Efficient Fmoc-based solid-phase synthesis (SPPS) of sulfotyrosine (sY) peptides is achieved by incorporating the sY residue(s) as a dichlorovinyl-protected (DCV) sulfodiester(s) and using 2-methylpiperidine for Fmoc removal. After removal of the other protecting groups, the DCV group could be cleaved by mild hydrogenolysis giving the sY peptides in good yield.

The trichloroethyl (TCE) group is shown to be a viable protecting group for sulfonates. TCE-protected sulfonates were found to be particularly stable to acid, a key characteristic that led to a straightforward enantioselective synthesis of L-FmocPhe(p-CH2SO3TCE)OH. This was used as a building block for the solid phase synthesis of an octapeptide corresponding to P-selectin glycoprotein ligand-1 residues 43-50 (PSGL-143-50) in which sulfotyrosine residues 46 and 48 were replaced with (sulfonomethyl)phenylalanine (SmP), an important hydrolytically stable sulfotyrosine mimic.

A series of sulfuryl imidazolium salts (SISs) were prepared and examined as reagents for incorporating trichloroethyl-protected sulfate esters into carbohydrates. The SIS that contained a 1,2-dimethylimidazolium moiety (SIS 9) proved to be a superior sulfating compared to SISs bearing no alkyl groups or bulkier alkyl groups on the imidazolium ring. Difficult O-sulfations that required prolonged reaction times and a large excess of the SIS bearing a 1-methylimidazolium group (SIS 5) were achieved in high yield using less than half the amount of SIS 9 in less time. Certain N-sulfated compounds that were practically inaccessible using SIS 5 were obtained in excellent yield using SIS 9

Tyrosine sulfation is a post translationalmodification that occurs on integral membrane and secreted proteins, and is required for mediating crucial biological processes. Until recently the synthesis of sTyr peptides, especially those containing multiple sTyr residues, were among the most challenging peptides to prepare. We recently described an efficient strategy for Fmocbased solid phase synthesis of sTyr peptides in which the sulfate group in the sTyr residue(s) is protected with a DCV group (FmocTyr(SO3DCV)OH, 1). After cleavage of the peptide from the support the DCV group is removed by hydrogenolysis. Here, we demonstrate that sTyr peptides containingMet or Trp residues can be prepared using our sulfate-protecting group strategy by preparing peptides corresponding to residues 1–20 of chemokine receptor CXCR6 and 8–42 of chemokine receptor DARC. Removing the DCV groups at the end of the syntheses was readily achieved, without any reduction of the indole ring in Trp, by performing the hydrogenolysis in the presence of triethylamine. These conditions were found to be particularly efficient for removing the DCV group and superiour to our original conditions using H2, ammonium formate, Pd/C. The presence of Met was found not to interferewith the removal of the DCV group. The use of pseudoproline dipeptides and N-backbone protection with the 2,4-dimethoxybenzyl group were found to be very effective tactics for preventing aggregation and aspartimide formation during the synthesis of thesepeptides.Wealso report an alternative andmorecost effective synthesis of amino acid 1.

Photoswitchable distance constraints in the form of photoisomerizable chemical cross-links offer a general approach to the design of reversibly photocontrolled proteins. To apply these effectively, however, one must have guidelines for the choice of cross-linker structure and cross-linker attachment sites. Here we investigate the effects of varying cross-linker structure on the photocontrol of folding of the Fyn SH3 domain, a well-studied model protein. We develop a theoretical framework based on an explicit-chain model of protein folding, modified to include detailed model linkers, that allows prediction of the effect of a given linker on the free energy of folding of a protein. Using this framework, we were able to quantitatively explain the experimental result that a longer, but somewhat flexible, cross-linker is less destabilizing to the folded state than a shorter more rigid cross-linker. The models also suggest how misfolded states may be generated by cross-linking, providing a rationale for altered dynamics seen in nuclear magnetic resonance analyses of these proteins. The theoretical framework is readily portable to any protein of known folded state structure and thus can be used to guide the design of photoswitchable proteins generally.

. Chloroquine a potent antimalarial also demonstrates potency against selected cancers. In this study, we demonstrate pulmonary delivery of encapsulated chloroquine phosphate (CQP) using three poly(glycerol adipate-co--pentadecalactone) microparticles, PGA-co-PDL, PEG1500-PGA-co-PDL and PEG4500-PGA-co-PDL, and its effects on lung cancer cell growth. Methods. Microparticles encapsulated with CQP were prepared using spray drying-double emulsion technique. Encapsulation efficiency, water content, density, morphology and in-vitro release (PBS, pH 7.4) was characterised. Furthermore, aerosolisation behaviour was performed using the next generation impactor at 60L/min via HandiHaler® containing 20mg powder in HPMC capsule (size 2). MTT assay quantified the effect of encapsulated CQP and carrier-free CQP on human lung cancer bronchial epithelial cell (NCI-H727) proliferation. Results: PGA-co-PDL (11.4KDa), PEG1500-PGA-co-PDL (12.4KDa) and PEG4500-PGA-co-PDL (13.6KDa) resulted in low encapsulation efficiency (13.11±1.23, 10.52±0.86, 9.25±1.02). Furthermore, a biphasic release profile was predominant for all formulations, with ~40-45% of payload released after 24h. The aerosolisation performance (%FPF) was significantly greater for PEG1500-PGA-co-PDL (13.05±0.46%) and PEG4500-PGA-co-PDL( 14.66±0.46%) compared to PGA-co-PDL (9.52±0.50%) microparticle carriers. The higher FPF achieved with PEG4500-PGA-co-PDL microparticles was due to the significantly lower water content and true density (0.76±0.12%, 0.98±0.07g/cm3) respectively, as opposed to PEG1500-PGA-co-PDL (1.04±0.25%, 1.17±0.12g/cm3) and PGA-co-PDL (1.73±0.84, 1.24±0.35g/cm3), indicating less aggregation and enhanced flow properties. Treatment, 240-0.25µM, using carrier-free CQP (61.71±9.25-9.78±2.59) and encapsulated CQP (PGA-co-PDL: 63.88±11.13-19.38±8.55, PEG1500-PGA-co-PDL: 56.73±9.41-26.75±13.58, PEG4500-PGA-co-PDL: 62.46±8.57-25.24±5.41) resulted in a decrease in cell viability. Conclusion: Results from this investigation indicate CQP encapsulated with in PGA-co-PDL and PEG-PGA-co-PDL microparticles show promise as a pulmonary delivery system for treatment of lung cancers.