Increasing the bioactive space of peptide macrocycles by thioamide substitution

Abstract

Thioamide substitution into macrocyclic peptides increases the conformational rigidity of the backbone resulting in enhanced biological activity and metabolic stability.

Fluorescent Probes for Studying Thioamide Positional Effects on Proteolysis Reveal Insight into Resistance to Cysteine Proteases

“…[2] As ingle thioamide near the scissile bond provided up to 750-fold enhancement of peptides tabilityc ompared to the all-amide peptide withouts ignificantly altering cellular activity,a nd at hioamide modifiedG LP-1 was active in rats.Arecent report by Chatterjee and co-workersd emonstrated that thioamide incorporation in macrocyclic integrin antagonists can increase their serum half-life by threefold while maintaining, or even increasing, potencyt oward cancer cell lines. [3] Studies such as these show that thioamides can be highly useful for the stabilization of peptides toward proteolytic events for in vivo applications (although it should be noted that renal clearance and other factors impact in vivo half-life). [4] However,d espite numerous investigations of proteases using thioamide peptides, few systematics tudies have been performed to study the positional effects of thioamide substitution on proteolysis.…”

Increasing protein stability by engineering the n → π* interaction at the β-turn

“…[53][54][55][56][57] On the other hand, thioamide substitution at a site where the amide oxygen is involved in a longer hydrogen bond or is solvent exposed, leads to minimal perturbation of the secondary structure. 53,55,[57][58][59] Therefore, we chose to substitute the solvent exposed C]O i+1 in the type-II 0 b-turn of 2 by C]S i+1 . The NBO analysis of the C]O i+1 to C]S i+1 substituted type-II 0 bturn in 2, 3, 4, and 5 clearly indicated a signicant enhancement in E n/p* , due to the amplied C]S i+1 / C]O i+2 n / p* interaction (Table S3 †).…”

Convenient synthesis of thioamidated peptides and proteins

“…We obtained a crude conversion of 65% for 9 ( Figure 5A F I G U R E 5 Crude HPLC chromatogram (monitored at 270 nm) after direct solid-phase synthesis and global deprotection of (A) thioamidated Pin1 WW domain (9), and (C) thioamidated GB1 (11). The electrospray ionization-mass spectrometry (ESI-MS) of purified (B) 9 and (D) 11 showing the multiply charged species, which were deconvoluted to obtain the molecular weights.…”

“…Various laboratories have utilized different Fmoc‐deprotection cocktails to circumvent the problem either by using dilute piperidine 25 or DBU 26,28 with minimal exposure to the thioamidated peptide or by reversible protection of the thioamide as a thioimidate 27 . Although dilute piperidine 42 shows minimal epimerization of the thioamidated peptide and can be successfully utilized for the synthesis of macrocyclic peptides, 9 the strategy suffers from incomplete Fmoc‐deprotections in longer peptides, giving rise to amino acid deletions. Thus, we reasoned that a cocktail that exhibits rapid Fmoc‐deprotection kinetics should provide high yield of C‐terminal thioamidated peptide with minimal epimerization.…”

“…This ultimately results in longer C═S bond length (1.71 Å) than the corresponding amide (1.23 Å), 8 weakened hydrogen bond accepting property, 7 and enhanced nucleophilicity of the thioamide sulfur (>C═S) than the carbonyl oxygen (>C═O). Thioamides have been utilized to enhance the affinity of macrocyclic peptides targeting integrins, 9 as spectroscopic labels and fluorescence quenchers, 10,11 photoswitches, 12,13 protease inhibitors, 14,15 for peptide synthesis 16 and cyclization, 17 and several other biophysical applications. [18][19][20][21][22][23] However, despite these exciting utilities, the synthesis of thioamidated peptides and protein stays a difficult task that continues to be addressed by several groups, including ours.…”