Protein chemical synthesis by serine and threonine ligation Yinfeng Zhang, Ci Xu, Hiu Yung Lam, Chi Lung Lee, and Xuechen Li1 Department of Chemistry, The University of Hong Kong, Hong Kong, People’s Republic of China Edited* by Samuel J. Danishefsky, Columbia University/Memorial Sloan-Kettering Cancer Center, New York, NY, and approved March 15, 2013 (received for review December 3, 2012) An efficient method has been developed for the salicylaldehyde (17, 35). Subsequently, an approach involving reductive amination ester-mediated ligation of unprotected peptides at serine (Ser) or was developed to facilitate acyl transfer (36, 37). Using such an threonine (Thr) residues. The utility of this peptide ligation ap- imine-induced intramolecular acyl transfer concept, Tam et al. have proach has been demonstrated through the convergent syntheses demonstrated that a peptide containing a C-terminal glycoaldehyde of two therapeutic peptides––ovine-corticoliberin and Forteo––and can couple with a second peptide, containing an N-terminal Cys, ∼ the human erythrocyte acylphosphatase protein ( 11 kDa). The req- Thr, or Ser residue, to generate a pseudoproline structure (thia- uisite peptide salicylaldehyde ester precursor is prepared in an epi- zolidine or oxazolidine) at the site of ligation (14, 38, 39). However, merization-free manner via Fmoc–solid-phase peptide synthesis. the resultant oxazolidine, being quite stable, has not been dem- onstrated to be cleavable to restore the natural peptidic linkage. synthetic protein | acyl transfer | chemoselective ligation In a preliminary disclosure, our laboratory recently described the coupling of an O-salicylaldehyde ester of a single amino acid or andmark advances in the field of synthetic protein chemistry dipeptide with the 1,2-hydroxylamine moiety as found in serine have enabled the preparation of complex, homogeneous L and threonine, to form an N,O-benzylidene acetal linked interme- proteins (1–3), including those that carry specific posttranslational fi – fi diate, which, without isolation, is readily converted to the natural modi cations (PTMs) (4 11). Of particular signi cance, Dani- – shefsky and coworkers have obtained glycosylated human follicle- peptidic linkage (i.e., Xaa Ser/Thr, Fig. 1) (40). stimulating hormone (7) and erythropoietin (11), solely through Having established the validity of this approach, we sought to chemical synthesis. In general, protein synthesis consists of two expand upon the general concept, with the ultimate goal of de- fi key phases: (i) solid-phase peptide synthesis (SPPS) allows for the veloping a versatile and ef cient solution to the challenge of native fi fi generation of moderately sized peptide segments (up to ∼30 amino Ser and Thr peptide ligation (Fig. 1). We identi ed two signi cant acids) (12); and (ii) chemical ligation serves to chemoselectively issues that must be addressed before our concept could be suc- join these synthetic peptide fragments (13–16). In the 1970s, Kemp cessfully extended to peptide synthesis applications. First, we would and coworkers conceptually devised a promising peptide ligation need to develop a robust method for the preparation of peptide strategy, involving prior capture followed by acyl transfer, which salicylaldehyde esters, which would be compatible with standard has laid the foundation for the development of chemical ligation SPPS strategies. In our previous study, we had used direct cou- in the convergent peptide synthesis (17–22). A milestone advance pling to prepare salicylaldehyde esters of Fmoc-protected single in the field was the discovery, by Kent and coworkers, of native amino acids and pseudoproline-containing dipeptides. However, chemical ligation (NCL) (13), in which a C-terminal peptide the analogous esterification of large peptide fragments via direct thioester and an N-terminal cysteine-containing peptide––both in coupling would not be feasible, due to the potential for epime- –– side-chain unprotected forms are selectively coupled to generate a rization of the C-terminal amino acid. Second, we would need to natural peptidic linkage (Xaa-Cys) at the site of ligation. Because demonstrate that the coupling reaction between peptide salicy- CHEMISTRY the Kent NCL approach exploits the unique nucleophilicity of the laldehyde esters and N-terminal Ser- or Thr-containing peptides cysteine thiol group, the relatively low abundance of cysteine could be compatible with fully unprotected peptide substrates. We residues in natural proteins can pose a significant challenge to were well aware that the diverse functionality present in large side- NCL-based protein synthesis efforts. In an effort to expand the chain unprotected peptides could potentially complicate efforts scope of NCL, a number of research groups have developed var- fi iants, wherein β-orγ-thiol containing amino acids are temporarily to achieve the high levels of chemoselectivity, ef ciency, and installed on the peptide N terminus, thus promoting NCL-like preservation of chirality required for peptide ligation reactions. ligation. Subsequent desulfurization serves to restore the natural The objectives outlined above have now been met, and a work- amino acid at the site of ligation (23–26). Whereas this NCL– able thiol-free Ser/Thr peptide ligation method has been developed. fi desulfurization strategy has been widely used in protein synthesis, We describe herein the identi cation of epimerization-free con- a menu of complementary thiol-independent ligation approaches ditions for the synthesis of peptide salicylaldehyde esters via (27–34) may offer new opportunities for convergent protein syn- Fmoc-SPPS, and the successful ligation of these substrates with thesis. Nevertheless, these thiol-independent ligations require the unprotected N-terminal Ser or Thr peptide fragments. Moreover, installation of unique reaction functionalities, often tediously, on the utility of this protocol has been demonstrated in the context of both sides of the N-terminal peptide segment and the C-terminal several biologically compelling target systems, including the human peptide segment to induce a chemoselective coupling reaction. erythrocyte acylphosphatase protein. Along these lines, our laboratory has been pursuing the deve- lopment of methods for ligation at N-terminal serine and threo- nine residues to generate natural Xaa–Ser/Thr linkages directly, Author contributions: Y.Z., C.X., H.Y.L., and C.L.L. performed research; X.L. designed re- using natural serine or threonine residues at the reacting N ter- search; Y.Z., C.X., H.Y.L., C.L.L., and X.L. analyzed data; and X.L. wrote the paper. minus. In an encouraging precedent, Kemp and coworkers have The authors declare no conflict of interest. examined the hemiaminal-mediated acyl transfer of 8-acetoxy-1- *This Direct Submission article had a prearranged editor. naphthaldehyde and 2-acetoxybenzaldehyde reacting with primary 1To whom correspondence should be addressed. E-mail: [email protected]. O → N amines. Under the optimal conditions, acyl transfer to the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. less-hindered amine (i.e., glycine and benzylamine) was observed 1073/pnas.1221012110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1221012110 PNAS | April 23, 2013 | vol. 110 | no. 17 | 6657–6662 Downloaded by guest on October 2, 2021 Previous work: peptide Nbz is generated via Fmoc-SPPS, under the conditions Me CHO developed by Blanco-Canosa and Dawson (42). The resin-bound HO O Py/AcOH, 2h peptide Nbz is then suspended in dry dichloromethane (DCM)/ FmocHN + O H N CO Bn 100% conversion THF (1/3, vol/vol), and salicylaldehyde dimethyl acetal and Na2CO3 2 2 are added to the flask. The reaction mixture is stirred at room Ala SAL ester Ser temperature for 16 h and the concentrated filtrate is treated with TFA-based mixture to remove the side-chain protecting groups, BnO2C Me TFA/H O/i-Pr SiH Me 2 3 H affording the desired peptide SAL ester. Alternatively, the peptide N O 5 min N CO Bn FmocHN FmocHN 2 Nbz may be cleaved from the Rink resin, before in-solution phe- 95% yield O OH O nolysis. In the original Blanco-Canosa and Dawson report (42), the OH N,O-benzylidine peptide thioester was prepared via in-solution thiolysis of the peptide acetal Nbz. However, in our experience, the on-resin phenolysis approach provided peptide SAL esters more efficiently and cleanly. The liquid This work: SPPS SPPS chromatography-mass spectrometry (LC-MS) trace of the crude filtrate, following on-resin phenolysis, reveals the desired peptide O HO R SAL esters, along with excess salicylaldehyde (Fig. 3). H unprotected peptide O + Using this method, we prepared and purified, by reverse-phase H2N unprotected peptide OH CHO HPLC, a series of peptide SAL esters of various lengths (6–21 SAL ester Ser or Thr amino acids) in overall yields of 20–30%, based on resin loading. Ligate To ascertain whether epimerization occurs during on-resin phe- nolysis, we synthesized epimeric Z-DTTADA-SAL ester and O Z-DTTADa-SAL ester. We were pleased to observe no epime- H unprotected peptide unprotected peptide OH N rization of the C-terminal Ala residues of these SAL esters, as O R determined by HPLC analysis. This strategy thus provides a re- liable and facile solution to the SPPS-compatible preparation of OH cleave peptide SAL esters. N,O-benzylidine acetal Serine/Threonine Peptide Ligation. With a menu of peptide SAL R OH esters in hand, we were now poised to investigate the extension O of our salicylaldehyde ester-induced coupling to the significantly H unprotected peptide N unprotected peptide OH more challenging context of convergent peptide synthesis. Thus, H peptide segment 1, incorporating a C-terminal SAL ester, was Ser (R = H) or Thr (R = Me) exposed to peptide 3, presenting a Ser residue at its N terminus, Fig. 1. Principle of salicylaldehyde ester-mediated peptide ligation at serine/ in pyridine acetate buffer. In the event, the two side-chain un- threonine sites. protected peptide segments reacted smoothly to produce the coupled peptide in nearly quantitative conversion, within 8 h.