid-phase synthesis is a convenient and suitable method
for the synthesis of peptides and small molecules.1 A key
feature of the solid phase approach is the linker,2 and
therefore, recent years have witnessed an explosion in the
research and development of novel linkers for polymer-
supported synthesis3 as well as in the publication of several
reviews4 covering this area. Originally, solid-phase synthesis
focused on the synthesis of peptides and nucleotides, but
the advent of combinatorial chemistry techniques triggered
the demand for a wider range of linkers. The ideal linker
should allow easy attachment of the starting material to the
support, be stable under a broad variety of reaction condi-
tions, and enable selective cleavage at the end of a synthesis
without causing damage to the product or cleavage of the
proper linker, which could be reincorporated into the final
product. This is more important in the case of a nonintegral
linker that is attached to the resin core through a chemical
bond.2
† Almirall-Barcelona Science Park Unit.
‡ Almirall & Prodesfarma.
§ Barcelona Science Park, University of Barcelona.
| Department of Organic Chemistry, University of Barcelona.
(1) (a) Solid Phase Synthesis: A practical guide; Albericio, F., Kates,
S. A., Eds.; Marcel Decker: New York, 2000. (b) Handbook of Combi-
natorial Chemistry; Nicolau, K. C.; Hanko, R.; Hatwig, W., Eds.; Wiley-
VCH: Winheim, 2002.
In our group, different acid-labile linkers were used for
the synthesis of small organic molecule libraries,5 and in
some cases, unexpected results due to the cleavage of the
linker during the synthesis6 or in the cleavage stage7 were
observed. The side product formed can contaminate the crude
(2) Linkers, as defined by Bradley et al. (see ref 4d), can be considered
simply as immobilized protecting groups and will be classified into one of
two types: (i) Integral linkers in which part of the solid support core forms
part or all of the linker and (ii) nonintegral (or grafted) linkers in which the
linker is attached to the resin core. A linker which has been prepared in
solution will be defined as a unloaded linker.
(3) (a) Okayama, T.; Burritt, A.; Hruby, V. J. Org. Lett. 2000, 2, 1787-
1790. (b) Gu, W.; Silverman, R. B. Org. Lett. 2003, 5, 415-418. (c) Liley,
M. J.; Jonson, T.; Gibson, S. E. J. Org. Chem. 2006, 71, 1322-1329. (d)
Kumar, A.; Ye, G.; Ahmadibenil, Y.; Parang, K. J. Org. Chem. 2006, 71,
7915-7918. (e) Kurosu, M.; Biswas, K.; Crick, D. C. Org. Let. 2007, 9,
1141-1144.
(4) (a) Blaney, P.; Grigg, R.; Sridharan, V. Chem. ReV. 2002, 102, 2607.
(b) Wills, A. J.; Balasubramanian, S. Curr. Opin. Chem. Biol. 2003, 7, 346-
352. (c) Guillier, F.; Orain, D.; Bradley, M. Chem. ReV. 2000, 100, 2091-
2157. (d) Gil, C.; Bra¨se, S. Curr. Opin. Chem. Biol. 2004, 8, 230-237.
10.1021/ol701879s CCC: $37.00
© 2007 American Chemical Society
Published on Web 09/15/2007

product, and even more damaging, back alkylation of the
target compound can occur.
alcohol is the 2-methoxybenzyl alcohol,14 which can be
masked by the use of the corresponding aldehyde. The
carboxylic acid can be attached to another phenyl ring, and
both rings can be bound through a Suzuki coupling reaction.
Thus, these two moieties can be coupled through two
different positions to give two regioisomer linkers (Scheme
1).
This side reaction is more severe when strong acid
conditions are used to cleave the final product.8 However,
undesired cleavage can take place with a Wang-type resin,
which requires milder TFA conditions for liberating the final
product (Figure 1). Thus, in peptide synthesis, Tsikaris et
Scheme 1. Possible Structures for the Linkers Proposed
Figure 1. Dual cleavage of Wang-type resin.
al.9 have described the incorporation of the p-hydroxybenzyl
moiety cleaved from the Wang resin into the N of the
C-terminal amide of a peptide during TFA cleavage.
Similarly, Martinez et al.10 have described the alkylation of
the indol ring of Trp-containing peptides by the p-hydroxy-
benzyl moiety. Furthermore, Stanger and Krchnak have
reported formation of O-(4-hydroxy)benzyl derivatives.11 The
use of the Wang resin for the solid-phase preparation of small
molecules has led to the introduction of impurities due to
the undesired cleavage from the resin (no cleavage at the
benzyl position) or from a back alkylation of the p-
hydroxybenzyl cation in the case of furopyridine and
furoquinoline target derivatives.12
First of all, linker 1 was synthesized from the commercially
available 3-formyl-4-methoxyphenylboronic acid and the
3-(4-bromophenyl) propanoic acid, which was first protected
in the form of the tert-butyl ester, through a Suzuki reaction.
Protection of the carboxylic acid is translated into a better
yield for the coupling reaction. Finally, the acidolysis of the
t-butyl ester followed by the reduction of the aldehyde render
the target linker 1 with an overall yield of 64% (Scheme 2).
Scheme 2. Synthesis of Linker 1a
The goal was to develop a more stable Wang-type linker
able to liberate the compound with 20-95% TFA in DCM.
As the Wang linker is based on a p-alkoxybenzyl alcohol,
the new linker should contain a benzyl alcohol activated by
a non cleavable electron-donating group in either the ortho
or para position and then a carboxylic group for easy
attachment to an amino resin.13 Furthermore, the linker
should be easy to prepare. The simplest activated benzyl
(5) (a) Fernandez, J.-C.; Sole-Feu, L.; Fernandez-Forner, D.; de la
Figuera, N.; Forns, P.; Albericio, F. QSAR Comb. Sci. 2006, 25, 961-965.
(b) Fernandez, J.-C.; Sole-Feu, L.; Fernandez-Forner, D.; de la Figuera,
N.; Forns, P.; Albericio, F. Tetrahedron Lett. 2005, 46, 581-585. (c) Sevilla,
S.; Forns, P.; Fernandez, J.-C.; de la Figuera, N.; Eastwood, P.; Albericio,
F. Tetrahedron Lett. 2006, 47, 8603-8606.
a
i) Isobutylene, DCM, -78 °C; ii) Pd(PPh3)4, toluene-EtOH
(9:1), 3-formyl-4-methoxyphenylboronic acid, 24 h, 90 °C; iii)
DCM-TFA (1:1); iv) NaBH4.
(6) Colombo, A.; Fernandez, J. C.; de la Figuera, N.; Forns, P.; Albericio,
F. QSAR Comb. Sci. 2005, 24, 913-922.
Synthesis of linker 2 was performed in the same way as
linker 1 (Scheme 3), but because the boronic acid required
is not commercially available, it was synthesized from
2-methoxy-4-bromobenzaldehyde (6). Linker 2 was prepared
with an overall yield of 80%.
(7) Yraola, F.; Ventura, R.; Vendrell, M.; Colombo, A.; Fernandez, J.-
C.; de la Figuera, N.; Fernandez-Forner, D.; Royo, M.; Forns, P.; Albericio,
F. QSAR Comb. Sci. 2004, 23, 145-152.
(8) For instance, Gu and Silverman (see ref 3b) designed a backbone
linker stable to reflux TFA.
(9) Stathopoulos, P.; Papas, S.; Tsikaris, V. J. Pept. Sci. 2006, 16, 227-
232.
Linkers 1 and 2 were loaded onto aminomethyl-polysty-
rene resin with HBTU and DIEA [until no primary amine
(10) Giraud, M.; Cavalier, F.; Martinez, J. J. Pept. Sci. 1999, 5, 457-
461.
(11) Stanger, K. J.; Krchnack, V. J. Comput. Chem. 2006, 8, 652-654.
(12) Cironi, P.; Tulla-Puche, J.; Barany, F.; Albericio, F.; AÄ lvarez, M.
Org. Lett. 2004, 6, 1405-1408.
(13) Gu and Silverman (ref 3b) incorporated the precursor of their
backbone linker to the resin through a metal-catalyzed coupling reaction.
(14) Phenylmethylethers are rather stable to acid conditions, and even
in the case of cleavage, the methyl cation formed is less reactive in
comparison with benzyl cations.
4320
Org. Lett., Vol. 9, No. 21, 2007

Cleavage of the anchored Fmoc-Leu-OH to resins 9 and
10 was studied with different TFA concentrations at room
temperature and carrying out one or two treatments. As
shown in Table 2, resin 10 is more labile than 9. Thus, 25%
Scheme 3. Synthesis of Linker 2a
Table 2. Yields and Purities of Cleaved Products
entry
resin
% TFA
time
% yield
% puritya
1
2
3
4
5
6
7
8
9
9
95
95
50
50
95
95
50
50
25
25
1 h
2 × 1 h
1 h
81
95
53
70
98
98
92
95
97
95
83
85
87
85
98
100
99
100
97
2 × 1 h
a
10
1 h
i) bis(pinacolato) diboron, PdCl2(dppf), dioxane, 24 h, 90 °C;
ii) 4, Pd(PPh3)4, toluene-EtOH (9:1), 24 h, 90 °C; iii) DCM-
TFA (1:1); iv) NaBH4.
2 × 1 h
1 h
2 × 1 h
1 h
was detected by the Kaiser test15 (12 h)], obtaining resin 9
from linker 1 and resin 10 from linker 2. Resins were
analyzed by HRMAS observing the total disappearance of
the proton of the CH2-NH2 from the aminomethyl resin and
the appearance of the proton corresponding to CH2OH and
OCH3, confirming the colorimetric test. Once the resins were
characterized, the stability of the linkers in acid conditions
was evaluated using 50% or 95% TFA at 60 °C during 1 h
by analyzing the filtrates by HPLC and HPLC-MS. No
cleavage of the linker was observed in any of the cases, but
a small percentage of linker (1 or 2) was observed in all
four along with HOBt coming for the anchoring to the resin.
For this reason, extensive washings of the resin were done.
Once the stability of the linkers anchored to the resin was
validated, the scope and limitations of these linkers were
tested. Thus, Fmoc-Leu-OH was incorporated onto resins 9
and 10 by several methods for 4 h, and the yield of the
piperidine-dibenzofulvene adduct was calculated by means
of UV (Table 1).16
10
2 × 1 h
100
a
Purity measured by HPLC.
TFA is enough for the total cleavage of the amino acid (#10)
instead of 95%, which is required for resin 9 (#2). Further-
more, a greater purity of Fmoc-Leu-OH is observed in the
cleaved samples performed on resin 10 (97-100% purity
for resin 10 vs 83-87% purity for resin 9).
These results are in line with those expected because in
resin 10 the electron-donating phenyl group is at the para
position with respect to the hydroxymethyl, increasing the
stability of the benzylic carbocation.
Next, to compare the stability of resins 9 and 10 to
commercial Wang resin, solid-phase synthesis of 2-ami-
nopyridine derivatives as well as of several model peptides
was performed.
For the synthesis of 2-aminopyridine derivatives (Scheme
4),17 the monitoring of the bromination and the loading of
Table 1. Coupling Conditions Tested with Both New Resins
Scheme 4. Synthesis of the 2-Amino-5-phenylpyridine17
%
entry resin
coupling conditions
treatments coupling
1
2
3
4
5
6
9
DIC/Fmoc-Leu-OH/HOBt/DMAP
(5:5:5:0.03)
1 × 1 h
2 × 1 h
1 × 1 h
2 × 1 h
1 × 1 h
2 × 1 h
1 × 1 h
2 × 1 h
1 × 1 h
2 × 1 h
1 × 1 h
2 × 1 h
66
100
96
100
90
100
17
23
73
100
75
98
DIC/Fmoc-Leu-OH/DMAP
(3:6:0.03)
HBTU/Fmoc-Leu-OH/DIEAa
(5:5:10)
10 DIC/Fmoc-Leu-OH/HOBt/DMAP
(5:5:5:0.03)
DIC/Fmoc-Leu-OH/DMAP
(3:6:0.03)
HBTU/Fmoc-Leu-OH/DIEAa
(5:5:10)
a
i) PBr3, DCM, 16 h; ii) 2-amino-5-bromopyridine, CsI, proton
a
DMAP was not used.
sponge, 24 h, 80 °C; iii) phenylboronic acid, Pd(PPh3)4, K2CO3,
DMF, 24 h, 90 °C; iv) DCM-TFA.
Table 1 shows that the symmetrical anhydride is the best
method for both linkers (entries 2 and 5) and that linker 9 is
acylated more easily than linker 10.
the 5-bromopyridin-2-amine was carried out by HRMAS to
confirm the total conversion in each step. The cleavage of
(15) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 594-598.
(16) Ma, Y.; Souveaux, E. Biopolymers 1989, 28, 965-973.
(17) Zhu, S.; Shi, S.; Gerritz, S. W.; Sofia, M. J. J. Comb. Chem. 2003,
5, 205-207.
Org. Lett., Vol. 9, No. 21, 2007
4321

the three resins was performed by two treatments with
different TFA solutions for 1 h each, at different temperatures
(Table 3).
Table 4. Coupling Conditions for the Synthesis of
Leu-enkephalina
coupling
conditions for
no. resin the Fmoc-Leu-OH
coupling
conditions for
the other aa
%
% mass
purity recovered
Table 3. Cleavage after the Synthesis of the
2-Amino-5-phenylpyridine
1
2
3
4
9
2×(DIC/AA/DMAP)
HBTU/DIEA/AA 62
(5:10:5)
43
48
98
99
(5:10:1)
2×(HBTU/DIEA/AA)b HBTU/DIEA/AA 63
no. resin cleavage solution temp yield (%) 13 (%)a 14 (%)a
(5:10:5)
(5:10:5)
10 2×(DIC/AA/DMAP)
DIC/AA/HOBt
93
1
2
3
4
5
6
Wang TFA-DCM (95:5) rt
Wang TFA-DCM (1:1) rt
100
76
15
60
22
40
45
97
96
98
95
60
55
-
-
-
(5:10:1)
(5:10:1)
2×(HBTU/DIEA/AA)b HBTU/DIEA/AA 92
(5:10:5)
(5:10:5)
9
TFA-DCM (95:5) rt
TFA-DCM (95:5) 60 °C
TFA-DCM (95:5) rt
TFA-DCM (95:5) 60 °C
a
b
9
Purity measured by HPLC at 254 nm. DMAP was not used.
10
10
100
-
a
Purity measured by HPLC at 210 nm.
Table 5. Results of the Synthesis of Peptides 15-17
no.
peptide
yield (%)
purity (%)a
As shown in Table 3, no byproducts were detected with
1
2
3
15
16
17
93
88
97
97
98
92
the two linkers (#3-6), and the target compounds were
obtained with good purity. On the other hand, Wang resin
released the unwanted compound 14 through an undesirable
cleavage by the benzylic ether (#1-2). Furthermore, Wang
resin showed a better lability allowing a total cleavage of
the compound at room temperature. This could be related to
the dual cleavage point. Resin 10 was optimal because total
cleavage was obtained at 60 °C.
A model peptide Leu-enkephalin (H-Tyr-Gly-Gly-Phe-
Leu-OH) was synthesized on the two resins (9 and 10) using
the Fmoc/tBu strategy. Final cleavage was performed with
TFA-TIS-H2O (95:2.5:2.5) (2 × 1 h). Table 4 shows that
resin 10 (#3-4) renders better purities and yields. Resin 9
(#1-2) resulted in a impurity of the Leu deletion peptide.
Finally, model peptides, H-Tyr-Leu-Ser-Gly-Ala-Asn-
Leu-Asn-Leu-OH (15), H-Arg-Pro-Gly-Leu-Leu-Asp-Leu-
Lys-OH (16), and H-Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-
Lys-OH (17), were synthesized with excellent purity and
yields on resin 10 (Table 5).
a
Purity measured by HPLC at 254 nm.
of anchoring of the first protected amino acid; (ii) cleavage
yield of the final product; and (iii) higher purity of the final
product. Any of them render undesired cleavage, which can
jeopardize the synthesis. This strategy can be used for
developing new acid-labile linkers.
Acknowledgment. This work was partially supported by
funds from CICYT (CTQ2006-03794/BQU), Laboratorios
Almirall, and the Barcelona Science Park. Aina Colombo
thanks Almirall-Prodesfarma for providing a predoctoral
fellowship. We thank Dr. Maria Antonia Molins for the
HRMAS experiments.
In summary, two non acid degradable linkers were
synthesized and tested for the solid phase of both peptides
and small molecules. Although both linkers form the same
carbocation during the cleavage stage, the different position
of the phenyl moiety confers different stabilities of this
carbocation to both. In this sense, the resin derived from
linker 2 gives better results in several parameters: (i) yield
Supporting Information Available: Synthesis and NMR
spectroscopic data of the compounds and the new resins,
protocols used for peptide synthesis, and details of HPLC
measurements. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL701879S
4322
Org. Lett., Vol. 9, No. 21, 2007