Hypervalent Iodine‐Mediated Late‐Stage Peptide and Protein Functionalization

Abstract Hypervalent iodine compounds are powerful reagents for the development of novel transformations. As they exhibit low toxicity, high functional group tolerance, and stability in biocompatible media, they have been used for the functionalization of biomolecules. Herein, we report recent advances up to June 2021 in peptide and protein modification using hypervalent iodine reagents. Their use as group transfer or oxidizing reagents is discussed in this Minireview, including methods targeting polar, aromatic, or aliphatic amino acids and peptide termini.


Introduction
Peptides and proteins are increasingly considered as drug candidates by established pharmaceutical companies. [1] Around 80 peptide therapeutics are currently on the market, more than 150 peptides in clinical development, and 400-600 in preclinical studies. [1i] Therefore,t he need for novel bioconjugation strategies is constantly in demand to improve the properties of biomolecules or to study their biological function. [2] However,the number of chemical transformations suitable for effective peptide and protein functionalization is still limited. They represent unique challenges for synthetic chemists,because of the range of functional groups present. In addition, these transformations need to be selective at asingle site,p roceed with fast reaction rates,o perate under biologically compatible conditions,a nd should provide stable bioconjugates with near complete conversion. [3] Hypervalent iodine reagents have recently emerged as powerful tools for the functionalization of peptides and proteins.T hese reactive compounds have high functional group tolerance,are stable in biocompatible media, [4] and are relatively nontoxic. Over the years,avariety of acyclic and cyclic hypervalent iodine reagents have been developed. Many of them are known for their oxidizing character,while others have been used as electrophilic group transfer reagents.T herefore,t hey can react with nucleophilic and oxidizable amino acids and can be used for the development of new methods for biomolecule functionalization (Figure 1). [5] This Minireview depicts the recent advances up to June 2021 in the field of hypervalent iodine-mediated modification of peptides and proteins.T he functionalization of single amino acids and hypervalent iodine-mediated peptide syntheses or cleavages will not be discussed. This Minireview is divided into four parts based on the targeted residue:sulfur-containing,aromatic, or aliphatic amino acids or peptide termini.

Cysteine
Cysteine (Cys) is one of the most targeted residues for the modification of biomolecules. [6,7] Its low natural abundance and high nucleophilicity enable fast and efficient site-selective bioconjugation methods,particularly in basic media. [7] Hypervalent reagents 1, 2,a nd 3 have been used by the Togni and Zhang groups as well as our group,respectively,for the fluoroalkylation, thiolation, and alkynylation of thiols. [8][9][10] Them ethods have been then extended to peptides and proteins.

Fluoroalkylation of Cys
Theintroduction of fluorinated groups onto biomolecules in amild and selective manner has attracted growing interest, as an improved in vivo stability can be achieved. [11] To perform trifluoromethylation reactions,t he Togni group developed cyclic hypervalent iodine reagents:t he trifluoromethyl benziodoxol(on)es 1a and 1b (Scheme 1). [5d, 8, 12] In ac ollaboration with the Seebach group,C ys residues in peptides (up to 13-residues long) containing a-a nd b-amino acids were trifluoromethylated in good yields using 1a (9, Scheme 1a). [13] When applicable,i tw as necessary to reduce the disulfide bridge before the addition of the hypervalent iodine reagent, as it was inert to the reaction conditions.T his transformation was also applied to the reduced (ring-opened) Hypervalent iodine compounds are powerful reagents for the development of novel transformations.Ast hey exhibit lowt oxicity, high functional group tolerance,and stability in biocompatible media, they have been used for the functionalization of biomolecules.Herein, we report recent advances up to June 2021 in peptide and protein modification using hypervalent iodine reagents.T heir use as group transfer or oxidizing reagents is discussed in this Minireview,including methods targeting polar,aromatic, or aliphatic amino acids and peptide termini. peptide-based drug octreotide 10 (Scheme 1b). [13] Reagent 1b was used along with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to yield compound 11 as the only observed product. In the absence of DBU,t rifluoromethylation of the tryptophan side chains was observed, while unprotected nucleophilic residues,i ncluding free amino and carboxylic groups, stayed intact.
Them echanism was investigated by experimental and computational studies using reagent 1a and thiophenol under standard reaction conditions (without additive;Scheme 2). [14] Ar adical pathway is the most plausible,a sthe CF 3 and thiyl radical species were intercepted by scavenging and trapping experiments.T he disulfide and CF 3 Hb y-products were also observed. Protonation of 1a is essential for the reaction to proceed. In the most plausible mechanism, the protonation would occur simultaneously with I À Sb ond formation (intermediate I), thereby yielding II.AHammett plot, which shows the formation of as mall negative charge on the sulfur atom during the rate-determining step,supports this pathway.After homolytic cleavage,g eneration, and recombination of CF 3 (IV)a nd thiyl (V)r adicals,t he desired compound would be generated. Other possible mechanisms start by the protonation of 1a by an external proton source.T he S-trifluoromethylated compound would then be formed after homolytic cleavage,recombination, and deprotonation.
TheT ogni and Beier groups later developed as eries of hypervalent iodine reagents bearing functionalized perfluoroethyl groups (Scheme 3). [15] These compounds present ar e-activity similar to their trifluoromethylated analogues and react with Cys residues.Awide range of reagents were synthesized, among them 1c and 1d bearing pyrene-or coumarin-based chromophores. [15,16] Glutathione (12)  Scheme 2. Most plausible mechanism for the trifluoromethylation of thiols.

Scheme 3.
Selected examples of perfluoroethyl-substituted reagents and application to glutathione labeling.T he yield of the isolated product as atrifluoroacetate salt is given in brackets. labeled in moderate yields (34 %b y 19 FNMR spectroscopy and 8% of the isolated product) using reagent 1d in an aqueous basic media at room temperature.
As 1c and 1d required am ultistep synthesis,amodular reagent 1e was developed as ap recursor to aw ide range of bioconjugation reagents (Scheme 4). [17a] 1e features as econdary amine that can be involved in classical linking methods compatible with the hypervalent iodine moiety.F or example, amide,s ulfonamide,o rt ertiary amine bonds can be easily generated, yielding reagents 1f-h.
In addition to the studies by the Togni and Beier groups on fluoroalkylation, Zhang and co-workers described the synthesis of the trifluoromethylthiolating reagent 2 based on an N-acetylbenziodazole core. [9] Cys-containing dipeptides were converted in high yields and chemoselectivity in 30 minutes into disulfides 21-26 (Scheme 7).
[21a] Moreover,afluorophore tag was added in at wo-step process:r emoval of the TIPS group and subsequent CuAACt ogive 33 (Scheme 8b). [10] Ther eaction mechanism was investigated using experimental and computational methods. [21] Thef irst step is believed to be the formation of the thiolate I,s ince in the absence of abase,only traces of the thioalkyne product were observed (Scheme 9). I can then interact with the Lewisacidic iodine (II). From here,there are two possible reaction pathways.I nt he case of a-addition of the thiolate I, computational studies revealed low energy transition state III (path a). III corresponds to ac oncerted a-addition/ elimination to give the thioalkyne product. In contrast, attack of thiolate I on the b-carbon atom occurs via the transition state IV (path b) leading to b-addition and formation of the vinyl anion V.T he latter then undergoes an a-elimination to form VI,w hich, after a1 ,2-shift, yields the thioalkyne. Alternatively,i np rotic solvents,i ntermediate V can be protonated, leading to vinylbenziodoxolone (VBX) products.
Calculating the free energy values of the transition states for different hypervalent iodine reagents revealed that the R 2 group on the alkyne has as trong influence on the reaction pathway.W henR 2 is an electron-withdrawing group,t he route going through an a-addition is the lower energy pathway,s ince the partial negative charge is stabilized. In contrast, when R 2 is an electron-donating group,s uch as an alkyl substituent, the preferred pathway is the b-addition. For aryl and silyl substituents,b oth pathways are possible,s ince the energy difference is small.
In joint efforts by Adibekiansa nd our group,t he thioalkynylation methodology was applied to in vitro and in situ proteomic profiling of Cys residues. [22] To avoid the need for cleavage of the TIPS group,b ut still allow functionalization of proteins through aC uAACr eaction, the EBX reagent 3c,c arrying an azide,w as used (Scheme 10 a).
Ther eactivity of 3c was tested on purified catalase and complete cell lysates under physiological conditions.U nder these neutral conditions,o nly more acidic-hyperreactive-Cys residues were deprotonated and reacted. It is believed that, due to the high reactivity and lipophilic environment of the Cys residues,i nduced by neighboring amino acids, thioalkynes rather than VBXs were obtained as major products,w hereas the reverse is observed for Cys with regular reactivity (see Section 2.1.3.). [23] 3c displayed higher efficiency,l ower cytotoxicity,a nd higher chemoselectivity compared with the commonly used Cys probe iodoacetamide (IAA) or IAA-alkyne.R eagent 3c was used for target discovery of the anticancer and anti-inflammatory agent curcumin. It was demonstrated to be complementary to IAAalkyne,s ince out of the 42 detected targets,1 6w ere exclusively modified by 3c.
In 2020, our group developed reagents bearing aT MS group and various aromatic substituents,w hich under aqueous media undergo fast TMS cleavage (Scheme 10 b). [24] For example,reagent 3e provided improved Cys labeling in HeLa cell lysates compared to 3c (Scheme 10). After digestion of the labeled proteins,4325 labeled peptides were detected for 3e,while 2257 were observed for 3c.The terminal thioalkynes were formed as the only products after rearrangement of the VBX intermediates with high Cys selectivity.The method was also extended to in situ labeling of living HeLa cells.
Next, modification of an antibody approved for breast cancer treatment, trastuzumab (34), was explored. Theuse of 8equivalents of 3f at pH 7.5 and 25 8 8Cf or 2minutes (Scheme 11) gave 60 %c onversion of the reduced trastuzumab (34)toyield TAMRA-labeled 35 with an average degree of conjugation (DoC) value of 1.2 after CuAAC. Under these conditions no side-reactivity was observed, also with nonreduced trastuzumab.V arying the reaction parameters led to DoC values ranging from 0.1 to 4.4. Whereas some side reactivity with the non-reduced antibody was observed under most conditions,itwas outcompeted by cysteine alkynylation of the reduced antibody.
Later, amphiphilic hypervalent iodine reagents were introduced for the lipidation of Cys residues. [25] Reagents bearing aT IPS group (3g)o raC 14 alkyl chain (3h)w ere synthesized (Scheme 12). Water solubility was achieved by introduction of a p-sulfonate group on the aromatic core.The presence of the sulfonate group led to thioalkynes as the major products,e ven with alkyl substituents.B oth reagents were applied to av ariety of hexapeptides,a nd yielded, for example, 37 and 40 in yields of 89 and 72 %i na queous buffers.Reversibility of the functionalization was achieved by hydration of the thioalkyne (38,41)and subsequent cleavage with hydroxylamine.T he method was further extended to longer peptides 42 and 45,a sw ell as to aH is6-Cys-ubiquitin protein 48 at micromolar concentration (Scheme 13).
In 2020, doubly functionalized reagents were developed for peptide stapling. [26] Bis-hypervalent iodine reagents, linked by phenyl or various silicon groups,w ere synthesized with Cys cross-linking.I nparticular, reagent 3i provided the i,i + 4s tapled peptide 51 in high yield (Scheme 14 a).
Installation of an activated ester allowed Cys-Lys stapling to be performed. Reagents bearing phenyl linkers with various substitution patterns were reported. Fore xample, excellent reactivity was observed with the para-substituted reagent 3j (Scheme 14 b), particularly when applied for i,i + 7 stapling of peptide 52 derived from p53 protein. Theobtained product 53 showed increased helicity and binding affinity to MDM2 protein-a native binder of p53 protein and aknown cancer target. Interestingly,since the reaction with Cys occurs first, proximity-driven Lyss electivity was observed. Ap oststapling modification for the Cys-Lys cross-linked products was achieved by performing RuAACw ith the thioalkyne present on the linker.T he triazole product 54 was generated in high yield and regioselectivity using either isolated stapled product (Scheme 14 b) or in aone-pot manner (Scheme 14 c).

Vinylation of Cys
In 2019, the application of the reagent 3c was extended from hyperreactive Cys (Scheme 10 a) to the less acidic surface-exposed Cys. [23] Ab asic buffer (pH 8.2) was used to efficiently convert less-reactive Cys residues,thereby leading to VBX products (Scheme 15). 3c was applied to the human Scheme 13. Lipidation of longer peptides and aprotein using 3g and 3h.

Scheme 14.
Selected examples of a) Cyc-Cys stapling, b) Cys-Lys stapling and the subsequent click reaction. c) One-pot Cys-Lys stapling followed by aclick reaction. Yields of isolated products are given in brackets.
Thep resence of ah ypervalent moiety enabled an additional modification, as demonstrated with the functionalized neuropeptide 60 (Scheme 16). Thep hotoprotection compound Tr olox was introduced through aS uzuki coupling between aboronic acid and the VBX moiety on 60 to give 61. Then aC y5 dye was added using strain-promoted cycloaddition to afford 62.T he bleaching time of the dye was increased by af actor of 3c ompared to when the Tr olox moiety was absent.

Methionine
Methionine (Met) has an important role in many biological processes.H owever,a saresult of its low nucleophilicity and high hydrophobicity,ithas been modified less often than Cys. [27] Nevertheless,h igh Met selectivity can be achieved under acidic conditions,t hus making it useful for the development of site-selective bioconjugation methods. [28] TheGaunt group used the fine-tuned acyclichypervalent iodine reagents 4a-c for peptide and protein labeling at Met residues (Scheme 17). [29] These types of compounds have been reported as efficient electrophilic diazo transfer reagents, [30] and allowing the generation of high-energy sulfonium conjugates.A dditives,s uch as thiourea, TEMPO,a nd formic acid (pH % 3) were required to minimize decomposition of the starting biomolecules,o xidized side-product formation, and achieve high selectivity.T he authors established in one case that the helical structure of the peptide was conserved after labeling, but no extensive studies were performed on the structural integrity and activity of other conjugates.T he reaction was tested on several peptides and proteins using diazo motifs bearing different substituents. High conversions were obtained in less than five minutes. Disulfide linkages and af luorescein-derived ester were tolerated (64, 66). As the N-terminal Met residue has only moderate surface exposure in ubiquitin 67,t he reaction was performed under deoxygenated conditions to minimize competitive oxidation. High conversion into the labeled product 68 with agood 10:1 labeling/oxidation ratio was obtained. As demonstrated with exenatide 65,reversibility and an excellent conversion was achieved using the tertiary phosphine tris(2carboxyethyl)phosphine (TCEP,S cheme 17 b). [31] Thes ulfonium products are stable enough for further derivatization. Irradiation led to the formation of radical ylides I,which can react with Hantzsch esters (Scheme 18). [32] Reduced trialkylsulfonium motifs 72 and 74 were obtained with high conversions using 70,without affecting the disulfide bridge of 73 (Scheme 18 a). 77 was formed with high conversion when the C4-benzylated Hantzsch ester 76 was used (Scheme 18 b). It is noteworthy that the Met bioconjugation and photoreduction steps can be carried out in ao nepot procedure,w ithout compromising the yield or purity of the products.

Aromatic Amino Acids
Although Cys bioconjugation is broadly applied, this residue is usually in the form of disulfide bridges or is an essential component of the active site of natural enzymes. Therefore,modification of other amino acids is also attractive. Forexample,the functionalization of tryptophan (Trp) [33] and tyrosine (Tyr) [34] can provide site-specific modifications,a s Tr pi st he rarest amino acid and Ty ri srarely exposed on the surface.

Fluoroalkylation of Trp
Functionalized tetrafluorinated hypervalent iodine reagents were used for the modification of Tr pr esidues in peptides and proteins.T he Novµka nd Beier groups discovered that sodium ascorbate triggers the generation of tetrafluoroethyl radicals and their reaction with Trpr esidues (Scheme 19, conditions A). [35] Later, visible light was used as the radical initiator (conditions B). [36] Preference for the Tr p C2-position was observed, but modification of the phenyl ring also occurred. Tolerance to ar ange of functional groups, including aromatic and nucleophilic residues,a sw ell as ad isulfide bridge,w as observed (78, 80). However,w hen the reaction was applied to ap eptide containing af ree Cys, both the Tr pa nd Cys residues were functionalized. [36] The sodium ascorbate initiated reaction was applied to myoglobin 81 using azide-substituted reagent 1n (Scheme 19 b). The more exposed Tr p14 was primarily functionalized, while Tr p7 was modified to alesser degree.Acycloaddition reaction with dibenzocyclooctyne-amine (DBCO-amine) was performed, which afforded labeled protein 83.

Alkynylation of Trp
In 2009, our group described the gold-catalyzed alkynylation of the C3-position of indoles,when the C2-position was blocked, using TIPS-EBX 3a. [37] Theexact mechanism of this reaction is still unknown, but has been proposed to involve alkyne transfer from iodine to gold without oxidative addition. [38] In 2016, the scope of the reaction was extended to Trpi ni ndependent studies by our group [39] and Hansen et al. (Scheme 20). [40] Using AuCl in acetonitrile,o ur group applied the method up to tripeptides,t hereby providing the desired alkynylated product 84 in moderate yield (Scheme 20 a). Hansen et al. demonstrated that the reactivity can be improved by the addition of aw eakly coordinating ligand (Me 2 S) and 2% TFA. [40] Full conversion was achieved with melittin 85-a peptide consisting of 26 amino acids,including Lys, Thr, Arg, and Ser,u sing ac atalytic amount of gold (86, Scheme 20 b). Thea lkyne-TIPS group was removed to perform ac lick reaction with dansyl-TEG-azide,t hereby yielding fluorescent-labeled melittin 87.The method was also applied to apomyoglobin 81,although asignificant amount of acetonitrile and 5equivalents of the gold complex were needed to achieve ag ood conversion and to obtain 67 %o f the dialkynylated and 25 %ofthe monoalkynylated products 88.

Arylation of Trp
In metal-catalyzed reactions,diaryliodonium salts behave as more reactive versions of aryl halides. [41] Using symmetrical or unsymmetrical reagents 4d-i,t he Ackermann group developed al ate-stage peptide C À Ha rylation of the C2position of Tr pr esidues (Scheme 21). [42] Peptides (up to 6 amino acids long) were arylated in good to excellent yields at ambient temperature using 5mol %Pd(OAc) 2 and acetic acid or pure water as the solvent to give 89-95.T he reaction tolerated other aromatic residues,b ut protecting groups on the nucleophilic ones were required. An unusual peptide coupling was performed using an asymmetric iodonium reagent bearing ap henylalanine residue to give 94.

Tyrosine
Recently,W ang et al. used (diacetoxyiodo)benzene (PI-DA, 5)f or the oxidation of phenol in Tyrr esidues,t hrough formation of the reactive intermediate 4-hydroxycyclohexadienone 96 (Scheme 22). [43] Subsequent reaction with arylhy-drazine yielded trans isomers of azobenzene-functionalized peptides.T he reaction was applied to arange of unprotected peptides containing up to 11 amino acids to give azobenzenes such as 97-99,t hus demonstrating broad functional group tolerance (Scheme 22 a). In addition, functionalized hydrazines were successfully introduced. Ah ydrazine-substituted peptide was utilized, which yielded 98.R eaction with aclickable alkyne moiety resulted in the biotin-functionalized peptide 100 after CuAAC (Scheme 22 b).

Angewandte Chemie
Theo xidation of Ty rr esidues using PIDA( 5)h as been also used for intramolecular coupling with Tr pt os ynthesize natural products. [44]

Aliphatic Amino Acids
Thel ate-stage derivatization of peptides and proteins targeting non-activated C À Hbonds is challenging due to their low reactivity and the difficulty of achieving selectivity. Nevertheless,h ighly reactive hypervalent iodine reagents have been successfully applied for the functionalization of leucine (Leu) and alanine (Ala) residues.
Ther eaction is believed to be initiated by ah omolytic cleavage of the weak I À N 3 bond of 6,thereby generating azido and iodanyl I radicals (Scheme 23 c). Then, I can perform ahydrogen abstraction on the substrate to yield intermediate II.T hisr adical can then attack reagent 6 to form the CÀN 3 bond, regenerating I,a nd propagating the radical chain. Interestingly,t he chlorination reaction seems to go through the same initiating step.T he chlorinated hypervalent analogue 105 is believed to be generated in situ by an exchange of azide with chloride.H owever,t he reaction did not proceed when only the isolated reagent 105 was used, since the I À Cl bond is harder to cleave.T he ABX reagent 6 is required for radical chain initiation. 105 is,however,more reactive than 6 towards nucleophilic attack of the C-tertiary radical II,a nd generates the chlorinated products.
Thes ame research group later described aC (sp 3 ) À H hydroxylation method. [47] Hydroxyperfluorobenziodoxole (PFBI-OH, 7a)w as used as it has as trong H-abstraction ability.T he reaction proceeded under similar photocatalyzed conditions with excellent selectivity.H ydroxylated dipeptide 107 was isolated in 44 %y ield, while lactone formation was observed with C-terminal Leu residues (109,Scheme 24).
Later, the Leonori group described ap hotoinduced remote H-atom transfer (HAT) strategy for the functionalization of amines and amides,s uch as Leu-containing dipeptide 110,b yu sing hypervalent iodine reagents as SOMOphiles (Scheme 25). [48] An electrophilic amidyl radical is proposed to be generated from activated precursor 110. After 1,5-HAT, the tertiary radical can add on the EBX reagent 3k,thereby generating the alkynylated dipeptide 111 in moderate yield.

Alanine
TheY ug roup described the acetoxylation of Ala-containing tripeptides with ap hthalimide group at the Nterminus using PIDA( 5)a sa no xidant. [49] Thes ide chains of N-terminal Ala residues were oxidized using 10 mol %P d-(OAc) 2 in acetic anhydride at 100 8 8C( Scheme 26). Although moderate yields were obtained for products 112-115,s electivity for the N-terminal Ala residue,even in the presence of aC -terminal Ala residue,w as observed (112).

Peptide Termini
Direct and selective functionalization of peptide termini is an attractive method to achieve biomolecule modification. [20,50] It allows as ingle site-selective modification of native peptides without the need to introduce or change an amino acid residue.

C-terminus
Photoredox-catalyzed decarboxylative transformations are especially attractive for the functionalization of Ctermini. [3e] They allow the rapid generation of reactive species under mild reaction conditions with high selectivity for the Cterminus,a st his site is easier to decarboxylate under oxidative conditions compared to side chains.
EBX reagents have been used for the decarboxylative photoredox-catalyzed alkynylation of amino acids. [51] Our group developed an iridium-catalyzed method [51b] that was later extended to adecarboxylative cyanation of amino acids and peptides. [52] Cyanobenziodoxolone (CBX, 8), developed by Zhdankin et al., [53] was used along with catalytic amounts of Ir[dF(CF 3 )ppy] 2 (dtbbpy)PF 6 in the presence of cesium benzoate and molecular sieves (Scheme 27). Thereaction was applied to dipeptides to give cyano amides such as 116 and 117.
Experimental and computational studies reveal that the alkynylation and cyanationr eactions are not going through the same intermediates (Scheme 28). [51b,52] Thec ommon

Angewandte
Chemie catalytic cycle starts with the excitation of 118 (Scheme 28 a). Asingle-electron transfer (SET) between 118* and the in situ generated carboxylate I occurs,t hereby generating the nucleophilic radical II after decarboxylation. Thea lkynylation then undergoes af ull radical pathway via the a-o rbaddition of II to the reagent 3a (Scheme 28 b). In contrast, another SETfrom radical II to CBX 8 is believed to yield the radical anion IV and iminium V (Scheme 28 c). Collapse of the radical anion to form cyanide VI,f ollowed by recombination with the carbocation VII,t hen leads to the cyanated product.
In 2019, our group extended the scope of decarboxylative alkynylation to peptides. [54] Ther eaction proceeded under mild, metal-free conditions in 30 minutes at room temperature in aDMF/water mixture.Ingeneral, awide range of Cand N-terminal amino acids were compatible with the reaction (119-133,S cheme 29). Reactivity in other cases can be improved using either side-chain protections (128, 132)or ad ifferent catalyst (131). In the presence of Cys,b oth the thiol group and the C-terminus were alkynylated (133). Functionalization of bioactive hexapeptide quantitatively yielded products containing reactive handles,s uch as an aldehyde or an azide (134, 135).
In 2021, our group introduced ap hotoredox-catalyzed oxidative decarboxylative reaction using acetoxybenziodoxole (BI-OAc, 7b;Scheme 30). [55] This allowed the transfer of the acetoxy group to peptides,thereby forming intermediates 136.T he generated N,O-acetals could not be isolated, but were trapped with phenol or indole nucleophiles.F or example,d ipeptides containing ap roteinogenic phenol or indole were introduced, which provided unprecedented unnatural peptides 137 and 138.Alternatively,inthe presence of an alcohol, for example,s erine or threonine residues, structurally diverse and stable N,O-acetals were formed.
In 2019, Tada, Itoh, and co-workers introduced the H-EBX reagent 3n,s tabilized by MeCN. [56] 3n was used to functionalize O-methylhydroxamic acids derived from car-boxylic acids (Scheme 31). [57] Fore xample,C -terminal-modified dipeptide 139 was used to yield cis-b-N-MeO-amide-VBX 140 in the presence of water and ac atalytic amount of base.I nt he presence of deuterium oxide,d euterium incorporation occurred at the vinyl positions,y ielding 141.

N-terminus
Besides C-terminal modification, 3n also reacted with at osyl-protected N-terminus to yield alkynylated dipeptides 142 and 143 when an excess of base was used (Scheme 32 a). [56] Although the scope was first limited to unsubstituted EBX, this drawback was addressed by the use of ac opper catalyst. [58] Reagents bearing various substituents were added at the N-terminus of amino acids,while TIPS-EBX 3a was used to obtain functionalized dipeptide 144 (Scheme 32 b).

Summary and Outlook
This Minireview summarizes applications of hypervalent iodine reagents for the modification of peptides and proteins. They have been used as oxidants or electrophilic transfer reagents.Awide range of reagents have been designed and applied to av ariety of amino acid side chains for bioconjugation, peptide stapling,o rp roteome-wide Cys profiling. Many methods achieve high site-selectivity and are performed under biocompatible conditions.T his has allowed applications on complex substrates,s uch as antibodies, proteins,a nd in living cells.
Although great advances have been achieved in the field, challenges still remain. Fore xample,m any methods are limited to amino acids or short peptides and can be only used in organic solvents.I na ddition, these biomolecule modifications are currently limited to oxidation, fluoroalkylation, thiolation, alkynylation, cyanation, azidation, and alkoxylation. Thedevelopment of new reagents is needed to introduce further diversity.This requires new synthetic methods to give easy access to novel hypervalent iodine compounds that combine aqueous solubility,r eactivity,and stability.
Nevertheless,these discoveries are still very recent. From the advantages offered by hypervalent iodine reagents, improvements can be expected in the near future,l eading to an increased use of these reactants for applications in biochemistry.