G. Subra et al.
treatment, the mechanism involving an oxazolinium-5-one
intermediate cleaved an amide or an ester bond and re-
leased amines or alcohols. The linker was demonstrated to
effectively and simply anchor different functional groups
(amines and alcohols). Further examples of the reverse
SPPS, a pseudo-peptide synthesis, and amino acid side-chain
anchoring shows the versatility of that new linker. Kinetic
cleavage studies clearly indicated the acid lability of this
linker, which can be used as an alternative to the trityl
linker in Fmoc-based synthesis strategies. Regarding the
straightforward anchoring, the attachment of other nucleo-
philes to a solid support through the Pip handle is under in-
vestigation and will be reported in due course.
Scheme 8. Preparation of C-terminal hydrazide peptides. TIS=triisoprop-
yl silane.
Experimental Section
Preparation of the Pip linker functionalized PS resin 1 (Pathway A, Two-
step procedure; Scheme 3)
100% TFA, 10% TFA in CH2Cl2, 1% TFA in CH2Cl2, and
10% AcOH in CH2Cl2. Aliquots of the solution were col-
lected at different times over 24 h and were analyzed by
HPLC. Ester and amide linkages behaved almost in the
same way under acidic conditions. The cleavage curves of
resin 8 are presented in Figure 1.[32]
Attachment of cyclohexane-1,4-dicarboxylic acid (resin 6): cis/trans-Cyclo-
hexane-1,4-dicarboxylic acid (cis/trans 77:23; 5.16 g, 30.0 mmol,
8.45 equiv) and HOBt (4.05 g, 30.0 mmol, 8.45 equiv) were dissolved in
DMF (20 mL), then DIC (4,69 mL, 30.0 mmol, 8.45 equiv) were added
and the mixture was gently stirred for 10 min. It was then added to ami-
nomethyl-PS resin (5 g, 0.71 mmolgÀ1, 3.55 mmol) preswollen in CH2Cl2,
and the reaction mixture was gently shaken for 6 h. After filtration, the
resin was washed with DMF (2ꢃ), MeOH, and CH2Cl2 (2ꢃ), and finally
dried at room temperature, under vacuum, to yield resin 6. The coupling
efficiency was checked by Kaiser and 2,4,6-trinitrobenzyenesulfonic acid
(TNBS) tests.
Attachment of d/l-pipecolic acid methyl ester (resin 7): d/l-Pipecolic acid
methyl ester hydrochloride (46; 2.15 g, 12 mmol, 3.4 equiv) was coupled
to resin 6 by using BOP (5.30 g, 12 mmol, 3.4 equiv), as activating agents,
in the presence of DIEA (5.22 mL, 30 mmol, 8.45 equiv). The reaction
mixture was allowed to react for 2 h in DMF, and the process was repeat-
ed once more. Finally, the resin was washed with DMF (2ꢃ), MeOH,
and CH2Cl2 (2ꢃ), and dried at room temperature under vacuum to yield
resin 7.
On-resin hydrolysis of pipecolic acid methyl ester (resin 1): A mixture of
a 2m aqueous solution of lithium hydroxide and tetrahydrofuran (200 mL
30:70 v/v) was added to resin 7. The resin was allowed to agitate on an
orbital shaker at room temperature for 24 h. The resin was washed with
H2O (3ꢃ), MeOH (2ꢃ), and CH2Cl2 (3ꢃ) to yield Pip-PS resin 1. The re-
sulting resin was dried under vacuum for 24 h.
~
*
Figure 1. Cleavage of resin 8. &: 100% TFA, : 10% TFA, : 1% TFA,
+: 10% AcOH.
Example of amine anchoring on Pip-PS resin: After the Pip-PS resin 1
(200 mg, 98 mmol, 0.49 mmolgÀ1) was swollen in CH2Cl2 for 30 min, and
washed with DMF (2ꢃ), it was added to DMF coupling solution
(5.4 mL) containing BOP (239 mg, 100 mm, 540 mmol, 5.5 equiv), DIEA
(140 mg, 188 mL, 200 mm, 1.08 mmol, 11 equiv), and Fmoc-1-amino-3-
aminopropane (160 mg, 100 mm, 540 mmol, 5.5 equiv) (Scheme 4). The
resin was gently stirred for 2 h and then washed with DMF (2ꢃ), MeOH,
and CH2Cl2 (2ꢃ). Then the resin was treated with a mixture of piperi-
dine/DMF (6 mL 20:80 v/v) for 3 min, and subsequently for additional
12 min. After removal of the deprotection solution, the resin was washed
by following a standard washing protocol (DMF (2ꢃ), MeOH, and
CH2Cl2 (2ꢃ)). In the next stage, the resin was treated with a solution of
m-toluoyl chloride (39 mg, 100 mm, 540 mmol, 5.5 equiv) and DIEA
(140 mg, 188 mL, 200 mm, 1.08 mmol, 11 equiv) in CH2Cl2 (5.4 mL). The
resin was gently stirred for 2 h and then washed (DMF (2ꢃ), MeOH,
and CH2Cl2 (2ꢃ)), and air dried. Cleavage with 100% TFA at room tem-
perature was carried out over 2 h with gentle stirring. The resin was fil-
tered and the TFA solution was evaporated under nitrogen. The residue
was dissolved in acetonitrile/water (1:1 v/v) and freeze dried. After lyo-
In general, the Pip linker was not stable under acidic con-
ditions, that is, after one hour treatment with TFA of the
amide and ester linkage, more than 80% of the product was
released from the resin, and the product was liberated from
the support after 12 h treatment with neat TFA. The linker
was not completely stable in 1% TFA either, since after one
hour 10% of the product was released from the support.
Conclusion
On the basis of the side reaction observed during arylpipera-
zine library generation, we developed a new linker for a
solid-phase synthesis, based on pipecolic acid. Upon acid
7552
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 7547 – 7553