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C. Echalier et al.
Starting from resin 4, Fmoc-Ala-O-2-chlorotrityl PS
in basic conditions at the stage of Fmoc removal from the
dipeptidyl-resin or during the subsequent coupling step.
The intramolecular attack of the free amino group on the
C-terminus ester bond leads to the cyclization-cleavage of
the DKP and the release of the resin linker as the alcohol
counterpart (Gisin and Merrifield 1972). For structural
reasons, DKP is favored by Gly/Pro containing sequences,
N-alkyl amino acids and D/L alternation. Benzyl ester
related linkers such as hydroxymethylphenoxy, and par-
ticularly electron withdrawing hydroxymethylbenzamido,
also favor this side reaction. Bulkier 2-Chlorotrityl linkers
have been designed to prevent this side reaction and gave
satisfactory protection against this premature cleavage. But
we cannot exclude that heating, which favors intramole-
cular reaction, could lead to DKP formation on trityl lin-
ker. This was verified by stopping the MW synthesizer at
the stage of resin 6, after 3 min of DMF/pip (8/2, v/v)
solution treatment (10 mL), which removed the Fmoc
protecting group at the N-terminus of phenylalanine resi-
due. An aliquot (20 ll) of the solution was taken and the
reactor was emptied. Then, a second deprotection cycle of
20 min was carried out and an aliquot was also taken.
A complete DKP formation on the remaining resin in the
reactor would have resulted in a 48 mM concentration of
c[Ala-Phe] when a 1 % DKP formation would have cor-
responded to 480 lM concentration in the sample. The two
LC/MS analyses did not reveal any significant amount of
c[Ala-Phe] in any DMF/pip solution (\0.1 %). This indi-
cated that the 2-Clorotrityl linker was probably bulky
enough to prevent DKP formation at 70 °C on a dipeptide
sequence composed of amino acids with the same
stereochemistry.
(1 g, 0.80 mmol/g, 0.8 mmol), tripeptide 7 was prepared
using standard MW-SPPS conditions (‘‘Materials and
methods’’). Before each deprotection step, a small amount
of resin was freeze dried and weighted to perform an UV
DBF-pip adduct titration (see ‘‘Materials and methods’’).
Compared to theoretical maximum loading, the experi-
mental values were lower (0.66 vs. 0.72 mmol/g and 0.59
vs. 0.66 mmol/g at the stage of di and tripeptides, respec-
tively). These measurements were in accordance with our
hypothesis of loading loss. Along with this titration, two
aliquots of 100 mg of resin were collected and poured into
4 mL of DMF. Samples were heated at 70 °C or left at
room temperature on the bench for 400 min. Thanks to
calibration curves, compounds eventually released in
solution from the resin beads were detected and quantified.
In the samples left at RT (Fig. 2, filledsquare, red curves),
very low concentrations of peptides were detected after
400 min. A maximum of 1.4 9 10-4 M was measured,
which corresponded to less than 1 % cleavage (curves pre-
sented in Supporting Information). On the contrary,
increasing concentrations of Fmoc-protected peptides were
detected at 70 °C (Fig. 2, filleddiamond). The thermal
cleavage phenomenon was significant enough to lead to a
loss of half the loading within about 220 min (50 % after
215 min for dipeptide 50 and 48 % after 225 min for tri-
peptide 70). This cleavage was even faster in the case of
resin 4 for which 87 % of Fmoc-Ala-OH 40 was released
after 150 min. Noteworthy, after 2 h heating, cleavage of the
peptide from the resin was concomitant with some extend of
Fmoc removal, which led to increase the DBF concentration
in the sample (Fig. 2, 9, purple curves). In sample con-
taining resin 4, a white turbidity and a slight precipitate
appeared after two hours. In the case of resin 7, a decrease of
protected peptide concentration was detected after 300 min
heating along with increasing concentrations of unprotected
tripeptide 700 (Fig. 2, filledtriangle).
To the best of our knowledge, neither the effect of
temperature on hydrolysis of trityl esters nor the study of
peptidyl trityl ester on solid support has been investigated.
However, the mechanism of trityl acetate hydrolysis in
aqueous solutions has been determined (Bunton and Kon-
asiewicz 1955). Bunton et al. used 18O as isotopic tracer in
aqueous dioxane to highlight the fission of the alkyl-oxy-
gen bond leading to the stabilized trityl carbocation. In
addition, solvolysis rates of trityl acetate were determined
in MeOH, EtOH, acetone and acetic acid (Swain et al.
1960) and more recently in water/acetonitrile (1/1 v/v) at
25° C (Horn and Mayr 2010a, b). These studies demon-
strated that trityl ester hydrolysis occurred as a classical
SN1 reaction: carbocations are formed as short-lived
intermediates, immediately trapped by the solvent.
Complementary experiments were also performed with
higher DMF qualities (anhydrous DMF, amine-free DMF)
and N-methyl pyrrolidone as well (See ‘‘Materials and
methods’’). No significant differences were observed
compared to standard DMF (data not shown).
We also checked that this cleavage was specific of the
2-Chlorotrityl linker. For that purpose, Fmoc-Ala-OH was
anchored on hydroxymethylphenoxy PS (Wang) resin
(0.75 mmol/g) and Rink amide aminomethyl PS
(0.96 mmol/g). Resin samples were submitted to the same
treatment as resin 4. Even after 400 min, the release of
Fmoc-Ala-OH (or Fmoc-Ala-NH2 in the case of Rink
amide linker) was not detected (data not shown).
Even if we could reasonably propose that the same
mechanism operates on peptidyl trityl ester bonds on solid
support, we cannot exclude that the peptide cleavage pro-
ceeds through oxazolone formation (Scheme 2).
To complete this study, we investigated the extent of a
putative cyclization-release of the peptide through the
intermolecular DKP formation. DKP cyclization happens
We could hypothesize that basic conditions during
coupling and deprotection cycles promote oxazolone
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