The design of enzyme-like complexity within metal–organic frameworks (MOFs) requires multiple reactions to be performed on a MOF crystal without losing access to its interior. Here, we show that seven post-synthetic reactions can be successfully achieved within the pores of a multivariate MOF, MTV-IRMOF-74-III, to covalently incorporate tripeptides that resemble the active sites of enzymes in their spatial arrangement and compositional heterogeneity. These reactions build up H2N-Pro-Gly-Ala-CONHL and H2N-Cys-His-Asp-CONHL (where L = organic struts) amino acid sequences by covalently attaching them to the organic struts in the MOFs, without losing porosity or crystallinity. An enabling feature of this chemistry is that the primary amine functionality (−CH2NHBoc) of the original MOF is more reactive than the commonly examined aromatic amines (−NH2), and this allowed for the multi-step reactions to be carried out in tandem within the MOF. Preliminary findings indicate that the complexity thus achieved can affect reactions that were previously accomplished only in the presence of enzymes.

Posttranslational modifications (PTMs) play a vital role in regulating the structure and dynamics of chromatin as well as DNA-driven cellular processes by covalently modifying the primary structures of the histone proteins (H2B, H2A, H3, H4) with various chemical entities.[1] One such example is the modification of the histone H2B by ubiquitin (Ub) at multiple lysine residues. Of these, ubiquitination at Lys120 (H2K120Ub) is best studied. This PTM was suggested to modulate gene expression, trigger H3 methylation, and activate DNA damage response pathways.[2] In contrast, little is known about the effects and functions of the other sites of ubiquitination in H2B, K34, K46, K108, and K116. [3] Notably, the preparation of any of these ubiquitinated histone proteins for biochemical analyses has been a challenge in the field and has thus hampered a variety of studies aiming to understand the effect of ubiquitination on chromatin. Only recently, novel nonenzymatic approaches were introduced and enabled the preparation of homogeneously native H2K120Ub on a milligram scale for structural and functional analyses.[4] These approaches proved to be successful for the incorporation of this single modification or any other modification within the short C-terminal synthetic peptide.In order to investigate the role of ubiquitination at other sites and to study the interplay of multiple modifications, a new synthetic strategy is needed. For example, it has been reported recently that ubiquitination of H2B at Lys34 directly regulates H3K4 and K79 methylation through trans-tail crosstalk both in vitro and in cells.[5] Hence, the preparation of such an analogue will enable studies aiming to understand how this modification regulates H3 methylation and potentially affects the structure of chromatin, as well as help to delineate mechanisms of K34 ubiquitination and deubiquitination. Here, we report our endeavors toward the total chemical synthesis of the H2B protein, and the successful sitespecific ubiquitination at Lys34 (H2K34Ub) for initial functional characterization.If one considers the preparation of H2K34Ub semisynthetically through the expression of a large C-terminal H2B fragment that bears an N-terminal Cys or through total chemical synthesis by applying a ligation approach with three fragments, the 57mer H2B(1-57), which bears the K34 modification, has to be prepared synthetically. Because H2B lacks any Cys residue, an Ala to Cys mutation for native chemical ligation (NCL) combined with desulfurization [6] should be performed at Ala58 (Scheme 1). Even if the semisynthetic method were applicable, it would not allow the insertion of different PTMs along the sequence but only at the N-terminal fragment. [7] Our initial studies with a threefragment approach revealed that the synthesis of the 57mer peptide via Fmoc-SPPS was very difficult. We thus concluded that a total chemical synthesis of the H2B protein from four fragments is inevitable. Hence, we divided the H2B sequence, taking into consideration the position of Ala residu...

The combination of native chemical ligation and desulfurization is considered a powerful strategy in protein synthesis. Homogeneous desulfurization conditions based on a radical induced reaction have been widely used in the syntheses of various challenging proteins and their analogues. However, the presence of aryl thiols in the reaction mixture as a ligation catalyst hampers one-pot ligation/ desulfurization, hence mandating additional purification/lyophilization steps prior to desulfurization. This significantly reduces the yield and prolongs the ligation process. Here we report that the use of preformed peptide-aryl thioester allows for efficient one-pot ligation and desulfurization. This approach was tested successfully for various model peptides including the synthesis of ubiquitin from two fragments. However, in the case of the synthesis of di-ubiquitin chains, where the ligation is mediated by d-mercaptolysine to form an isopeptide bond, excess aryl thiol was required for efficient ligation, necessitating purification prior to desulfurization. To overcome these obstacles, we found that functionalization of the aryl thiol with a hydrazide moiety enabled, after the ligation step, its capture by resin-aldehyde to permit direct desulfurization. Altogether, these approaches should facilitate protein synthesis with improved efficiency in yields and time.