The research in stapled peptide has been under the spotlight of
peptide based drug discovery in recent years and has successfully
emerged its potential in the clinical trial of p53 stapled peptide.1
Such peptides represent potential therapeutics against
intracellular targets previously thought to be unreachable1a. The
all-hydrocarbon staple could endue the stapled peptide with
significant improvement in the stability of secondary structure,
binding affinity, and metabolic stability2. The most prevalent
strategy to achieve single3 or double hydrocarbon stapled
procedures require separation of the free amino acid by ion-
exchange before N-protection with Fmoc group8-9. On the other
hand, for the synthesis of 1g, reported procedure of bis-alkylation
of N-diphenylmethylene-glycine ethyl ester involved tedious
protection strategy and low-temperature condition2. To establish a
more feasible synthesis, the synthesis of the chiral complex and
asymmetric alkylation process were modified and optimized. The
synthesis of the α,α-disubstituted amino acid 1g was also
achieved by introducing achiral complex analogously (Scheme
1).
2
peptide4 as well as “stitched peptide” is the olefin metathesis
between α-alkenyl Alanine derivatives (Figure 1, 1a-1e) or with
additional α-bisalkenyl substituted glycine (Figure 1, 1g).
Alternative strategies including stapling by Husigen click
reaction5 and photo-induced thiol-yne click reaction6 are
facilitated by α-alkyne amino acids (Figure1, 1f). Apparently,
these unnatural α-alkyl amino acids are essential for those
described approaches.
Reagents and conditions: (a) 2-aminobenzophenone, EDCI, DMAP, DCM,
reflux, 12h; (b) HCl/EtOAc, rt, 2h. (c) 2-fluorobenzyl bromide, K2CO3,
.
acetonitrile, 60~70˚C,8h; (d) Gly, Ni(NO3)2 6H2O, KOH, MeOH, 65˚C, 1.5h;
(e) From 5a: iodoalkene, tBuONa, DMF, rt, 0.5h.; From 5b: iodoalkene or
bromoalkyne, NaOH, acetonitrile, r.t, 0.5h; (f) i) 3mol·l-1 HCl, MeOH, reflux;
ii) EDTA-2Na, Fmoc-OSu, Na2CO3, H2O/ acetonitrile, r.t., 16h,.
Figure 1. Unnatural amino acids commonly involved in synthesis of stapled
peptides.
Scheme 2. Synthesis of Fmoc protected α-alkenyl and α-alkyne amino acids
For the synthesis of chiral Ni(II)-complex, Boc-Pro-OH was
coupled with (2-aminophenyl)phenylmethanone to give the
relatively hydrophobic intermediate which could be purified by
simple extraction and washing with 1mol.L-1 HCl aqueous
solution. Considering low nucleophilicity of the amino group of
the (2-aminophenyl)-phenylmethanone, various coupling
reagents were screened for their efficiency. We found the readily
available EDC/DMAP combination could achieve ideal yield
comparable with MsCl/N-methylimidazole (Table 1, entry 11).
Upon removing Boc group by HCl/EtOAc, alkylation by 2-
fluorobenzyl bromide and complexation with Ni(NO3)2·6H2O
and Glycine or Alanine were carried out according to the
reported procedure 9b,10. Therefore, the chiral auxiliary 5a and 5b
were synthesized with 87% and 89% yields from Boc-Pro-OH
with feasible reagents and easy workup procedure (Scheme 1).
The optical purity of 4a and its enantiomer 4b were confirmed
(ee >99%) by chiral HPLC chromatography.
Scheme 1. Synthesis strategies for compound 4a and 1g
As for the alkylation step, glycine complex 5b could be
deprotonated by sodium hydroxide at room temperature and
successively reacted with alkyl bromide in good yield and
diastereoselectivity in the solvent of CH3CN. However, due to
the lower acidity of second α-proton of 5a, the established
method utilized four equivalents of NaOH as the base under
heating with four equivalents of alkyl bromide. It could be
presumed that more active alkyl halide and stronger base might
be needed to achieve better enolization under milder condition. In
order to lower the excess of base and alkyl halides, reaction
temperature, and time consumption, initially, we transform the
alkyl bromide to the more active alkyl iodide by simply heating
alkyl bromide with NaI in acetone. Next, we screened several
typical bases at 1.2 equivalents in 30min reaction time at room
temperature. As shown in Table 2, tBuONa was found to get
nearly quantitative yield in the synthesis of 6a. Other solvents
including CH3CN, THF, Dioxane, tBuOH and DCM were also
During our undergoing stapled peptide research, we found
these amino acids were of high price and some of them were
even not commercially available. To tackle this, upon studying of
literature, glycine/alanine derived chiral Ni(II)-complex
auxiliary provided an accessible starting point to access
asymmetric synthesis of α-alkyl amino acids7. However, the
reported procedure8 for the synthesis of the preferable chiral
auxiliary
pyrrolidine-2-carboxamide
(S)-N-(2-benzoylphenyl)-1-(2-fluorobenzyl)-
(2-FBPB, 4a) required
methylsulfonylchloride (MsCl) which was a tightly regulated
violent toxic reagent to achieve efficient coupling. Furthermore,
the hydrophilicity of the benzylproline intermediate had to be
isolated by isoelectric precipitation and column purification was
required for each workup step which made the whole process
quite tedious for large scale preparation in the lab. After
alkylation of the Ni(II) complex Schiff bases, most reported