Chemical Synthesis of Peptides and Proteins Bearing Base‐Labile Post‐Translational Modifications: Evolution of the Methods in Four Decades

The S‐palmitoylation on Cys residue and O‐acetylation on Ser/Thr residues are two types of base‐labile post‐translational modifications (PTMs) in cells. The lability of these PTMs to bases and nucleophiles makes the peptides/proteins bearing S‐palmitoyl or O‐acetyl groups challenging synthetic targets, which cannot be prepared via the standard Fmoc‐SPPS and native chemical ligation. In this review, we summarized the efforts towards their preparation in the past 40 years, with the focus on the evolution of synthetic methods.


Introduction
Post-translational modifications (PTMs) of proteins increase the complexity of protein structures by more than one order of magnitude. [1]Within more than 400 covalent PTMs, the Spalmitoylation has been predicted to cover 10 % of the proteome. [2]The reversible nature of the thioester bond and the hydrophobicity of the lipid chain make S-palmitoylation a key regulator of the protein subcellular trafficking and membrane localization, [3] and an important factor affecting protein functions. [4]For example, the S-palmitoylation of the C-terminal region of Ras proteins, in collaboration with S-farnesylation in the same region, affects the recycling of Ras between cytoplasm membrane and other cellular compartments and its functions. [5]-palmitoylation has also been identified from bacterial and viral proteins and involved in the pathogen-host interactions. [6]ompared to S-palmitoylation, the O-acetylation has only been identified in limited examples, with their functions largely unknown. [7]Though biologically quite different, from the synthetic point of view, S-palmitoylation and O-acetylation shares one chemical property, the base lability.These two PTMs are generally sensitive to bases and nucleophiles, which lead to the β-elimination and acyl group removal respectively (Figure 1).This base-labile (also nucleophile-labile) nature makes the peptides/proteins bearing S-palmitoyl or O-acetyl groups challenging synthetic targets.The standard Fmoc-SPPS [8] and native chemical ligation [9] , which are currently the most popular tools for peptide and protein synthesis, are not readily applicable.Since the synthesis of homogeneous S-palmitoylated and O-acetylated peptides and proteins are essential to biological functional studies, synthetic chemists have devoted many efforts to tackle the challenges.
In this review, we summarize the synthetic efforts toward peptides and proteins bearing base-labile S-palmitoylation and O-acetylation PTMs.Both the extensive early endeavors that set the basis for lipidated peptide synthesis in solution/solid phase and the recent protein chemical syntheses that demonstrate the state of the art will be described, with the focus on the methodological evolution.Some of the works have been summarized in several reviews [5b,10] and book chapters [11] .Other base-labile PTMs such as the O-crotonylation, [12] Oubiquitination, [13] S-ubiquitination [14] beyond the scope of this review, since the synthetic efforts toward related peptides and proteins are limited. [15]We hope this summary could help the scientists entering the field to get an overview and inspire the development of novel synthetic methods to further improve the synthesis of S-palmitoylated and O-acetylated proteins of biological interest.

Direct S-palmitoylation
A direct way to synthesize S-palmitoylated peptides and proteins is the installation of palmitoyl group onto unprotected peptides or proteins via selective S-acylation (Figure 2).The key to achieving this selectivity is the superior nucleophilicity of the thiol group under pH ranging from mild acidic to mild basic conditions.The first attempt of this approach was reported by Cronan and Klages in 1981, in which a series of acyl imidazole reagents including palmitoyl imidazole 1 were applied to the Sacylation of native acyl carrier protein (ACP) from E. coli. [16]nder the optimized conditions (0.5 M imidazole buffer at pH 6.5), the 0.4-0.6 palmitoyl group was installed on ACP, without competitive palmitoylation on Lys and Tyr residues.
In 1987, O'Brien et al. [17a] and Bizzozero et al. [17b] discovered that the S-palmitoylation of disc membrane rhodopsin and Opalmitoylation of myelin proteolipid protein were mediated by palmitoyl-Coenzyme A 2 without enzyme participation, respectively (Figure 2).Inspired by this observation, in 1991, Stults et al. reported the direct palmitoylation of recombinant lung surfactant protein C (SPÀ C, 33-35 amino acids) using palmitoyl-Coenzyme A 2 via thioester exchange. [18]Two palmitoyl groups were installed on the two neighboring Cys residues in human and bovine SPÀ C, while one palmitoyl group was installed on the single Cys in canine SPÀ C. The highly hydrophobic products were purified by reversed-phase HPLC on C4 column with npropanol/water (0.1 % TFA) as eluent.In 2005, based on the same principle, Leung and Silvius investigated the S-palmitoylation using thioester reagents simpler than 2. [19] Using short model peptides derived from S-farnesylated H-Ras protein and P o -glycoprotein, they identified thioesters 4 and 5 as the optimal reagents, giving much higher reactivity than 2 and high selectivity for Cys S-palmitoylation over Lys and Ser modification (Figure 2).The application of the (3-(3cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS) micelle was found to significantly enhance the reactivity.
In 1999, Strömberg et al. reported a new method for direct S-palmitoylation of peptides using palmitoyl chloride 3 as the acyl donor (Figure 2). [20]Unlike the above methods using aqueous buffers as the reaction media, 100 % TFA was used in this case to achieve fast (< 10 min) and selective reaction.As a bonus, the hydrophobic peptides derived from SPÀ C were well dissolved in TFA after the installation of two palmitoyl groups.
The product was purified by RP-HPLC on C18 column with isopropanol/ethanol as eluent.Unfortunately, no differentiation between Cys and Ser/Thr was observed, as the model SPÀ C peptide with Cys mutated to Ser gave the same level of di-Opalmitoylated product.
Though all the methods mentioned above have been demonstrated to be powerful in several cases, they share the same limitation.No selectivity can be achieved between differently located Cys residues unless using protecting group, which is difficult for recombinant proteins.

Solution-phase peptide synthesis
One way to tackle the selectivity problem is the stepwise chemical synthesis, during which the modification or protection can be installed sequentially.For the synthesis of S-palmitoylated peptides, solution phase synthesis has become one appealing method since 1995. [21]In this area, most of the endeavor was focused on the synthesis of C-terminal peptides derived from Ras proteins, which bear both S-palmitoylation and S-farnesylation (Figure 3  (palmitoylation) group, the appropriate protecting groups of the N-and C-terminus of the S-Palm containing peptide building blocks are inevitable for the successful synthesis.The widely used Fmoc and Cbz N-terminal protecting groups fail in this case as the thioester will be destroyed by high concentration piperidine treatment and hydrogenolysis.Boc group is compatible with S-Palm modification, but the S-Far (farnesylation) will undergo carbocation induced cyclization under acidic conditions. [22]For C-terminus, esters that need saponification or hydrogenolysis for removal are all excluded.
21b] Based on this N-terminal protecting method, Waldmann et al. reported the synthesis of N-Ras derived heptapeptide fluorescent probe bearing both S-Palm and S-Far modifications and discovered that only the coexistence of the two modifications could lead to plasma membrane location of the probe in NIH-3T3 fibroblast cells. [23]nother N-terminal protecting group 4-phenylacetoxybenzyloxy (PhAcOZ) that could be removed by Penicillin G acylase treatment at pH 6.8 [24] was also developed for the synthesis of peptides bearing S-Palm modifications (Figure 3a). [25]or C-terminal protection, the allyl ester that can be removed by Pd(PPh 3 ) 4 /morpholine [21,23] and Pd(PPh 3 ) 4 /N,N'dimethylbarbituric acid (DMB) [25,26] was well tolerated in the synthesis.Moreover, a positively charged ester group with solubilizing effect derived from choline (Cho) was introduced by Waldmann et al. as an enzymatically cleavable C-terminal protection (Figure 3a). [27]The cleavage was facilitated by butyrylcholine esterase catalyzed hydrolysis at pH 6.5.By using the protecting groups mentioned above, Waldmann et al. finished the synthesis of a series of lipidated peptides bearing S-Palm modifications (Figure 3b), including N-Ras hexapeptide (S-Palm/S-Far), [28] biotinylated N-Ras heptapeptide (S-Palm/S-Far), [29] R-Ras pentapeptide (S-Palm/S-Far), [30] N-terminal hexapeptide of Gαo protein (S-Palm/N-terminal myristoylation), [31] H-Ras octapeptide (two S-Palm/S-Far), [32] influenza A virus hemagglutinin derived fluorescent hexapeptide probe (two S-Palm), [33] and endothelial NO-synthase (eNOS) N-terminal lipopeptide (two S-Palm/N-terminal myristoylation, 29 amino acids). [25]uring the synthesis of the R-Ras derived C-terminal peptide, to get compatibility with the S-Far group, the Lys side chain was protected by 4-methyltrityl (Mtt) group, while the Cys side chain was protected by 4-methoxytrityl (Mmt).The two groups could be removed by 1 % TFA in dichloromethane (DCM) containing Et 3 SiH. [34]n all the above synthesis, the S-Palm was installed via palmitoylation of the Cys side chain.During this process, Cys side chain serves as a nucleophile.In 2005, a new synthetic route of N-Ras hexapeptide was reported by Schmidt et al., in which the S-Palm was installed in an inversed way.The βbromoalanine containing peptide prepared in solution phase was treated with thiopalmitoic acid to give the thioester via S N 2 substitution. [35]n the above successful solution phase syntheses, the protected S-Palm dipeptides like AcOZ-Gly-Cys(Palm)-OH were used as building blocks for peptide elongation, which could avoid undesired S-to-N palmitoyl transfer during the N-terminal deprotection.However, the amide coupling of the dipeptide might lead to C-terminal epimerization since Cys has been regarded as racemization prone.In reported cases, optimized coupling conditions like N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI)/1-hydroxybenzotriazole (HOBt)/ Et 3 N, [30] N,N'-diisopropylcarbodiimide (DIC)/HOBt, [21b] DIC/HOBt/ Et 3 N 23 and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ)/Et 3 N 33 were used to give good results, without detailed study of the epimerization issue.

Solid-phase peptide synthesis
The successful syntheses of S-Palm peptides in solution phase demonstrate that the S-Palm peptides in both side chain protected or unprotected forms are handleable to synthetic chemists.However, the tedious column purification after each coupling step makes the solution phase approach laborious which limits its application.The solid phase peptide synthesis with minimized purification efforts becomes the goal to pursue.
For the success of solid phase synthesis, choosing appropriate immobilization linkers to generate special C-terminal modifications and facile strategies to introduce S-Palm are key questions to consider, which is showcased in the synthetic studies toward S-palmitoylated Ras peptides (Figure 4a and 4b).Since the C-termini of native Ras proteins are methyl ester of Cys residue, the application of a suitable linker that can facilitate methyl ester formation without destroying the S-Palm is needed.In 2002, Waldmann et al. reported the first solid phase synthesis of Ras peptide C-terminal methyl esters bearing S-Palm and S-Far, by using a hydrazine-based linker and latestage on-resin S-palmitoylation strategy (Figure 4a). [36]The 4hydrazinylbenzoic acid linker developed before [37] was installed onto the AM NovaGel resin to give rise to 6, from which a standard Fmoc-SPPS was conducted to assemble the peptide.To overcome the difficult coupling of S-Far Cys, the S-Trt protected Cys was installed first, followed by the assembly of tripeptide 8.The S-Far group was then installed on resin after removing S-Trt.After installing the next four amino acids, the S-Mtt protection on the second Cys residue in 11 was removed under milder acidic conditions (compatible with acid-labile S-Far group), and S-Palm was installed by palmitoyl chloride 3. The subsequent elongation used the Alloc protected amino acid building block.Finally, the linker in 12 was oxidized by Cu(OAc) 2 and O 2 to afford N-acyl diazene intermediate, which was treated with pyridine (30 equiv)/HOAc (50 equiv)/MeOH (215 equiv) to give rise to methanolysis product 13 without Cterminal epimerization.The late-stage on-resin S-Palm installation strategy can avoid the possible S-to-N palmitoyl transfer during the N-terminal deprotection and the risk of epimerization during the coupling of S-Palm Cys residue.Without the need to form C-terminal ester, this strategy has been applied on different resin types in the synthesis of several lipopeptides, including S-Palm modified antigen peptides derived from gonadotropin-releasing hormone and VP2 protein of canine parvovirus, [38] single S-Palm modified SPÀ C protein, [39] S-Palm Tcell epitopes of myelin proteolipid protein, [40] Src oncoprotein derived tripeptide probe bearing both N-terminal myristoylation and S-Palm, [41] and Trp-flanked ion channel with S-Palm on N-terminal Cys. [42]n 2004, Waldmann et al. reported the synthesis of Ras peptide C-terminal methyl ester by using the second strategy, In which the Cys building blocks with S-Palm and S-Far installed were used in the Fmoc-SPPS (Figure 4b).[43] Starting from resin 6, the first Cys residue 14 bearing S-Far was installed using DIC/ HOBt in DCM/DMF 1 : 1 to avoid racemization.Theses conditions were also used in the coupling of Fmoc-Cys(S-Palm)-OH 17 to give 18.Next, to avoid the S-to-N palmitoyl transfer as well as the aminolysis of the labile thioester by piperidine, a special Fmoc removing condition, 1 % 1,8-diazabicyclo[5.4.0]undec-7ene (DBU) in DCM for 2 × 30 s followed by 6 × 5 s fast DMF washing, were used.Further elongation was then conducted immediately with preactivated amino acid building block in DCM/DMF 4 : 1 or even 7 : 1 to suppress the palmitoyl transfer.Further modification of the N-terminus of 20 by maleimide derived acid 21 followed by linker oxidation and methanolysis afforded product 23.With this optimized protocol, several S-Palm/S-Far peptides containing up to 16 amino acids were efficiently synthesized.In 2006, another type of linker, the Ellman sulfonamide linker, [44] was used by Waldmann et al. to the S-Palm peptide synthesis (Figure 4c).[45] After the assembly of the full peptide sequence, the N-peptidyl sulfonamide was activated by N-alkylation via ICH 2 CN/N,N'-diisopropylethylamine (DIPEA)/N-methylpyrrolidin-2-one (NMP) treatment, followed by hydrolysis or methanolysis to give rise to the C-terminal free acid or methyl ester respectively.With this method, the S-Palm peptides derived from N-Ras, H-Ras, K-Ras, phospholipase D and eNOS were achieved in good yields.The fluoride-labile (2phenyl-2-trimethylsilyl)ethyl (PTMSEL) linker developed by Kunz et al. [46] was also used by Waldmann et al. for S-Palm peptide synthesis (Figure 4c).[47] The mild cleavage condition, 2 equiv tetrabutylammonium fluoride trihydrate (TBAF-3H 2 O) in DCM, released the C-terminal free acid without destroying the thioester.

Protein semisynthesis via peptide ligations giving nonnative linkages
Armed with the solution phase and solid phase syntheses, the assembly of S-Palm peptides containing up to 29 amino acids has been achieved.However, the investigation of the function of S-palmitoylation on protein location and activity needs the modified full protein beyond the peptide probe to be synthesized.The protein semisynthesis via joining the chemically synthesized short S-Palm peptides and expressed long fragments together in site specific manner becomes an appealing choice.
In 2000, Waldmann and Kuhlmann et al. reported the semisynthesis of H-Ras protein bearing different C-terminal S-Palm/S-Far patterns (Figure 5a). [48]The recombinant H-Ras 1-181 24 was ligated with N-terminal maleimide modified S-Palm/ S-Far peptide 23 synthesized in solution phase via the Michael addition of C-terminal Cys181 side chain and with the maleimide group (MIC ligation) under physiological pH to afford the hybrid protein mimic 25.The hybrid proteins H-Ras G12 V bearing activating mutation were also constructed in the same way.Microinjection of the mutated hybrid protein into rat pheochromocytoma cell line PC12 successfully induced the differentiation phenotype in comparable level as the full-length H-Ras G12 V mutant, and the hybrid bearing both S-Palm and S-Far gave stronger inducing effect than the one bearing only S-Far but lacking the Cys residue for S-Palm formation.Changing the hydrolysable S-Palm to a lipid thioether led to the decrease of activity, which indicated the importance of the dynamic feature of thioesters for the protein function.In 2005, using the semisynthetic N-Ras mimic proteins bearing S-Palm/S-Far and Shexadecyl/S-Far modifications, Wittinghofer and Bastiaens et al. revealed the essential role of the S-deacylation/reacylation cycle for the rapid exchange of Ras proteins between plasma membrane and Golgi apparatus. [49]The S-depalmitoylation redistributes the S-Far Ras to all the membranes, and the Srepalmitoylation leads to the trapping of Ras at Golgi and redirecting to plasma membrane via secretory pathway.
In 2006, a new reaction called Diels-Alder ligation was developed by Waldmann et al. to construct the Rab7 protein mimic bearing S-Palm and S-Far modifications (Figure 5b). [50]he truncated Rab7 fragment 26 was obtained from the recombinant thioester via native chemical ligation (NCL) with the diene containing linker modified with N-terminal Cys.The Cys side chain after NCL was protected in disulfide bond form by the Ellman's reagent to avoid the competitive MIC ligation.The Diels-Alder ligation with 100 equiv peptide derived maleimide dienophile at pH 5.5-6.5 followed by disulfide cleavage with dithioerythritol (DTE) gave product 28.

Protein chemical synthesis via peptide ligations giving native linkages
The proteins formed via semisynthesis have been successfully applied to biological studies.However, the potential effects of the non-native structures on the bioactivities need to be evaluated case by case.Thus, the preparation of proteins bearing S-Palm and O-Ac modifications without incorporating non-native structures like thiol-maleimide adduct (MIC ligation) and cyclohexene (Diels-Alder ligation) becomes the next goal.Till today, it has only been achieved via protein chemical synthesis.
In 2020, Zheng et al. reported the total chemical synthesis of S-palmitoylated influenza virus M2 channel protein via two strategies (Figure 6). [51,52]Since the S-Palm is not compatible with the most popular native chemical ligation due to the S-to-S palmitoyl transfer to the N-terminal Cys followed by S-to-N transfer, the authors applied the serine/threonine ligation (STL) developed by Li et al. [53] for the synthesis of S-Palm M2 (1-97).To tackle the challenge from the hydrophobicity of the Spalmitoylated protein and peptide fragments, Zheng et al. introduced the removable backbone modification (RBM) [54] to the most hydrophobic transmembrane domain.The phenolic hydroxyl group of the RBM was acylated by Boc protected γaminobutyrate, so the RBM GABA can survive the global deprotection and release of free phenol under mild condition compatible with S-palmitoylation.
In the first strategy, the full sequence protein was formed via a single serine ligation at the Ala30-Ser31 site within the transmembrane domain. [51]The fragment 29 was synthesized on Wang resin, and the RBM GABA was installed on-resin via reductive amination and amide coupling.A Lys 6 tag was also installed to further increase the solubility.After removing Mmt from the Cys50 and S-palmitoylation, 31 was subjected to global deprotection.Due to the strong acidic conditions, the γaminobutyrate on the TBM GABA group was kept in the protonated form, which helped the solvation of the peptide 32 and facilitated HPLC purification.Dissolving 32 in pyridine/HOAc buffer triggered the cyclization of the γ-aminobutyrate and formation of 33, which was then ligated with peptide salicylaldehyde (SAL) ester 34 to give the S-palmitoylated full sequence M2 channel 35.The correct folding of M2 channel was confirmed by CD spectrum in the n-octyl-β-glucopyranoside vesicles.This strategy was also used in the synthesis of rabbit S-palmitoyl sarcolipin (SLN). [51]n the second strategy, Zheng et al. developed the N-to-C sequential NCL/STL to achieve membrane protein synthesis. [52]nlike the N-to-C sequential STL/STL and STL/NCL developed by Li et al. in 2016, [55] the new strategy needs the preparation of the peptide SAL ester (or its protected form) with N-terminal free Cys residue.Since the preparation of the N-terminal free Cys peptide with C-terminal SAL ester protected in semicarbazone form failed due to the formation of side products, they synthesized the peptide with C-terminal SAL ester protected in dithioacetal form via semicarbazone-dithioacetal exchange with 1,3-propanedithiol (PDT).This PDT protected SAL ester can survive the typical NCL reaction, and the release of SAL ester is facilitated by AgNO 3 /N-chlorosuccinimide (NCS) treatment.With the established protocol, the S-palmitoylated M2 channel was synthesized via the sequential NCL (at Gly16-Cys17 site) and STL (at Ala30-Ser31 site) of fragments 36, 37 and 33.After the NCL, the free thiol at the ligation site was transformed into disulfide with Cys19 by 4,4'-dithiodipyridine (DTDP) 38 treatment, and the PDT group was deprotected to give SAL ester 34.The next serine ligation was the same as the first strategy to give full sequence of M2 channel.This sequential ligation strategy further expanded the application of STL reactions in the synthesis of large proteins.It was also applied to the synthesis of S-palmitoylated interferon-induced transmembrane protein 3 (IFITM3). [52]n 2021, Hojo et al. reported the chemical synthesis of Cys133/143/156 triple S-palmitoylated caveolin-1 (Cav-1) protein (Figure 7). [56]As a membrane protein, the location of Cav-1 to the cytoplasm side of the cell membrane is controlled by the triple S-palmitoylation.Thus, preparation of this triple Spalmitoylated protein is important to further understand its function.To achieve this goal, Hojo et al. developed a convergent synthesis based on the aminolysis of peptide thioesters (developed by Hojo and Aimoto in 1991 [57] ) followed by the late-stage S-palmitoylation. [56]Since Cav-1 was very hydrophobic even without S-Palm groups, four O-acyl isopeptide [58] units were introduced to the C-terminal wing of Cav-1 with the α-amino groups protected by hydrophilic 4pyridylmethoxy carbonyl (iNoc) groups Based on the same strategy, the O-palmitoylated histone H4 protein was synthesized. [59]n 2022, Li et al. reported the synthesis of histone H3 protein bearing Ser10/Thr22 O-acetylation modification (Figure 8). [60]his type of PTM is relatively rare in proteins, and the function of this PTM is still less understood.Compared to the Opalmitoylation, the O-acetyl group was more sensitive to bases (β-elimination) and nucleophiles (acetyl removal), which was difficult to be introduced into peptide sequence via standard Fmoc-SPPS.The Boc-SPPS approach was suitable for its syn-   thesis.However, due to the lack of soft cleavage conditions for the Boc-SPPS, the "n + 1" approach [55] and direct coupling with protected salicylaldehyde dimethyl acetal for SAL ester synthesis could not be used.To this end, Li et al. developed a new method to prepare peptide SAL esters via Boc-SPPS, based on the semicarbazone chemistry [55] without using oxidative conditions (e. g., O 3 ). [61]Based on this method, the peptide SAL ester 50 bearing base-labile PTMs such as O-acetylation and Spalmitoylation was readily prepared from semicarbazonemodified AM resin 49.The O-acetylation was installed via coupling of Boc-Ser(OAc)-OH and Boc-Thr(OAc)-OH to the preneutralized resin-bound peptide by DIC in DMF, followed by Boc removal and subsequent peptide elongation under HATU/ DIPEA conditions, without observing the O-to-N acetyl transfer.
For the assembly of the peptide fragment, a C-to-N sequential cysteine/penicillamine ligation (CPL) [62] strategy was used.Compared with STL, CPL involves C-terminal SAL ester and N-terminal Cys/Pen instead of Ser/Thr for ligation with the formation of similar N,S-benzylidene acetal intermediate.CPL generally showed much faster ligation kinetics, which can take place under lower concentration at hinder ligation sites and show good compatibility with both pyridine/HOAc buffer and acidic aqueous buffer.As illustrated in Figure 8, through three sequential CPL reactions, the fragments 50, 51, 52 and 54 were joint together to give 56, and the three N,S-benzylidene acetal groups were removed via acidolysis using 0.2 M HCl in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in the presence of triisopropylsilane (TIPS) and 1,2-ethanedithiol (EDT).The three thiol groups released were then removed via radical desulfurization, [63] and the two native Cys residues were released via PdCl 2 mediated Acm removal [64] to give 57.The synthetic histone H3 (S10ac, T22ac) protein was tested in mononucleosome reconstitution together with recombinant histone H2A, H2B and H4.
The Boc-SPPS based peptide SAL ester synthesis can also fit the demand of the synthesis of S-palmitoylated peptide SAL ester, in which the thioester is more base-labile than O-acetyl moiety.Thus, Li et al. applied their Boc-SPPS of peptide SAL esters to develop the synthesis of S-palmitoylated caveolin-1 (131-155) fragment 63 (Figure 9). [60]In this work, the 131-139 fragment derived SAL ester 60 was synthesized via Boc-SPPS from 58, and a Lys 6 solubilizing tag was installed onto the phenyl ring of salicylaldehyde to facilitate both ligation and product purification.The S-Palm was installed during the Boc-SPPS using the S-palmitoylated building block Boc-Cys(Palm)-OH 59 by DIC/HOBt coupling to minimize the racemization.When installing the next amino acid, the coupling was done in 35 % THF/NMP to avoid the S-to-N acyl transfer.The CPL reaction between 60 and 61 worked smoothly under acidic condition (pyridine/HOAc 1 : 9) and showed full compatibility with S-Palm modification without observing palmitoyl group transfer, which was quite different from NCL involving near neutral to slightly basic condition.Purified 62 was subjected to acidolysis to remove the N,S-benzylidene acetal together with the Lys 6 tag, and the highly hydrophobic peptide 63 was characterized by Tricine-SDS-PAGE and ESI-MS.

Conclusion and Perspectives
The base-labile post-translational modifications like S-palmitoylation and O-acetylation have been identified in many proteins with biological interests.Detailed investigation of their roles in the biological processes needs the homogeneous peptide probes and proteins bearing the designated PTMs.Due to the high complexity of the protein structures, especially the existence of multiple Cys and Ser/Thr residues with the potential to be modified and largely unknown "writers" for these PTMs, generation of S-palmitoylated or O-acetylated proteins directly via biological approaches is almost impossible.Chemical synthesis, including both semisynthesis and total chemical synthesis, becomes an appealing choice.In this short review, we summarized the synthesis of peptides and proteins bearing S-Palm and O-Ac modifications, with the focus on the evolution of the methods from the direct palmitoylation of the peptides/proteins which is almost nonselective, to the stepwise peptide synthesis (both solution phase and solid phase) and protein synthesis via modern ligation reactions.
The huge progress in the past two decades, especially the development of ligation methods and solubilizing technologies, facilitates the assembly of short to medium sized membrane proteins (currently up to 177 amino acids and will increase further) via joining the hydrophobic and less reactive peptide fragments.For larger sized proteins like GPCR and immune checkpoint proteins, the semisynthesis using the expressed non-modified long fragment and the chemically synthesized short PTM-bearing fragment will be more powerful.The semisynthesized H-Ras and N-Ras protein mimics bearing S-Palm and S-Far modifications help scientists to identify the role of the two lipid moieties on its membrane location and reveal the importance of the dynamic nature of S-Palm modification.Since the S-palmitoylated Ras proteins with native sequences are still not available due to the incompatibility of NCL with side chain thioester group, [65] the semisynthesis of the native proteins bearing S-Palm via STL or other ligation methods will be of great interest and a valuable goal to pursue.
The synthetic challenges not only come from the base lability, but also the hydrophobicity.The S-palmitoylated peptides are generally poorly soluble in aqueous buffer and several other solvent systems, which impede their purification and further ligation.Increasing the solubility and reactivity of these "difficult peptides" is long-lasting question in peptide chemistry. [66]Some recently developed technologies like ligation embedding aggregation disruptor (LEAD), [67] tunable backbone modification (TBM) [68] and reducible solubilizing tags (RSTs) [69] are worth to be tested.We hope this short review could be helpful for the scientists who plan to start their adventure in this field and inspire the development of new methods to tackle the challenges in both synthetic protein chemistry and chemical biology.
). Due to the base-lability of S-Palm Wenjie Ma received her Ph.D degree in 2022 from the University of Hong Kong.She is currently a postdoctoral researcher in Prof. Xuechen Li's group.Her research interests include the chemical synthesis of proteins and bioactive cyclic peptides.Han Liu received his B.S. and Ph.D degrees from Peking University.Then he did postdoctoral research at the University of Hong Kong and Aarhus University.Currently, he is a research assistant professor at the Department of Chemistry, the University of Hong Kong.His current research interests include total synthesis of peptide antibiotics and development of synthetic glycoconjugate vaccines.Xuechen Li received his PhD degree from Harvard University in 2007, then as a postdoctoral researcher at Memorial Sloan Kettering Cancer Center, US.Currently, he is a professor in the Department of Chemistry, the University of Hong Kong.His research fields include chemical protein synthesis, bioconjugate chemistry, carbohydrate chemistry, chemical biology and drug discovery.
. The full sequence (2-178) was divided into five fragments 40-44, and the Pro or Gly were chosen for the thioester formation to avoid the risk of epimerization.The aminolysis between 43 and 44 (with Nterminal and Lys5 side chain capped by acetyl) was mediated by 3-hydroxy-1,2,3-benzotriazin-4-one (HOOBt)/DIPEA to give 45 under kinetic control, with the less reactive alkyl thioester group kept intact.The assembly of the full sequence 46 then started from the C-terminal fragment 40.The fragments 41, 42 and 45 were installed sequentially in C-to-N manner mediated by AgCl/HOOBt/DIPEA.All the aminolysis reactions were conducted in DMSO, which was beneficial for the synthesis of hydrophobic peptides.To further increase the solubility of fragments 41 and 42, a short Arg 3 tag was introduced to the thioester groups.The three S-Palm groups were installed onto Cys133/143/156 by Palm-OSu 47 after the removal of three Acm groups by AgNO 3 .The iNoc groups were removed by SmI 2 treatment in DMF, and the O-to-N acyl transfer was facilitated by 2,2,2-trifluoroethanol (TFE)/sodium phosphate buffer at pH 7.3.Though the HPLC analysis and MALDI-TOF characterization of the triple S-palmitoylated Cav-1 48 were prohibited by its highly hydrophobic feature, the synthetic protein successfully folded into bicelle and gave correct CD spectrum.