Recycling the versatile pipecolic linker

Article abstract of DOI:10.1021/cc100099z

The Pipecolic linker is a new highly versatile handle which immobilizes on solid support through a carboxylic acid function a wide range of amines, alcohols, and hydrazines. The anchoring step on pipecolic resin is very easy and efficient, and compounds are released with high purities upon acidic treatment. During this treatment, an oxazolonium intermediate is hydrolyzed, yielding the cleavage of ester or amide bond and the release of free carboxylic acid of the starting linker. In this study, we report the possibility of recycling the pipecolic resin after the use of several trifluoroacetic acid (TFA) cleavage cocktails. We demonstrate that it can be reused up to five times without significant loading decrease.

Full text of DOI:10.1021/cc100099z

J. Comb. Chem. 2010, 12, 747–753
747
Recycling the Versatile Pipecolic Linker
†
,‡
†
†
‡
Pawel Zajdel, Nicolas Masurier, Pierre Sanchez, Maciej Pawlowski,
†
†
†
†
†
Aude Kreiter, Ga e¨ l Nomezine, Christine Enjalbal, Muriel Amblard, Jean Martinez,
,
†
and Gilles Subra*
IBMM, Institut des Biomol e´ cules Max Mousseron, UMR 5247, UniVersit e´ s Montpellier I and II, CNRS,
Facult e´ de Pharmacie, 15 aV. C. Flahault, BP 14491, 34093 Montpellier Cedex 05, France, and Department
of Medicinal Chemistry, Jagiellonian UniVersity Medical College, 9 Medyczna Street,
30-688 Krak o´ w, Poland
ReceiVed June 11, 2010
The Pipecolic linker is a new highly versatile handle which immobilizes on solid support through a carboxylic
acid function a wide range of amines, alcohols, and hydrazines. The anchoring step on pipecolic resin is
very easy and efficient, and compounds are released with high purities upon acidic treatment. During this
treatment, an oxazolonium intermediate is hydrolyzed, yielding the cleavage of ester or amide bond and the
release of free carboxylic acid of the starting linker. In this study, we report the possibility of recycling the
pipecolic resin after the use of several trifluoroacetic acid (TFA) cleavage cocktails. We demonstrate that
it can be reused up to five times without significant loading decrease.
Introduction
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.
According to the proposed mechanism, during TFA
treatment, the oxazolonium ion is then attacked by a water
molecule yielding epimerized pipecolic acid residue (Scheme
Unexpected hydrolysis of the amide bond involving the
carboxylic function of pipecolic acid residue was reported a
few times in the literature, in particular during trifluoroacetic
6
1
,2
acid (TFA) treatment of peptides as well as in small
3
molecules libraries. The proposed mechanism is very similar
to the acid catalyzed hydrolysis of peptides containing
N-alkylated aminoisobutyric acid (AIB) residues involving
the formation of oxazolonium-5-one intermediate and the
release of free amino group (Scheme 1).
During the preparation of a library of compounds contain-
ing pipecolic acid residues, we observed that the reaction
rate of this acid promoted-hydrolysis is favored by electron-
1
, left) after the release of the target compound from the
solid support. In contrast to most TFA-labile linkers for
which a carbocation is generated on the resin upon cleavage,
4
Scheme 1. Cleavage of Amide Bond of Pipecolic Acid
Containing Compounds
3
donating substituents on the amino group. These observa-
tions are in accordance with results obtained by Sobolewski
et al. showing that protonated N-terminal peptides containing
pipecolic residue are less sensitive to hydrolysis than acylated
5
ones. This can be explained by a decrease in the electron
density of the carbonyl group of the pipecolic amide bond.
We took advantage of this acid-catalyzed mechanism to
6
design the pipecolic linker which consists of racemic
pipecolic acid linked to amino methyl polystyrene resin
through the electron donating and bulky cyclohexane dicar-
boxylic acid spacer (Scheme 2). This new linker was
demonstrated to effectively and simply anchor primary,
secondary, aromatic amino group as well as the alcohol and
phenol function. Further examples of the reverse solid-phase
peptide synthesis (SPPS), a pseudopeptide synthesis, amino
acid side-chain anchoring and preparation of C-terminal
hydrazide peptide showed the versatility of that new linker.
Scheme 2. Principle of Pipecolic Resin
*
To whom correspondence should be addressed. E-mail: gilles.subra@
univ-montp1.fr. Phone: +33 4 67 54 86 37. Fax: +33 4 67 54 86 54.
†
Universit e´ s Montpellier I.
Jagiellonian University Medical College.
‡
1
0.1021/cc100099z  2010 American Chemical Society
Published on Web 08/17/2010

7
48 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 5
Zajdel et al.
Scheme 3. Recycling Pip-PS Resin
Table 1. Successive Anchoring/Cleavage from Pip-PS Resin
a
[M+H]+ m/z found
resin no.
compound released
purity (%)
RT (min)
1.36
recovery yield (%)
2
3
4
5
6
Fmoc-aminoethanethiol
H-Ala-OMe, TFA
N-1 Fmoc-diaminopropane, TFA
N-1 Fmoc-diaminoethane, TFA
N-1 Fmoc-diaminobenzene, TFA
<90
300.4
104.0
297.3
283.3
331.2
51
75
79
73
90
b
b
99
0.46
99
99
99
1.37
1.35
1.54
b
a
The purity was calculated with integration of areas of peaks in the HPLC chromatograms at λ ) 214 nm. This polar compound was analyzed
using Atlantis C18 column in modified isocratic mode (see Experimental Section), purity was checked by H NMR analysis.
1
pipecolic linker is converted into free carboxylic acid and
recovers its original state. We leveraged this to recycle the
resin and reuse it several times.
obtained were weighed to determine the recovery yield
calculated on the basis of initial resin loading (0.42 mmol/
g). The results are presented in Table 1.
Between each round of anchoring/cleavage, extensive
washing steps were performed to recycle the Pip-PS resin.
This point is particularly important to remove unbound
materials and to neutralize TFA solution. After recycling and
drying the resin, the weight of the resin does not vary
significantly during successive experiments. More impor-
tantly, LC/MS analysis of cleaved compounds revealed no
detectable contamination by previous chemical anchored to
resin. This result was checked by specific ion detection
(single ion recording (SIR)) of expected contaminants. As
an example, cleavage of resin 6 yields 99% pure N-1 Fmoc-
diaminobenzene (m/z 331.1) but no trace of previous
compounds like N-1 Fmoc-diaminopropane (m/z 297.1) or
N-1 Fmoc-diaminoethane (m/z 283.1) was detected. Recovery
Results and Discussion
Pip-PS resin was prepared according a described 3-step solid-
phase procedure. Briefly, the commercially available cis/trans
6
cyclohexane dicarboxylic acid was coupled to AM-PS resin.
Then pipecolic acid methyl ester was coupled, and the resulting
resin was saponified with 2 M LiOH in tetrahydrofuran (THF)
to yield Pip-PS resin 1 (0.42 mmol/g).
To investigate the recycling potency of pipecolic resin (300
mg), five compounds were successively anchored and
released from the resin by neat TFA treatment (Scheme 3).
LC/MS analysis was performed immediately after cleavage
to monitor the putative presence of contaminants from
previous anchoring. After lyophilization, the compounds

Recycling the Versatile Pipecolic Linker
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 5 749
Scheme 4. Tripeptide Synthesis Using Recycled Pip-PS Resin
8
yields of cleaved materials ranged from 51 to 90% depending
on the compound attached to the linker and are comparable
to those obtained using non-recycled Pip-PS resin. In
strategy was used (Scheme 5). Briefly, pipecolic linker was
synthesized on Rink amide-PS resin (resin 9). Then, N-1 Fmoc-
diaminopropane was anchored to Pip-Rink-PS resin. Two hours
of TFA treatment induced simultaneous release of Fmoc-
diaminopropane 4 from pip linker and cleavage of the pipecolic
linker itself from Rink amide resin.
Cleavage solutions containing amidated pipecolic linker
analogues were evaporated under nitrogen flux, solubilized with
aqueous acetonitrile (1/1 v/v solution), and analyzed by LC/
MS. Results are presented in Figure 1 and in Table 2.
As expected, LC/MS analysis of crude cleaved compound
from dual linker resin 10 in conditions (a) with neat TFA
revealed the presence of Fmoc-diaminopropane 4 (RT ) 1.36
6
another set of experiments (data not shown), N-1 Fmoc-
diaminobenzene was coupled on Pip-PS and cleaved up to
four times successively. Before each cleavage, UV titration
of released dibenzofulvene upon dimethylformamide (DMF)/
piperidine treatment showed that the loading did not vary
significantly ((6%) between each round of recycling. On
the contrary of most TFA labile linkers, we demonstrated
that Pip-PS can be recycled up to four times still yielding
non contaminated compounds after TFA cleavage and
subsequent washings.
In another experiment, a modified tripeptide as a model
was synthesized, using a Hoffmann rearrangement as the key
step (Scheme 4). After SPPS and cleavage with TFA,
compound 7 was obtained with 90% of purity. The Pip-PS
used for this synthesis was recycled, and the same reactional
scheme was carried out to offer gem-diamino peptide 7 with
comparable purity (89%). The 6-step synthesis of H-Ala-
Phe-gLeu-H 7 twice on the same batch of Pip-PS resin,
without loss of efficiency between the two experiments
justified the interest of recycling the resin.
9
min) and pipecolic amide 11 (RT ) 0.96 min). It is worth
noting that the cleavage was quantitative and no remaining
amide compound was detected. Cleavage in conditions (b) led
to a UV chromatogram similar to those obtained after neat TFA
treatment. It proves that no side product is obtained adding
triisopropylsilane scavenger. This is particularly interesting for
the recycling of the Pip-PS resin after Fmoc SPPS of sensitive
peptides necessitating neutralization of carbocations during
cleavage. Adding 5% isopropanethiol in TFA resulted in
appearance of thioester 12c. Free carboxylic acid 11 is still
present but in low quantity (14% compared to 12c). In
conditions (d) (5% ethanedithiol (EDT) in TFA), LC/MS
analysis showed the appearance of a new peak (RT ) 1.33
min). Surprisingly, LC/MS analysis reveals only traces of
expected mono protonated ion corresponding to the EDT
thioester (m/z 359.1). Instead, an ion m/z 341.1 was present with
a fair abundance at a very close retention time (RT) as displayed
in the Supporting Information section (the two ions were
detected at RT ) 1.34 and 1.33 min respectively). This
unexpected ion has a m/z value 18 units lower than the one
which could correspond to the ethanedithiol thioester described
Cleavage Cocktail Investigations. Several examples of
peptide and pseudo peptide synthesis were recently described
using the Pipecolic linker. Notably, C-terminal methyl ester
peptides through side chain anchoring of Ser, Thr, and Tyr and
6
C-terminal peptide hydrazide were successfully prepared.
7
Pipecolic resin was compatible with Fmoc SPPS methodology.
Final TFA treatment enables concomitant removal of side chain
protecting groups and cleavage of the linker. During this final
step, it is of high importance to neutralize carbocations released
by side chain protecting groups which may react with sensitive
residues such as Met, Tyr, and Trp. To that purpose, nucleo-
philes scavengers are added in TFA solution. In this particular
case, the oxazolonium ion generated during cleavage of
pipecolic linker is susceptible to reaction with other nucleophiles
than water. Upon cleavage of pipecolic acid containing peptides,
it has already been described that thiol reagents such as ethanedi-
thiol (EDT) may attack this cation intermediate to yield
2
by Rubini et al. Notably, these authors did not find the correct
m/z value for the compound they described during their study
and considered that a dehydration happened during the ioniza-
10
tion step in the source of the ESI-MS instrument. Although
applying high voltages in the ESI-MS tuning may lead to in-
source fragmentation for fragile molecules, soft ionization
conditions should allow collecting at least some protonated
molecular ions. To address this hypothesis, the ESI source
tuning was refined to prevent the formation of fragment ions.
We found that the dehydration was not occurring during the
analysis but referred actually to another side product (12d). After
the formation of thioester with EDT (12d′), the compound (m/z
2
corresponding thioester (Scheme 1, right). Even if the thioester
could be hydrolyzed afterwards if extra water is added to the
TFA solution, this side reaction is a constraint for the ease of
recycling. We then decided to study in detail the behavior of
pipecolic linker when thiol or triisopropylsilane scavengers are
added to TFA solution. To that purpose, a dual linker-probe

7
50 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 5
Zajdel et al.
a
Scheme 5. Dual Linker Strategy for Sub-Products Monitoring upon Cleavage
a
Reagent and conditions: (i) DMF/Piperidine 80/20 v/v; (ii) 1,4-dicyclohexylcarboxylic acid, BOP, DIEA, DMF; (iii) D/L Pip-OMe, BOP, DIEA, DMF;
O 95/2.5/2.5 v/v/v, (c)
(
iv) LiOH 2N, THF; (v) N1-Fmoc-diaminopropane, BOP, DIEA, DMF. Cleavage cocktails: (a) TFA, (b) TFA/TIPS/H
2
TFA/isopropanethiol 95/5 v/v, (d) TFA/EDT 95/5 v/v.
Figure 1. Chromatograms of cleavage results of resin 10.
3
59.1) underwent a quantitative dehydration in TFA leading to
detected (absence of m/z 341.1). All structures of the recorded
fragment ions are directly depicted on the MS/MS spectra in
Figure 2.
cyclic compound 12d. Tandem MS/MS experiments upon low
energy collision induced dissociation conditions were performed
on a Q-Tof Mass spectrometer (allowing higher resolution
measurement) on both ions (m/z 359.15 and m/z 341.13) as
reproduced in Figure 2. Fragmentation patterns were clearly
different for the two precursor ions indicating that the latter
Conclusion
In this study we evidenced another advantage of the pipecolic
linker over other handles. Indeed, we demonstrated that
recycling of this resin is not only possible but also very easy to
perform, yielding after a cycle of simple washing, a fully
operational resin. As already described, the cleavage mechanism
(
(
12d) was not obtained by in-source dehydration of the former
12d′) (Figure 2). In particular, the supposed loss of water by
dehydration from the expected compound 12d′ was not at all

Recycling the Versatile Pipecolic Linker
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 5 751
Table 2. Side Products Obtained upon Treatment of Resin 10
with Cleavage Cocktails a, b, c, and d
theoretical loading 1.1 mmol/g and Fmoc-Rink-amide AM
polystyrene 100-200 mesh, theoretical loading 0.49 mmol/
g, was purchased from Senn Chemicals. All reagents and
solvents were from Alfa Aesar and Acros and were used
without further purification. Protected amino acids and BOP
coupling reagent were purchased from Iris Biotech. All
reactions and washes were conducted at room temperature.
Analytical HPLC were run on a Waters Alliance 2690 HPLC
instrument, equipped with a photodiode array and Chromolith
SpeedROD (4.6 × 25 mm) reversed-phase column. Standard
conditions were eluent system A (water/0.1% TFA), system
B (acetonitrile/0.1% TFA). A flow rate of 5 mL/min and a
gradient of 0 to 100% of B over 3 min were used, detection
2
14 nm, unless otherwise stated. Retention times (RT) are
given in minutes. Mass spectrometry analyses - Samples were
prepared in acetonitrile/water (50/50 v/v) mixture. The LC/
MS/MS system consisted of a Waters Alliance 2695 HPLC,
coupled to a Quattro Micro mass spectrometer (electrospray
ionization mode ESI-triple quadrupole (QqQ)). Analysis of
H-Ala-OMe (cleaved from resin 3) was carried out using
Waters Atlantis T3 C18 column, 25 × 4.6 mm reversed-
phase highly polar column. A flow rate of 3 mL/min in 100%
A isocratic mode followed by gradient of (0-100)% B over
2.5 min (or over 15 min) were used. Eluent A: water/0.1%
a
b
Non applicable. The ratio was calculated with integration of areas
of peaks in the HPLC chromatograms at λ ) 214 nm.
involves the hydrolysis of an oxazolonium intermediate to
regenerate the carboxylic acid function. If scavengers are
required, we suggest using silane and water containing cocktail
to avoid nucleophilic attack on the oxazolonium that compete
with hydrolysis.
2 2
HCO H; eluent B: acetonitrile/0.1% HCO H. Retention times
(RT) are given in minutes. All other analyses were carried
out using a Phenomenex Onyx Monolithic C18, 25 × 4.6
mm reversed-phase column. A flow rate of 3 mL/min and a
gradient of (0-100)% B over 2.5 min (or over 15 min) were
Experimental Section
used. Eluent A: water/0.1% HCO
0.1% HCO H. Positive ion ESI mass spectra were acquired
at a solvent flow rate of 150 µL/min. Nitrogen was used for
2
H; eluent B: acetonitrile/
General Information. Commercially available amino-
methyl-PS resin cross-linked with 1% DVB, 100-200 mesh,
2
Figure 2. MS/MS analysis of m/z 341.1 (top) and m/z 359.1 (bottom).

7
52 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 5
Zajdel et al.
both nebulizing and drying gas. LC/MS data were obtained
by scanning the first quadrupole in 0.7 s in a mass range
from 100 to 1000 m/z; 10 scans were summed up to produce
the final spectrum. Mass chromatograms of specific ions were
recorded by tuning the first quadrupole on a targeted m/z
value known as single ion recording experiment. LC/MS/
MS Data were obtained upon low energy collision induced
dissociation (CID) conditions. The first quadrupole was fixed
to the m/z value corresponding to the precursor ion to be
dissociated, the collision cell (q) was filled with argon as
collision gas and the last quadrupole was scanned in 0.7 s
from 100 to 500 m/z; 10 scans were summed up to produce
the final daughter ion mass spectra.
swollen in DMF for 30 min, it was added to 2.2 mL of DMF
coupling solution containing BOP (98 mg, 100 mM, 220
µmol, 6.3 equiv), DIEA (87 mg, 117 µL, 300 mM, 0.67
2
mmol, 19 equiv), and H-Leu-NH .HCl (37 mg, 100 mM,
220 µmol, 6.3 equiv). The resin was gently stirred for 2 h
and then washed with DMF (3×), MeOH (2×), DCM (3×).
2
The resin was then swollen in 3 mL of DMF/H O (80/20,
v/v) solution containing pyridine (30 µL, 30 mg, 100 mM,
380 µmol, 11 equiv) and BTIB (45 mg, 35 mM, 105 µmol,
3 equiv) and was stirred for 1 h. The resin was then washed
with DMF (3×) MeOH (2×), and DCM (3×). The resin
obtained was swollen in 2 mL of DMF coupling solution
containing BOP (85 mg, 100 mM, 192 µmol, 5.5 equiv),
DIEA (50 mg, 67 µL, 192 mM, 385 µmol, 11 equiv), and
Fmoc-Phe-OH (75 mg, 100 mM, 193 µmol, 5.5 equiv). The
resin was gently stirred for 2 h and then washed with DMF
(3×), MeOH (2×), and DCM (3×). Fmoc deprotection was
carried out using 20% piperidine in DMF (2 mL) for 30 min
and repeated twice. The resin was then washed with DMF
Preparation of Resins (1) and (9). Pip-PS resin 1 and
Pip-Rink PS 9 were prepared according a described proce-
6
dure, starting from AM-PS resin (theoretical loading 1.1
mmol/g) and Fmoc-Rink amide AM-PS resin (theoretical
loading 0.49 mmol/g), respectively.
Yields and Resin Loading. When nothing was stated,
yields of the final compounds were calculated on the basis
of the initial loading of the starting resins and were the overall
yields of all reaction steps starting from these resins. Resin
loadings were determined by three successive UV titration
of released dibenzofulvene upon piperidine mediated Fmoc
cleavage from Fmoc-protected compounds anchored on Pip-
PS resin.
TFA Cleavage. Four milliliters of TFA were added to
the resin (≈300 mg). After 1 h of gentle stirring, TFA was
removed from the resin. Fresh TFA was added again (4 mL),
and resin was stirred again for 1 h. TFA filtrates were
gathered and evaporated under nitrogen flux. The remaining
oil or solid was dissolved in acetonitrile/water 1/1 v/v
(
3×), MeOH (2×) and DCM (3×). The resin obtained was
swollen in 2 mL of DMF coupling solution containing BOP
46 mg, 50 mM, 104 µmol, 3 equiv), DIEA (27 mg, 36 µL,
(
1
5
00 mM, 210 µmol, 6 equiv), and Fmoc-Ala-OH (33 mg,
0 mM, 105 µmol, 3 equiv). The resin was gently stirred
for 2 h and then washed with DMF (3×), MeOH (2×), and
DCM (3×) and air-dried. Resin was finally cleaved with TFA
during 120 min under gentle stirring. Resin was filtered off,
and the TFA solution was concentrated under nitrogen. The
residue was dissolved in acetonitrile/water 1/1 solution and
freeze-dried. After lyophilization, 19 mg (yield 84%, purity
9
0%) of compound 7 were obtained as TFA salt. RT ) 1.64
+
1
min, Calc. MW) 542.3, MS ESI+ [M+H] m/z 543.4, H
NMR (300 MHz, DMSO d ): δ 0.74 (d, 3H, J ) 5.7 Hz,
CH ), 0.84 (d, 3H, J ) 5.7 Hz, CH ), 1.14 (d, 3H, J ) 7.0
Hz, CH ), 1.40 (m, 2H, CH ), 2.89 (m, 2H, CH ), 4.04 (q,
H, J ) 7.0 Hz, CH ), 4.23 (m, 1H, CH ), 4.33 (m, 1H,
(
4 mL) before being lyophilized.
6
3
3
Procedure for the Pip-PS Resin Recycling. Resin was
3
2
2
stirred during 2 min with 8 mL of each solvent. Beads were
first washed with DCM (2×), then with DMF/DIEA (9/1
v/v), DMF (2×), MeOH and DCM. Resin was kept in the
same reactor and dried under vacuum overnight.
Example of Thiol Anchoring on Pip-PS Resin: Fmoc-
aminoethanethiol (2). After the Pip-PS resin 1 (200 mg,
2
2
2
CH), 4.60 (m, 1H, CH), 6.95 (brs, 1H, NH), 7.12 (brs, 1H,
NH), 7.21 (m, 5H, 5xHAr), 7.33 (t, 2H, J ) 7.5 Hz, 2xHAr),
7
2
8
d
.43 (t, 2H, J ) 7.5 Hz, 2xHAr), 7.72 (d, 2H, J ) 7.5 Hz,
xHAr), 7.89 (d, 2H, J ) 7.5 Hz, 2xHAr), 8.13 (brs, 2H, NH ),
.83 (d, 1H, J ) 8.0 Hz, NH), C NMR (75 MHz, DMSO
): δ 18.1 (CH ), 20.9 (CH ), 22.9 (CH ), 23.4 (CH), 37.8
), 46.7 (CH ), 50.1 (CH), 50.3 (CH), 53.5 (CH), 55.6
CH), 65.7 (CH ), 120.1 (2x CHAr), 125.3 (2x CHAr), 126.1
CHAr), 127.1 (2xCHAr), 127.7 (2xCHAr), 128.1 (2xCHAr),
2
1
3
9
8 µmol, 0.49 mmol/g) was swollen in DCM for 30 min,
6
3
3
3
and washed with DMF (2 min), it was added to 5.4 mL of
DMF coupling solution containing DIC (68 mg, 79 µL, 100
mM, 540 µmol, 5.5 equiv), HOBt (73 mg, 100 mM, 540
µmol, 5.5 equiv), and Fmoc-aminoethanethiol (161 mg, 100
mM, 540 µmol, 5.5 equiv). The resin was gently stirred for
(
(
(
CH
2
2
2
1
1
29.2 (2xCHAr), 136.9 (CAr), 140.7 (2xCAr), 143.8 (2xCAr),
55.7 (CO), 172.0 (CO), 172.7 (CO).
2
h and then washed with DMF (2×), and DCM (2×).
Example of Amine Anchoring on Pip-PS Resin: Fmoc-
Supporting Information Available. Extracted LC/MS
diaminopropane (4). After the Pip-PS resin 1 (200 mg, 98
µmol, 0.49 mmol/g) was swollen in DCM for 30 min, and
washed with DMF (2 min), it was added to 5.4 mL of DMF
coupling solution containing BOP (239 mg, 100 mM, 540
µmol, 5.5 equiv), DIEA (140 mg, 188 µL, 200 mM, 1.08
mmol, 11 equiv), and Fmoc-1-amino-3-aminopropane (160
mg, 100 mM, 540 µmol, 5.5 equiv). The resin was gently
stirred for 2 h and then washed with DMF (2×), MeOH,
DCM (2×).
chromatograms and HR-MS of compounds 12d and 12′,
NMR spectra of cleaved compounds from resins 4, 5, and
6, NMR spectra and LC/MS spectrum of compound 7 are
given. This material is available free of charge via the Internet
at http://pubs.acs.org.
References and Notes
(1) Urban, J.; Vaisar, T.; Shen, R.; Lee, M. S. Int. J. Pept. Protein
Res. 1996, 47, 182–189.
Preparation of the gem-diamino Derivative (7). After
the Pip-PS resin 1 (100 mg, 35 µmol, 0.35 mmol/g) was
(
2) Rubini, C.; Osler, A.; Calderan, A.; Guiotto, A.; Ruzza, P. J.
Pept. Sci. 2008, 14, 989–997.

Recycling the Versatile Pipecolic Linker
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 5 753
(
3) Zajdel, P.; Subra, G.; Bojarski, A. J.; Duszynska, B.;
(8) Giovannoni, J.; Subra, G.; Amblard, M.; Martinez, J. Tetra-
hedron Lett. 2001, 42, 5389–5392.
Pawlowski, M.; Martinez, J. J. Comb. Chem. 2004, 6, 761–
7
67.
(9) A contaminant at RT ) 1.53 min was detected in all
experiments which correspond to the direct coupling of Fmoc
diaminopropane on cyclohexane dicarboxylic acid witnessing
that the coupling of pipecolic acid during the preparation of
the linker was not complete.
10) See supplementary information of Rubini, C.; Osler, A.;
Calderan, A.; Guiotto, A.; Ruzza, P. J. Pept. Sci. 2008, 14,
(
4) Creighton, C. J.; Romoff, T. T.; Bu, J. H.; Goodman, M. J. Am.
Chem. Soc. 1999, 121, 6786–6791.
5) Sobolewski, D.; Prahl, A.; Kwiatkowska, A.; Slaninova, J.;
Lammek, B. J. Pept. Sci. 2009, 15, 161–165.
6) Zajdel, P.; Nomezine, G.; Masurier, N.; Amblard, M.;
Pawlowski, M.; Martinez, J.; Subra, G. Chem.sEur. J. 2010,
(
(
(
9
89–997.
1
6, 7547–7553.
(
7) Amblard, M.; Fehrentz, J. A.; Martinez, J.; Subra, G. Mol.
Biotechnol. 2006, 33, 239–254.
CC100099Z