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The canonic M2e epitope entailed considerable synthetic difficulties already as a simplemonomer, which were substantially overcome by the use of ChemMatrix resin, systematicdouble couplings and, especially, pseudoproline dipeptide units at the two Ser-Ser pairs [4].All these improvements, plus flexibilizing Ahx units at every branching point, wereincorporated to the all-SPPS synthesis of tetravalent construct C (Figure 2), resulting in aclean, easily purifiable and satisfactorily characterized end product (C 517 H 838 N 146 O 196 S 5 ,MW = 12387.86 Da; [M+H + ] = 12387.79; 4.8% yield).To evaluate the convergent (chemical ligation) approach, constructs D and E (seeFigure 3) were prepared [4]. For thioether ligations, a tetravalent core functionalized withchloroacetyl groups (ClAc 4 -Lys core), andeither the sM2e or the M2e monomers (for Dand E, respectively; each C-terminallyelongated with Cys) were prepared in highlypure form. Ligations were conducted inTris·HCl, pH 7.6, 2 M guanidinium chloride,at 50 ºC, using 10-12-fold molar excess ofpeptide. Reaction progress was monitored byanalytical HPLC and revealed in both casesrather complex processes (Figure 3) [4].MALDI-TOF MS analysis showed MAPproducts containing different numbers ofepitope copies, along with substantial amountsof the disulfide-bound dimers of the linearpeptides. HPLC resolution of the reactionmixtures was challenging and did not allowefficient separation of the target tetravalentconstructs from less substituted species.Additionally, in the ligation for E, an M2etetramer, coeluting with the disulfide dimerand identified as a non-covalent double dimer,further complicated the situation [4].In view of these difficulties with theligation approach, the all-SPPS route appearsFig. 3. Progress of thioether ligationmonitored by HPLC (triangles indicatethioether linkage).advantageous, particularly for epitopes such asM2e, where factors such as size (sterichindrance preventing access of the Cys thiol tounreacted ClAc groups), solubility andaggregation (both difficult to predict) can haveseverely limiting effects. Ligation is also more expensive and time-consuming than the all-SPPS approach, and in thiol-based chemistries tends to generate sizable amounts ofdisulfide dimer whose recycling back to the thiol form is costly in time and effort.Regarding the all-SPPS approach, on the basis of the above results we would proposetwo recommendations: (i) incorporating flexibility-enhancing units (e.g., Ahx) at branchingpoints can be synthetically beneficial; (ii) an exploratory synthesis of the linear epitope canidentify trouble spots to be smoothed before the synthesis of the considerably morecomplicated MAP structures is undertaken.AcknowledgmentsWork supported by MICINN, the Spanish Ministry of Science and Innovation (grants BIO2005-07592-CO2-02 and BIO2008-04487-CO3-02 to D.A.), by the regional government of Catalonia (SGR2005-00494). W.K. is a fellow in the Juan de la Cierva Program of MICINN.References1. Tam, J.P. Proc. Natl. Acad. Sci. U.S.A. 85, 5409-5413 (1988).2. Tam, J.P., In Goodman M (Ed.) Peptide Dendrimers and Protein Mimetics, Thieme, Stuttgart, 2000,p. 129.3. Cady, S.D., Luo, W., Hu, F., Hong, M. Biochemistry 48, 7356-7364 (2009).4. Kowalczyk, W., De la Torre, B.G., Andreu, D. Bioconj. Chem. 21, 102-110 (2010).161