Full details of the development of a direct coupling of catharanthine with vindoline to provide vinblastine are described along with key mechanistic and labeling studies. Following an Fe(III)-promoted coupling reaction initiated by generation of a presumed catharanthine radical cation that undergoes a subsequent oxidative fragmentation and diastereoselective coupling with vindoline, addition of the resulting reaction mixture to an Fe(III)-NaBH 4 /air solution leads to oxidation of the C15′-C20′ double bond and reduction of the intermediate iminium ion directly providing vinblastine (40-43%) and leurosidine (20-23%), its naturally occurring C20′ alcohol isomer. The yield of coupled products, which exclusively possess the natural C16′ stereochemistry, approaches or exceeds 80% and the combined yield of the isomeric C20′ alcohols is >60%. Preliminary studies of Fe(III)-NaBH 4 /air oxidation reaction illustrate a generalizable trisubstituted olefin scope, identified alternatives to O 2 trap at the oxidized carbon, provides a unique entry into C20′ functionalized vinblastines, and affords initial insights into the observed C20′ diastereoselectivity. The first disclosure of the use of exo-catharanthine proceeding through Δ 19′,20′ -anhydrovinblastine in such coupling reactions is also detailed with identical stereochemical consequences. Incorporating either a catharanthine N-methyl group or a vindoline N-formyl group precludes Fe(III)-promoted coupling, whereas the removal of the potentially key C16 methoxy group of vindoline does not adversely impact the coupling efficiency. Extension of these studies provided a total synthesis of vincristine (2) via N-desmethylvinblastine (36, also a natural product), 16-desmethoxyvinblastine (44) and 4-desacetoxy-16-desmethoxyvinblastine (47) both of which we can now suggest are likely natural products produced by C. roseus, desacetylvinblastine (62) and 4-desacetoxyvinblastine (59), as well as a series of key analogues bearing systematic modifications in the vindoline subunit. Their biological evaluation provided additional insights into the key functionality within the vindoline subunit contributing to the activity and sets the foundation on which further, more deep-seated changes in the structures of 1 and 2 will be explored in future studies.

Proline is unique among the natural amino acids in the similar propensity of its peptide bond to be in the cis or trans conformation. This attribute affects many processes, including the rate at which proteins fold, their structures, and their activities. Other aliphatic amino acids can serve as mimics for proline residues with trans peptide bonds. In contrast, chemical synthesis is needed to create surrogates for cis prolyl peptide bonds. Here, 1,5-disubstituted-[1,2,3]-triazoles were assessed as cispeptide bond surrogates. Huisgen's 1,3-dipolar cycloaddition reaction of amino alkynes and azido acids and a Ru(II) catalyst were used to synthesize a variety of Xaa-1,5-triazole-Ala modules in moderate-to-high yields. Two of these modules, along with their 1,4-triazole regioisomers, were installed in a turn region of bovine pancreatic ribonuclease by using expressed protein ligation. The resulting semisynthetic enzymes displayed full enzymatic activity, indicating the maintenance of native structure. The 1,5-triazole surrogates instilled conformational stability that was comparable to that of Xaa-cis-Pro segments, whereas the 1,4-triazoles conferred markedly less stability. The stability conferred by both surrogates was independent of the Xaa residue, eliminating an uncertainty in protein design. We conclude that Xaa-1,5-triazole-Ala modules can serve as viable mimics of Xaa-cis-Pro segments. The possibility of synthesizing this surrogate by the ligation of fragments in situ and the emergence of biocompatible catalysts for that process portends its widespread use.Proline is unique among the natural amino acids in the similar propensity of its peptide bond to be in the cis or trans conformation. 1 This attribute affects many processes, including the rate at which proteins fold, 2 their structures, 3 and their activities. 4 Other aliphatic amino acids can serve as mimics for proline residues with trans peptide bonds. In contrast, chemical synthesis is needed to create surrogates for cis prolyl peptide bonds. 1,5-7

NAD is an indispensable redox cofactor in all organisms. Most of the genes required for NAD biosynthesis in various species are known. Ribosylnicotinamide kinase (RNK) was among the few unknown (missing) genes involved with NAD salvage and recycling pathways. Using a comparative genome analysis involving reconstruction of NAD metabolism from genomic data, we predicted and experimentally verified that bacterial RNK is encoded within the 3 region of the nadR gene. Based on these results and previous data, the full-size multifunctional NadR protein (as in Escherichia coli) is composed of (i) an N-terminal DNA-binding domain involved in the transcriptional regulation of NAD biosynthesis, (ii) a central nicotinamide mononucleotide adenylyltransferase (NMNAT) domain, and (iii) a C-terminal RNK domain. The RNK and NMNAT enzymatic activities of recombinant NadR proteins from Salmonella enterica serovar Typhimurium and Haemophilus influenzae were quantitatively characterized. We propose a model for the complete salvage pathway from exogenous N-ribosylnicotinamide to NAD which involves the concerted action of the PnuC transporter and NRK, followed by the NMNAT activity of the NadR protein. Both the pnuC and nadR genes were proven to be essential for the growth and survival of H. influenzae, thus implicating them as potential narrow-spectrum drug targets.

The traceless Staudinger ligation is an effective means to synthesize an amide bond between two groups of otherwise orthogonal reactivity: a phosphinothioester and an azide. An important application of the Staudinger ligation is in the ligation of peptides at a variety of residues. Here, we demonstrate that the traceless Staudinger ligation can be achieved in water with a water-soluble reagent. Those reagents that provide a high yield of amide product discourage protonation of the nitrogen in the key iminophosphorane intermediate. The most efficacious reagent, bis(pdimethylaminoethylphenyl)phosphinomethanethiol, mediates the rapid ligation of equimolar substrates in water. This reagent is also able to perform a transthioesterification reaction with the thioester intermediate formed during intein-mediated protein splicing. Hence, the traceless Staudinger ligation can be integrated with expressed protein ligation, extending the reach of modern protein chemistry.