Journal of the American Chemical Society
Article
a
shortened dramatically by using 10 mol % HOBt as a catalyst
(for a comparative study of the HOBt catalysis effect, see the
epimerization occurred in the activation or aminolysis reaction.
Indeed, phenylglycine (Phg), which is extremely prone to loss
of chiral integrity upon activation (60 times more prone to
racemization than Ala), could be employed as either the
carboxylic acid or the amine coupling partner (10u,v).
Although no racemization occurred during the activation of
Phg (9o), it underwent 10% epimerization in the subsequent
aminolysis reaction at room temperature. Fortunately, the
epimerization could be completely suppressed by performing
the aminolysis reaction at low temperature (10u). The side
chains of Ser and Thr (−OH) and Trp (NH) were tolerated in
both the activation and the aminolysis reaction, and hence,
their protection was not necessary. Although His did not act as
a good carboxyl partner (10w), it worked well as an amine
partner (10x). While the peptide bond was formed in two
steps, allowing the efficiency of each step to be systematically
investigated, all of the dipeptides could be synthesized through
a one-pot reaction, with results comparable to or better than
those of the two-step strategy (Scheme 4). Even though free
amines were employed as the nucleophile, the HCl salt of
amines also worked well for this reaction in the presence of a
tertiary amine, such as N,N-diisopropylethylamine (see the
Allenone-Mediated Peptide Fragment Condensation.
Urethane-based amine protecting groups are known to be
beneficial for suppressing racemization during peptide bond
formation. To further demonstrate the robustness of the
allenone coupling reagent, peptide fragment condensation
involving the activation of a peptide acid, which is more prone
to racemization than the urethane-protected amino acid, was
studied. As shown in Scheme 5, dipeptide, tripeptide, and
tetrapeptide acids that are highly prone to epimerization are
viable substrates for the allenone-mediated peptide fragment
condensation. Although the time required for activation
increased with increasing peptide length, the aminolysis
reaction proceeded rapidly, with the reaction time ranging
from minutes to hours. The activation of peptide acids and the
subsequent aminolysis proceeded smoothly to furnish the
target tri-, tetra-, penta-, and hexapeptides in excellent yields
(14a−k). Importantly, no epimerization was observed for the
entire peptide fragment condensation process, thus providing
an attractive convergent peptide synthesis strategy.
Synthesis of Carfilzomib with Allenone 2a as the
Coupling Reagent. To illustrate the synthetic utility of this
method, carfilzomib, an anticancer peptide drug used for
treating refractory multiple myeloma, was synthesized using
allenone 2a as the coupling reagent for constructing the four
peptide bonds. It has been reported that the chiral keto-
epoxide warhead is unstable under the conditions of peptide
elongation (i.e., conditions for both coupling and depro-
tection) and hence must be introduced at the final stage of the
peptide synthesis.22 Although an N to C peptide elongation
strategy can fulfill this requirement, this strategy is rarely used
for peptide synthesis because of the serious epimerization.
Considering the remarkable superiority of allenone 2a in
suppressing the epimerization of peptide acids, we speculated
that allenone 2a could be an ideal coupling reagent for
carfilzomib synthesis via an N to C peptide elongation strategy.
To monitor the entire process carefully, all of the intermediates
were isolated and characterized. As shown in Scheme 6, all of
Scheme 3. Allenone-Mediated Amide Bond Formation
a
First step: allenone 2a (0.2 mmol), carboxyl acid (0.22 mmol), DCE
(2 mL). Second step: α-carbonyl vinyl ester (0.2 mmol), amine (0.22
mmol), DMF (1 mL), isolated yield. 10 mol % of HOBt as catalyst.
b
c
d
Two-step one-pot reaction. Two-step one-pot reaction with 10 mol
% of HOBt as the catalyst for the second step.
2a as the coupling reagent under the optimal reaction
conditions (Scheme 4). All of the proteinogenic amino acids,
except His, gave the corresponding target α-carbonyl vinyl
esters in excellent yields. The low yield with His might be
attributed to its imidazole side chain functional group (9p).
The 1,4-addition/isomerization reaction was highly efficient,
and no excess reactant was required, although 1.1 equiv of the
amino acid was typically used for the reactions. Conventional
urethane-based amine protecting groups such as carboxyben-
zoyl (Cbz), tert-butylcarboxyl (Boc), and 9-fluorenylmethylene
carboxyl (Fmoc) groups were compatible under the reaction
conditions employed. Moreover, steric hindrance had little
influence on the activation efficiency (9c,g). Even the
activation of the most sterically hindered, non-natural, α,α-
disubstituted amino acid α-aminoisobutyric acid (Aib)
proceeded smoothly to give the target active ester 9h in 91%
yield. In the next step, all of the proteinogenic amino acid
based α-carbonyl vinyl esters underwent aminolysis smoothly
at room temperature to furnish the target peptides in excellent
yields. Sterically hindered natural and non-natural amino acids
were well tolerated in both the electrophilic carboxyl and
nucleophilic amino coupling partners. The steric hindrance of
the carboxyl partner imposed a greater effect on the reaction in
comparison to that of the amino partner (10j vs 10k). In
general, the aminolysis reactions proceeded to completion
within a few minutes, while longer reaction times were
required for α-carbonyl vinyl esters of sterically hindered
amino acids such as Ile, Val, and Aib (10c,h,i,k,l). Even the
highly hindered dipeptide of N-methyl amino acid such as
Fmoc-MeLeu-MeLeu-OBn (10o) could be obtained in
excellent yield. As was observed for simple amides, the
reaction time for sterically hindered dipeptides could be
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J. Am. Chem. Soc. 2021, 143, 10374−10381