New stable backbone linker resins for solid-phase peptide synthesis.

Article abstract of DOI:10.1021/ol027212g

[reaction: see text] Two new 4-methoxybenzaldehyde backbone linker resins were developed for the solid-phase synthesis of peptides. The linkers are very stable during the cleavage of common protecting groups for amines (Fmoc, Boc) and carboxylic acids (Me, All, tBu) in peptide synthesis. Cleavage from the resin with refluxing TFA is sufficiently mild for peptides containing polar and nonpolar amino acids.

Full text of DOI:10.1021/ol027212g

ORGANIC
LETTERS
2
003
Vol. 5, No. 4
15-418
New Stable Backbone Linker Resins for
Solid-Phase Peptide Synthesis
Wenxin Gu and Richard B. Silverman*
4
Department of Chemistry and Drug DiscoVery Program, Northwestern UniVersity,
EVanston, Illinois 60208-3113
agman@chem.northwestern.edu
Received October 31, 2002
ABSTRACT
Two new 4-methoxybenzaldehyde backbone linker resins were developed for the solid-phase synthesis of peptides. The linkers are very
stable during the cleavage of common protecting groups for amines (Fmoc, Boc) and carboxylic acids (Me, All, tBu) in peptide synthesis.
Cleavage from the resin with refluxing TFA is sufficiently mild for peptides containing polar and nonpolar amino acids.
In 1963, Merrifield reported the concept and initial demon-
stration of solid-phase peptide synthesis (SPPS). SPPS has
coupling of N-protected amino acids. After elongation of the
peptide chains, the acid-labile linkage is cleaved with a TFA
solution to release the expected peptides. (4-Formyl-3,5-
1
developed into a convenient and popular method for the
synthesis of peptides and other small molecules. The key
feature of solid-phase synthesis is the linker element, which
is a molecule that keeps the intermediates bound to the
support during solid-phase synthesis. Linkers should allow
easy attachment of the starting material to the support, be
stable under a broad variety of reaction conditions, and yet
enable selective cleavage at the end of a synthesis without
damage to the product. Many linkers have been developed
for SPPS, among which the backbone linker strategy is highly
2
dimethoxyphenoxy)alkyl- (Figure 1, R ) R′ ) OMe), (4-
Figure 1.
2
,3
effective. This approach offers a simple and direct way to
prepare a variety of C-terminal modified and cyclic peptides.
The initial dipeptide is attached to the backbone linker in
two steps: reductive amination with amino acid esters and
formyl-3-methoxyphenoxy)alkyl- (Figure 1, R ) OMe, R′
3
)
H), and (4-formylphenoxy)alkyl- (Figure 1, R ) R′ )
3
d
*
Corresponding author. Phone: (847) 491-5653. Fax: (847) 491-7713.
H) linkers have been used for the synthesis of a variety of
peptide and nonpeptide compounds. The backbone linker
strategy is particularly useful for preparing C-terminal
modified peptides. However, this strategy suffers from
serious problems when synthesizing unprotected peptides and
cyclic peptides. These backbone linkers are very acid labile
as a result of the electron-donating substituents on the
benzylamines (di- or (trialkoxybenzyl)amines); therefore,
acid-labile protecting groups for both the amine (Boc) and
the acid (tert-Bu ester) cannot be utilized in this strategy,
which restricts the usefulness of these resins in peptide
(
1) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154.
(
2) (a) Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.;
Masada, R. I.; Hudson, D.; Barany, G. J. Org. Chem. 1990, 55, 3730-
3
743. (b) Jensen, K. J.; Alsina, J.; Songster, M. F.; Vagner, J.; Albericio,
F.; Barany, G. J. Am. Chem. Soc. 1998, 120, 5441-5452. (c) Alsina, J.;
Yokum, T. S.; Albericio, F.; Barany, G. J. Org. Chem. 1999, 64, 8761-
8
769.
(
3) (a) Harikrishnan, L. S.; Hollis Showalter, H. D. Synlett 2000, 1339-
1
7
7
341. (b) Fivush, A. M.; Willson, T. M. Tetrahedron Lett. 1997, 38, 7151-
154. (c) Sarantakis, D.; Bicksler, J. J. Tetrahedron Lett. 1997, 38, 7325-
328. (d) Swayze, E. E. Tetrahedron Lett. 1997, 38, 8465-8468. (e)
Bilodeau, M. T.; Cunningham, A. M. J. Org. Chem. 1998, 63, 2800-2801.
(
f) Kearney, P. C.; Fernandez, M.; Flygare, J. A. J. Org. Chem. 1998, 63,
1
96-200.
1
0.1021/ol027212g CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/23/2003

synthesis. Acid-stable Fmoc strategy can be used in peptide
synthesis, but it has been found that with backbone linker-
anchored dipeptides, base-promoted removal of Fmoc, even
at the dipeptidyl stage, is accompanied by almost quantitative
benzaldehyde afforded the 4-alkoxybenzaldehyde resin 6. To
determine the acid stability of the ether bond of 6, the resin
was treated with refluxing TFA for 3 h, which are conditions
employed for the removal of a 4-methoxybenzyl group in
amides and is a potential deprotection method for solid-phase
4
diketopiperazine formation. Although diketopiperazine for-
7
mation was not observed with tert-Bu ester protection, the
requisite acid deprotection conditions for the tert-Bu ester
causes cleavage of the peptide from the resin.
peptide synthesis. Unfortunately, cleavage of the resin was
observed. One problem occasionally encountered with back-
bone linkers attached to polystyrene as aryl benzyl ethers is
that cleavage of the entire linker can compete with C-N
A stable aldehyde linker that is compatible with acid-labile
protecting groups and can be utilized for solid-phase peptide
and cyclic peptide synthesis would fill this void. Recently,
8
bond cleavage, as we encountered.
To avoid this problem, we attached 4-methoxybenzalde-
hyde to the resin via an alkyl chain instead of an ether
linkage. As shown in Scheme 2, the iodination of 4-meth-
5
a 4-alkoxybenzyl-derived linker was reported. This linker
allows Boc strategy, but cleavage of the peptide from the
resin requires hydrofluoric acid protocols. Furthermore, the
initial dipeptide has to be synthesized in solution before being
attached to the linker on the solid support. Therefore, its use
is restricted. Here we report two new resin-bound linkers (1
and 2), which are very stable during removal of common
protecting groups for amines (Fmoc, Boc) and carboxylic
acids (Me, All, tert-Bu), and the product can be cleaved from
the resin with refluxing TFA.
Scheme 2. Synthesis of 4-Methoxybenzaldehyde Resin 1a
a
Reagents and conditions: (a) I
2
, F-TEDA, CH
3
CN, 12 h, rt,
8
3
7%; (b) 4, 9-BBN, THF, Pd(PPh
days.
3
)
4
, 2 M Na CO , DMF, 110 °C,
2
3
Our initial design was a 4-alkoxybenzaldehyde linker,
which was bound to polystyrene through an aryl alkyl ether.
As shown in Scheme 1, Merrifield resin 3 was treated with
oxybenzaldehyde (7) with F-TEDA and I
2
in acetonitrile
afforded 8. Suzuki coupling of 4 and 8, with Pd(PPh as
as the base, afforded the 4-meth-
oxybenzaldehyde resin 1. To avoid the presence of “black”
9
3 4
)
1
0
2 3
the catalyst and K CO
Scheme 1. Synthesis of 4-Alkoxybenzaldehyde Resina
1
1
Pd species in 1 after the Suzuki reaction, a similar linker
was synthesized by another approach (Scheme 3). Allyl-
2
ation of 4-hydroxybenzaldehyde 9 with allyl bromide fol-
lowed by Claisen rearrangement at 200 °C afforded 11,
which was treated with iodomethane using K CO as the base
2 3
Scheme 3. Synthesis of 4-Methoxybenzaldehyde Resin 2a
a
Reagents and conditions: (a) allylmagnesium chloride, toluene,
2 2
60 °C; (b) 9-BBN, H O , KOH, THF; (c) 4-hydroxybenzaldehyde,
DEAD, PPh , THF; (d) neat refluxing TFA, 3 h.
3
allylmagnesium chloride to afford 4.3a,6 Hydroboration-
oxidation of 4 with 9-BBN and H solution afforded
alcohol 5.3 A Mitsunobu reaction of 5 with 4-hydroxy-
2 2
O
a
(
4) Fresno, M.; Alsini, J.; Barany, G.; Albercio, F. Tetrahedron Lett.
998, 39, 2639-2642.
5) Bourne, G. T.; Meutermans, W. D. F.; Alewood, P. F.; McGeary, R.
P.; Scanlon, M.; Watson, A. A.; Smythe, M. L. J. Org. Chem. 1999, 64,
1
a
Reagents and conditions: (a) allyl bromide, K
2
CO
CO
3
, acetone,
, acetone,
(
refluxing, 97%; (b) 200 °C, 5 h, 86%; (c) MeI, K
refluxing, 92%; (d) 4, Grubbs Ru catalyst, CH Cl , refluxing, 24
2 2
2
3
3
095-3101.
6) Hu, Y.; Porco, J. A.; Ladadie, J. W.; Gooding, O. W. J. Org. Chem.
998, 63, 4518-4521.
(
h.
1
416
Org. Lett., Vol. 5, No. 4, 2003

Scheme 4. Solid-Phase Synthesis of Dipeptidesa
a
2
1
Reagents and conditions: (a) H-Leu-OR ‚HCl (10 equiv), NaBH(OAc)
3
(10 equiv), 1% HOAc in DMF, 5 h; (b) R -Leu-OH (10 equiv),
/DMF, 6 h; (c) neat TFA, rt, 1 h; (d) 37:2:1 CHCl /AcOH/NMM, Pd(PPh (5 equiv),
O/THF; (f) piperidine in DMF (1:4), 30 min; (g) 50% TFA in CH Cl , rt, 30 min; (h) neat TFA, refluxing 3
HATU (10 equiv), DIPEA (15 equiv), 9:1 CH
h; (e) 1 N LiOH in 1:7 H
2
Cl
2
3
3 4
)
3
2
2
2
h >80% cleavage yield of all dipeptides.
to give 12. Metathesis of 12 and 4 using the Grubbs Ru
catalyst afforded resin 2.12
as the coupling reagent provided support-bound di-
peptides.
14
To determine the stability of the 4-methoxybenzaldehyde
resins 1 and 2, five common protecting groups of amines
and carboxylic acids were employed in the support-bound
dipeptides 16-20. The tert-Bu ester of 16 was removed with
neat TFA for 1 h. The allyl ester of 17 was removed with
To examine the stability of the 4-methoxybenzaldehyde
resins 1 and 2 during solid-phase peptide synthesis, we
synthesized dipeptide H-Phe-Leu-OH using different pro-
tecting groups. As shown in Scheme 4, H-Leu-OR was
loaded onto the support by reductive amination employing
2
3 4
Pd(PPh ) for 3 h. The methyl ester of 18 was removed with
NaBH(OAc) in 1% acetic acid in DMF to afford 13-15.
3
8,13
2
1 M LiOH solution in THF for 12 h. The Fmoc group of 18
was removed with piperidine in DMF (1:4) for 30 min. The
Boc group of 19 was removed with 50% TFA in methylene
chloride for 30 min. After deprotection, the filtrates of every
reaction were analyzed, and it was shown that no cleavage
from the resin resulted. Compounds 18, 21, 22, and 20 were
then cleaved from the resin with refluxing TFA for 3 h to
afford dipeptides 23-26, respectively, in over 80% cleav-
age yield, based on the loading level of 4-methoxybenz-
aldehyde linkers 1 and 2. These cleavage reactions also
demonstrate that peptide bonds and Fmoc and methyl ester
protecting groups are stable to the refluxing TFA condi-
tions. Racemization was not observed during the reactions
as determined by chiral HPLC analysis of Fmoc-Phe-Leu-
OMe.
To minimize racemization, H-Leu-OR and the reductant
were premixed in 1% acetic acid in DMF followed by
addition of the resin-bound aldehyde. Acylation of the
1
resulting secondary amines 13-15 with R -Phe-OH using
the highly activated azabenzotriazole-based reagent HATU
(
7) Brooke, G. M.; Mohammed, S.; Whiting, M. C. Chem. Commun.
997, 1511-1512.
8) Boojamra, C. G.; Burow, K. M.; Ellman, J. A. J. Org. Chem. 1995,
0, 5742-5743.
9) Zupan, M.; Iskra, J.; Stavber, S. Tetrahedron Lett. 1997, 38, 6305-
306.
1
6
6
(
(
(
10) Lee, Y.; Silverman, R. B. Org. Lett. 2000, 2, 3743-3746.
(11) Mannhold, R.; Kubinyi, H.; Timmerman, H. Combinatorial Chem-
istry: A Practical Approach; Bannwarth, W., Felder, E., Eds.; VCH
Publishers: Weinheim, Germany, 2000; Chapter 3, p 125.
(
12) Schwab, P.; Grubbs, R. H.; Ziller, J. W. J. Am. Chem. Soc. 1996,
18, 100-110.
13) Boojamra, C. G.; Burow, K. M.; Thompson, L. A.; Ellman, J. A. J.
1
(
Org. Chem. 1997, 62, 1240-1256.
(14) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.
Org. Lett., Vol. 5, No. 4, 2003
417

To demonstrate that polar peptides also are stable to the
refluxing TFA cleavage conditions, three other dipeptides
were synthesized by the same methodology described in
Scheme 4: D-Ser-L-Trp-OMe, L-Thr-L-Asn, and L-Tyr-L-Asp,
from resin-bound Boc-D-Ser-L-Trp-OMe, Boc-L-Thr-L-Asn-
OtBu, and Boc-L-Tyr-L-Asp(OtBu)-OtBu, respectively, were
synthesized in >80% yield, based on the loading level.
Both new resins are comparable in efficiency; the differ-
ences are that higher loading levels are possible with 1, but
carboxylic acid protecting groups. The peptides can be
removed from the solid support with refluxing TFA and
conditions that do not hydrolyze Fmoc or methyl ester
protecting groups and do not destroy amide bonds or
common amino acid side chains.
Acknowledgment. The authors are grateful to the finan-
cial support from the Institute for Bioengineering and
Nanoscience in Advanced Medicine at Northwestern Uni-
versity.
2
does not contain small amounts of insoluble black Pd
species.
In conclusion, we have developed two new stable 4-meth-
Supporting Information Available: Complete experi-
mental details and product characterization. This material is
available free of charge via the Internet at http://pubs.acs.org.
oxybenzaldehyde backbone linker resins for solid-phase
peptide synthesis. The 4-methoxybenzaldehyde linkers are
bound to the resin through a carbon-carbon bond and are
compatible with both acid- and base-labile amine and
OL027212G
418
Org. Lett., Vol. 5, No. 4, 2003