1. Solid-Phase Total Synthesis of Bacitracin A
Jinho Lee, John H. Griffin, Thalia I. Nicas J Org Chem. 1996 Jun 14;61(12):3983-3986. doi: 10.1021/jo960580b.
An efficient solid-phase method for the total synthesis of bacitracin A is reported. This work was undertaken in order to provide a general means of probing the intriguing mode of action of the bacitracins and exploring their potential for use against emerging drug-resistant pathogens. The synthetic approach to bacitracin A involves three key features: (1) linkage to the solid support through the side chain of the L-asparaginyl residue at position 12 (L-Asn(12)), (2) cyclization through amide bond formation between the alpha-carboxyl of L-Asn(12) and the side chain amino group of L-Lys(8), and (3) postcyclization addition of the N-terminal thiazoline dipeptide as a single unit. To initiate the synthesis, Fmoc L-Asp(OH)-OAllyl was attached to a PAL resin. The chain of bacitracin A was elaborated in the C-to-N direction by sequential piperidine deprotection/HBTU-mediated coupling cycles with Fmoc D-Asp(OtBu)-OH, Fmoc L-His(Trt)-OH, Fmoc D-Phe-OH, Fmoc L-Ile-OH, Fmoc D-Orn(Boc)-OH, Fmoc L-Lys(Aloc)-OH, Fmoc L-Ile-OH, Fmoc D-Glu(OtBu)-OH, and Fmoc L-Leu-OH. The allyl ester and allyl carbamate protecting groups of L-Asn(12) and L-Lys(8), respectively, were simultaneously and selectively removed by treating the peptide-resin with palladium tetrakis(triphenylphosphine), acetic acid, and triethylamine. Cyclization was effected by PyBOP/HOBT under the pseudo high-dilution conditions afforded by attachment to the solid support. After removal of the N-terminal Fmoc group, the cyclized peptide was coupled with 2-[1'(S)-(tert-butyloxycarbonylamino)-2'(R)-methylbutyl]-4(R)-carboxy-Delta(2)-thiazoline (1). The synthetic peptide was deprotected and cleaved from the solid support under acidic conditions and then purified by reverse-phase HPLC. The synthetic material exhibited an ion in the FAB-MS at m/z 1422.7, consistent with the molecular weight calculated for the parent ion of bacitracin A (MH(+) = C(73)H(84)N(10)O(23)Cl(2), 1422.7 g/mol). It was also indistinguishable from authentic bacitracin A by high-field (1)H NMR and displayed antibacterial activity equal to that of the natural product, thus confirming its identity as bacitracin A. The overall yield for the solid-phase synthesis was 24%.
2. Optimized selective N-methylation of peptides on solid support
Eric Biron, Jayanta Chatterjee, Horst Kessler J Pept Sci. 2006 Mar;12(3):213-9. doi: 10.1002/psc.711.
Peptides containing N(alpha)-methylamino acids exhibit interesting therapeutic profiles and are increasingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by the high price and nonavailability of many N(alpha)-methylamino acids. An efficient and practical three-step procedure for selective N-methylation of peptides on solid support is described. The procedure was based on the well known solid-phase N-methylation of N(alpha)-arylsulfonyl peptides, which was improved by using dimethylsulfate and the less expensive DBU as base. Every step of the procedure, amine activation by an o-nitrobenzenesulfonyl group, selective N-methylation and removal of the sulfonamide group, was optimized in respect of time and economy. The described optimized three-step procedure is performed in 35 min without solvent changes, instead of 3 h. Tripeptides (Fmoc-Phe-MeXaa-Leu-OH) containing N-methylated common amino acids were also prepared using the optimized procedure to demonstrate its compatibility with these amino acids. The described procedure allows an efficient synthesis of N(alpha)-methylamino acid containing peptides in a very short time using Fmoc solid-phase peptide synthesis.
3. Contulakin-G, an O-glycosylated invertebrate neurotensin
A G Craig, et al. J Biol Chem. 1999 May 14;274(20):13752-9. doi: 10.1074/jbc.274.20.13752.
We have purified contulakin-G, a 16-amino acid O-linked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1-->) attached to Thr10. The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(beta-->3) GalNAc(alpha-->)Thr10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated con- tulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakín-G was found to be active at 10-fold lower doses than the nonglycosylated Thr10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr10 contulakin-G was approximately an order of magnitude lower than that of neurotensin1-13 for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.
