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We report the total chemical synthesis of human C3a by onepot native chemical ligation of three unprotected peptide segments followed by efficient in-vitro folding, which yielded the target molecule in high yield and excellent purity. The 10 synthetic material was fully active and facilitated determination of the C3a crystal structure at 2.1Å resolution.The anaphylatoxins C3a and C5a are key mediators of the complement system, which represents the first line of immunological defense for the recognition and elimination of 15 microbes and pathogens. 1 They selectively bind to their respective G protein-coupled receptors (C3aR and C5aR) triggering a variety of pro-inflammatory processes and have recently been linked to a number of infectious, inflammatory, neurodegenerative and autoimmune diseases. 2 Currently, C3a is 20 commercially produced in relatively low yields by biological means (recombinant expression or purification from plasma) 3, 4 resulting in a market value of approximately US$ 5000 per mg of protein. In addition, these approaches often require protein purification tags and additional steps for their removal and suffer 25 from the inherent lability of C3a in biological fluids. C3a is rapidly inactivated within seconds by carboxypeptidase-mediated cleavage of a single C-terminal arginine residue (Arg 77 ). 5,6 The resulting protein, C3a-desArg (also termed acylation-stimulating protein, ASP), does not bind C3aR, lacks any pro-inflammatory 30 activity but instead has been shown to stimulate triglyceride synthesis and glucose uptake in adipose tissue. [7][8][9] To circumvent the problems associated with the isolation of C3a from biological sources we sought a total chemical synthesis approach enabling preparation of homogenous full length C3a. 35Human C3a is a 77 residue protein containing three intramolecular disulfide bonds between C22-C49, C23-C56, and C36-C57. 10, 11 Because polypeptides of this size are difficult to obtain in high purity by standard stepwise solid phase peptide synthesis (SPPS), 12 we envisioned a fragment ligation approach 40 by employing Kent's native chemical ligation (NCL). 13 The 77 amino acid polypeptide chain is retro-synthetically split into three peptide segments of about similar length and the three polypeptides are then joined consecutively in the C-to Nterminal direction by NCL as shown in Scheme complete after 6h after which methoxyamine HCl was added and the pH adjusted to 4.0. After completion of the deprotection reaction, the mixture was readjusted to pH 7.1 and the second ligation initiated by adding C3a[1-22]-α-thioester (complete after 6 h). This one-pot approach afforded fully reduced C3a 60 without purification of intermediate products in short time (~24 h), good yield (41%) and high purity (SI Figure 1). The final folding of a cysteine-rich polypeptide chain into its 3D structure can often be problematic because they tend to yield multiple disulfide isomers that are difficult to separate and result 65 in lower overall yield. This often necessitates optimizati...

No abstract

A two-step synthesis of structurally diverse pyrrole-containing bicyclic systems is reported. ortho-Nitro-haloarenes coupled with vinylic N-methyliminodiacetic acid (MIDA) boronates generate ortho-vinyl-nitroarenes, which undergo a "metal-free" nitrene insertion, resulting in a new pyrrole ring. This novel synthetic approach has a wide substrate tolerance and it is applicable in the preparation of more complex "drug-like" molecules. Interestingly, an ortho-nitro-allylarene derivative furnished a cyclic β-aminophosphonate motif.

The dCTP pyrophosphatase 1 (dCTPase) is a nucleotide pool "housekeeping" enzyme responsible for the catabolism of canonical and noncanonical nucleoside triphosphates (dNTPs) and has been associated with cancer progression and cancer cell stemness. We have identified a series of piperazin-1-ylpyridazines as a new class of potent dCTPase inhibitors. Lead compounds increase dCTPase thermal and protease stability, display outstanding selectivity over related enzymes and synergize with a cytidine analogue against leukemic cells. This new class of dCTPase inhibitors lays the first stone toward the development of drug-like probes for the dCTPase enzyme.

Starting from o‐nitro‐aryl and ‐heteroaryl halides a two‐step sequence of Suzuki coupling and Cadogan—Sundberg cyclization yields indoles and pyrrolo‐annelated heteroarenes.

No abstract

In this study, we provide insight into the metabolic profile of a series of piperazin-1-ylpyridazines suffering from rapid intrinsic clearance in a metabolic stability assay using liver microsomes ( compound MLM/HLM = 2/3 min). Aided by empirical metabolite identification and computational predictive models, we designed the structural modifications required to improve intrinsic clearance by more than 50-fold ( compound MLM/HLM = 113/105 min).