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30. General solid-phase procedures: Attachment of the Fmoc-protected Rink linker
to the amino-functionalized PEGA800 resin was carried out by pre-mixing
Fmoc-protected Rink amide linker (3.0 equiv), NEM (4.0 equiv) and TBTU
(2.88 equiv) in DMF for 5 min. The volume of DMF was kept to a minimum, just
enough for full coverage and swelling of the beads. The resulting solution was
added to the resin, and allowed to react for 2 h. After removal of the reaction
mixture by suction, the resin was washed with DMF (ꢂ6), CH2Cl2 (ꢂ6) and then
dried under vacuum. Removal of the Fmoc group was carried out by treating
the resin with 20% piperidine in DMF, first for 2 min, followed by washing with
DMF (ꢂ2), and then for 18 min. The deprotected resin was then washed with
DMF (ꢂ6), CH2Cl2 (ꢂ6) and DMF (ꢂ6) again. Subsequent attachment of amino
acids was accomplished using the TBTU-mediated protocol as described above.
Release from the solid-phase was achieved by treatment of the resin with 95%
TFA (aq) for 2 h, and a wash with MeCN and H2O. After removal of the solvents
on a SpeedVac, the residue was re-dissolved in a minimum of MeCN and
precipitated with Et2O. The typically off-white crystalline peptide was then
lyophilized overnight.
Na(OAc)3BH. Mono-alkylation was achieved by treatment of the
resin with the aldehyde (10 equiv) for 1 h, followed by washing
with DMF to remove any aldehyde present, and final addition of
the reductant (5 equiv). Double alkylation was achieved in a simi-
lar manner, except that all reagents were added concomitantly
(Scheme 4). Both protocols provided the desired peptides 4ꢀTFA
and 20 in excellent purities after TFA-mediated release from the
solid support (both >95%).30 This route provides a new way of
accessing peptide 4ꢀTFA, but also provides an entry for the synthe-
sis of further appended versions of 4, as exemplified by the synthe-
sis of the doubly alkylated peptide 20. The approach enhances
considerably the scope of the strategy, since it allows for orthogo-
nal deprotection/unmasking of the amine functionality (under
azide disguise) by simple solid-phase reduction. The selectivity
by which the amine can be mono-alkylated holds the promise of
enabling the rapid construction of decorated versions of the origi-
nal target product. Synthesized peptide 4ꢀTFA has been subjected
to biological evaluation and was shown to exhibit the same activity
profile as previously reported.28
In summary, we have developed an efficient route for the solid-
phase synthesis of a known Smac mimetic tetrapeptide 4 using an
orthogonally protected proline-derived building block 10. In a sec-
ond strategy, a cis-4-azido-proline-containing tetrapeptide was
selectively reduced to an amine, thus providing an entry for reduc-
tive alkylation reactions on solid support and access to the target
peptide 4 and other derivatives. In general, products were obtained
in excellent crude purity after release from the solid phase (>95%).
Ongoing studies focus on the synthesis and biological evaluation of
larger Smac mimetic libraries incorporating diverse substituents
on the proline and phenylalanine residues.
Acknowledgments
The Technical University of Denmark, Danish Council for Inde-
pendent Research, Natural Sciences (T.E.N.), Carlsberg Foundation
(S.L.Q.), and Torkil Holm Foundation are gratefully acknowledged
for financial support. We are very thankful to Tina Gustafsson for
technical assistance.
The solid-phase azide reduction was carried out by treatment of the resin with
0.1 M DTT (4 equiv) and DBU (2 equiv) in DMF for 1 h. After removal of the
reaction mixture by suction, the resin was washed with DMF (ꢂ6), MeOH (ꢂ6),
CH2Cl2 (ꢂ6) and then dried under vacuum.
Supplementary data
The mono-alkylation of the solid supported amine was carried out by addition
of cyclohexanecarbaldehyde (10 equiv) in DMF to the resin. The reaction was
allowed to proceed for 1 h, whereupon the resin was washed with DMF (ꢂ6).
To the resin was then added freshly prepared Na(OAc)3BH (5 equiv) in DMF/
DMSO/AcOH (50:50:1), and the reaction allowed to proceed for 1 h. After
removal of the reaction mixture by suction, the resin was washed with DMF
(ꢂ6), H2O (ꢂ6), DMF (ꢂ6), MeOH (ꢂ6), CH2Cl2 (ꢂ6) and then dried under
vacuum.
Supplementary data (experimental synthetic procedures and
data for all synthesized compounds) associated with this article
The di-alkylation of the solid supported amine was carried out by concomitant
addition of cyclohexanecarbaldehyde (10 equiv) and freshly prepared
Na(OAc)3BH (5 equiv) in DMF/DMSO/AcOH (50:50:1) to the resin. The
reaction was allowed to proceed overnight. After removal of the reaction
mixture by suction, the resin was washed with DMF (ꢂ6), H2O (ꢂ6), DMF (ꢂ6),
MeOH (ꢂ6), CH2Cl2 (ꢂ6) and then dried under vacuum.
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Compound 4ꢀTFA: 1H NMR (500 MHz, DMSO-d6) d 9.16–9.06 (m, 1H), 9.06–8.97
(m, 1H), 8.97–8.83 (m, 2H), 8.75 (d, J = 8.1 Hz, 1H), 8.65 (d, J = 7.5 Hz, 1H), 7.47
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4.9 Hz, 1H), 4.40–4.33 (m, 2H) 4.07–4.02 (m, 1H), 3.94–3.86 (m, 3H), 2.99 (dd,
J = 14.0, 5.1 Hz, 1H), 2.91 (dd, J = 14.0, 8.4 Hz, 1H), 2.88–2.76 (m, 2H), 2.50 (br s,
3H), 2.46–2.42 (m, 1H), 2.13–2.06 (m, 1H), 2.05–1.98 (m, 1H), 1.76–1.66 (m,
5H), 1.65–1.59 (m, 1H), 1.33 (d, J = 6.8 Hz, 3H), 1.23–1.13 (m, 3H), 0.92 (d,
J = 6.6 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.99–0.86 (m, 2H); 13C NMR (50 MHz,
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55.8, 54.6, 51.1, 49.5, 37.1, 34.7, 30.7, 30.5, 29.9, 29.7, 29.6, 25.5, 25.0, 18.8,
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Compound 16: 1H NMR (500 MHz, DMSO-d6 d 8.93 (br s, 1H), 8.82 (br s, 1H),
8.72 (d, J = 8.1 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.32 (s, 1H), 7.27–7.17 (m, 5H),
7.08 (s, 1H), 4.41–4.37 (m, 3H), 4.35–4.31 (m, 1H), 4.09 (dd, J = 10.4 Hz,
J = 6.6 Hz, 1H), 3.91–3.85 (m, 1H), 3.42 (dd, J = 10.2 Hz, J = 6.1 Hz, 1H), 2.97 (dd,
J = 13.8 Hz, J = 5.6 Hz, 1H), 2.89 (dd, J = 13.8 Hz, J = 7.7 Hz, 1H), 2.51–2.48 (m,
3H), 2.46–2.41 (m, 1H), 2.04–1.96 (m, 1H), 1.86–1.81 (m, 1H), 1.32 (d,
J = 6.8 Hz, 3H), 0.94 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H); 13C NMR
(50 MHz, DMSO-d6) d 172.3, 170.0, 169.8, 168.6, 137.7, 129.2, 128.0, 126.2,