molecules

Review Review AmideAmide BondBond ActivationActivation of Biological Molecules Molecules Sriram Mahesh, Kuei-Chien Tang and Monika Raj * Sriram Mahesh, Kuei-Chien Tang and Monika Raj * Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849, USA; [email protected] of Chemistry (S.M.); and kzt0026@ti Biochemistry,germail.auburn.edu Auburn University, (K.-C.T.) Auburn, AL 36849, USA; [email protected]* Correspondence: [email protected]; (S.M.); [email protected] Tel.: +1-334-844-6986 (K.-C.T.) * Correspondence: [email protected]; Tel.: +1-334-844-6986 Academic Editor: Michal Szostak


AcademicReceived: Editor:7 September Michal 2018; Szostak Accepted: 9 October 2018; Published: date
 Received: 7 September 2018; Accepted: 9 October 2018; Published: 12 October 2018 Abstract: Amide bonds are the most prevalent structures found in organic molecules and various Abstract: Amide bonds are the most prevalent structures found in organic molecules and various biomolecules such as peptides, proteins, DNA, and RNA. The unique feature of amide bonds is their biomolecules such as peptides, proteins, DNA, and RNA. The unique feature of amide bonds ability to form resonating structures, thus, they are highly stable and adopt particular three- is their ability to form resonating structures, thus, they are highly stable and adopt particular dimensional structures, which, in turn, are responsible for their functions. The main focus of this three-dimensionalreview article is to structures, report the which, methodologies in turn, are for responsible the activation for their of functions.the unactivated The main amide focus bonds of this reviewpresent article in biomolecules, is to report thewhich methodologies includes the for enzy thematic activation approach, of the metal unactivated complexes, amide and bonds non-metal present inbased biomolecules, methods. whichThis article includes also the discusses enzymatic some approach, of the applications metal complexes, of amide and bond non-metal activation based methods.approaches This in articlethe sequencing also discusses of proteins some of and the th applicationse synthesis ofof amide peptide bond acids, activation esters, approachesamides, and in thethioesters. sequencing of proteins and the synthesis of peptide acids, esters, amides, and thioesters.

Keywords:Keywords: peptide bond cleavage; amide amide bond bond reso resonance;nance; twisted twisted amides; amides; enzymes; enzymes; metal metal complexes;complexes; catalystscatalysts

1.1. IntroductionIntroduction TheThe amideamide bondbond is one of the most abundant abundant ch chemicalemical bonds bonds and and widely widely exists exists in in many many organic organic moleculesmolecules andand biomolecules biomolecules [1– 6[1–6].]. Nature Nature has usedhas amideused bondsamide tobonds make theseto make important these biomoleculesimportant becausebiomolecules of the because high stability of the high of amide stability bonds of amide towards bonds various towards reaction various conditions reaction conditions (acidic and (acidic basic conditions),and basic conditions), high temperature, high temperature, and the presence and the ofpresence other chemicalsof other chemicals [7]. The high[7]. The stability high stability of amide bondsof amide is attributedbonds is attributed to its tendency to its tendency to form to a fo resonatingrm a resonating structure, structure, which which provides provides a double a double bond characterbond character to the to amide the amide CO-N CO-N bond bond (Figure (Figure1)[ 1)8– [8–10].10]. The The resonance resonance ofof thesethese amide bonds bonds forms forms aa planarplanar structure and and hinders hinders the the free free rotation rotation arou aroundnd the the CO-N CO-N bond, bond, thus, thus, it is itresponsible is responsible for 3D for 3Dstructures structures adopted adopted by byproteins proteins and and other other biomolec biomolecules.ules. These These 3D 3D structures structures of of biomolecules biomolecules are are responsibleresponsible forfor variousvarious importantimportant biological functions.

O O O R C O R' R' R' R N R N R N R' N R'' R'' R'' R'' = 0 o; N = 0 o

Figure 1. ClassicalClassical amide amide bo bondnd resonance. resonance.

HansenHansen etet al.al. carried carried out out the the rate rate studies studies on on the the hydrolysis hydrolysis of of amide amide bonds bonds at at various various pH pH conditionsconditions [[11].11]. The The study study concluded concluded that that at atpH pH 7, 7,the the rate rate of ofhydrolysis hydrolysis is due is due to the to thedirect direct attack attack of ofwater water on on peptide peptide and and measured measured as as kH kH2O.2O. The The rate rate constant constant showed showed th thatat the the half-life half-life of of the the amide amide bondsbonds isis 267267 years,years, similar to the value determined determined by by Radzicka Radzicka and and Wolfenden Wolfenden [12]. [12]. This This study study also also + − showedshowed thatthat thethe ratesrates ofof acidacid (kH(kH33O+)) and and base base hydrolysis hydrolysis (kOH (kOH−) are) are identical, identical, therefore, therefore, the the rate rate of of thethe hydrolysishydrolysis ofof thethe peptidepeptide bondbond is dominated by by kH kH22OO throughout throughout the the pH pH range range from from pH pH 5–9. 5–9.

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Molecules 2018, 23, 2615; doi:10.3390/molecules23102615 www.mdpi.com/journal/molecules Molecules 2018, 23, 2615 2 of 43 Molecules 2018, 23, x 2 of 43

Recently,Recently, variousvarious methods methods have have been been reported reported in thein literaturethe literature to activate to activate the amide the amide bonds towardsbonds atowards variety a of variety nucleophiles of nucleophiles or electrophiles or electrophiles for the synthesis for the synthesis of other of organic other organic compounds. compounds. This includes This theincludes use of the enzymes, use of enzymes, metal complexes, metal complexes, and non-metal and non-metal based methods based methods [13–15]. [13–15]. One widely One reportedwidely approachreported approach for the activation for the activation of amide of bonds amide involves bonds involves the distortion the distortion of amide of amide bonds, bonds, thus, thethus, amide the bondamide is bond no longer is no ablelonger to formable to a resonatingform a resonating structure, structure, loses its loses double its bonddouble character, bond character, and becomes and morebecomes susceptible more susceptible to nucleophilic to nucleophilic or electrophilic or electr attack.ophilic A attack. higher A distortion higher distortion of the amide of the bond amide from thebond planar from structurethe planar makes structure it more makes reactive, it more as reac evidencedtive, as evidenced by various by twisted various amide twisted bonds amide present bonds in cyclicpresent nonplanar in cyclic bridgednonplanar lactams, bridged as demonstratedlactams, as demonstrated by Stoltz [16 by,17 ],Stoltz Kirby [16,17], [18–20 Kirby], and others[18–20], [21 and–23 ] (Figureothers 2[21–23]). One of(Figure the special 2). One cases of the to achieve special maximumcases to achieve rotational maximum inversion rotational of the amide inversion bond of so the that itamide remains bond in so the that twisted it remains conformation in the twisted is the useconformation of N-acyl-glutarimides is the use of N [24-acyl-glutarimides–29] and N,N-substituted [24–29] amideand N, bondsN-substituted [30,31] (Figureamide 2bonds). It is [30,31] this strong (Figure distortion 2). It is ofthis amide strong bonds distortion that provides of amide amide bonds bonds that withprovides a high amide reactivity bonds toward with a ahigh variety reactivity of nucleophiles toward a variety and electrophiles. of nucleophiles and electrophiles.

H H H R N N O S Me N N O N Me BF4 N O H O O CO2H Lukes (1938) Woodward (1941) Kirby (1998) Stoltz (2006) Aube (2005) -lactam perpendicularly twisted medium bridged (  = 90 , N = 60o) ( = 50 ; N = 35o)

O O O O R' n O [M] n+2 N R-Y R' R'' R R N or N R [M] R R + R R'' acyl aryl O Ln N-acyl-glutarimide N-C(O) rotation electronic activation N-C amide activation by distortion = 88.6 ; N = 6.6o (twist tuneable reactivity)

FigureFigure 2. 2. TwistedTwisted amides amides for for activation activation of of amide amide bonds. bonds.

ThereThere are already some excellent review review articles articles in in the the literature literature covering covering the the reactivity reactivity of of twisted/activatedtwisted/activated amide amide bonds bonds for for the the synthesis synthesis of of th thee variety variety of of different different organic organic molecules molecules such such as as ketones,ketones, esters, esters, acids, acids, and and alcohols, alcohols, by cross-coupli by cross-couplingng reactions reactions [24–31]. [24 The–31]. main The focus main of focusthis review of this reviewis to summarize is to summarize the methods the for methods the activation for the of activation less reactive of lessamide reactive bonds present amide bondsin biomolecules present in biomoleculessuch as peptides, such proteins, as peptides, glycopeptides, proteins, glycopeptides,nucleotides in DNA nucleotides and RNA in DNA and various and RNA other and peptide various otherbioconjugates, peptide bioconjugates,toward attack towardby various attack nucleophiles. by various nucleophiles.This task was This accomplished task was accomplished by various bymethods various such methods as by using such biological as by using molecules, biological metal molecules, complexes, metal and complexes, non-metal and based non-metal methods basedand methodsis discussed and below. is discussed below.

2.2. BiomoleculesBiomolecules for the Activation of of Amide Amide Bonds—The Bonds—The Enzyme-Directed Enzyme-Directed Hydrolysis Hydrolysis of ofAmides Amides WeWe havehave summarized different kinds of of enzymes, enzymes, their their mechanisms mechanisms of of hydrolysis hydrolysis of of unactivated unactivated peptidepeptide bonds,bonds, and the point of cleavages in Table Table 11..

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Table 1. Enzymatic directed hydrolysis of peptide bonds.

Entry Enzyme Method of Hydrolysis Point of Cleavage Ref. Serine and Oxyanion binding hole with 1 -[32–37] Cysteine Proteases catalytic triad Internal peptide bonds on the Thermolysine with Zn2+ binds to 2 Metallo-endopeptidase N-terminal side of large [38–51] His 142, His 146, Glu 166 hydrophobic amino acids Carboxypeptidase A with Zn2+ C-terminus comprising large 3 Metalloexopeptidase [52–54] through Lewis acid activation hydrophobic amino acids Glycosylation followed by enzyme 4 O-GlcNAc transferase N-terminal glutamic acid [55,56] catalyzed pyroglutamate formation Enzyme Chelation to Zn2+ and 5 Nicotinamidase Nicotinamide [57–60] catalytic triad Flavin hydroperoxide intiated Unactivated amide bond 6 Flavoenzyme [61–63] oxidative mechanism in uracil

Catalyzes unactivated primary Primary amide bond of L-isomer 7 Antibody Fab-BL 125 [64,65] amide bond hydrolysis of peptides Unactivated alkyl amide of 8 RNA Mg2+ catalyzed mechanism [66] DNA analog

2.1. Serine Proteases Amide bonds are widely present in proteins due to their high stability and the tendency of amide bonds to exist in resonating structures, which is one of the key factors responsible for secondary structures adopted by proteins and their biological activities. Nature has developed some methods for the cleavage of highly stable amide bonds to control their functions. One such approach is the use of enzymes (serine proteases), which have active sites, and binding pockets for binding to particular amino acids followed by the activation of amide bonds for hydrolysis. These enzymes exist in various families such as trypsin, chymotrypsin, elastase, subtilisin, etc., but have a similar catalytic site containing oxyanion binding hole with Ser, His and Asp triad [32,33]. Some of the proteases have catalytic dyads with two amino acids at the active site, however, triads are the most common. All these enzymes based on the binding pocket prefer to bind to particular amino acids but the mechanism by which they hydrolyze the amide bond is similar. During the catalysis, these enzymes form an oxyanion hole made up of three amino acids—His, Asp, and Ser—which work in a synergistic manner to break the amide bond (Figure3). First, the side chain of Asp makes a hydrogen bond with histidine, thus making it more nucleophilic. Second, histidine forms a strong H-bond with the hydroxyl group of serine and abstracts the proton from the hydroxyl group (OH) of serine which in turn attacks amide bond to form a tetrahedral transition state (TS). This TS eventually collapses resulting in the hydrolysis of the amide bond by acid–base catalysis. Wells et al. demonstrated the importance of these residues at the active site by mutating it to alanine [32,33]. They showed that any mutation in the catalytic triad greatly reduces the turnover number which is a consequence of the changes in the enzyme mechanism. Residues in the catalytic triad function in a strongly synergistic manner and contribute a factor of 2 × 106 to the rate enhancement. The study concluded that enzymes increase the rate of amide bond hydrolysis at by least 109 to 1010 times that of the non-enzymatic hydrolysis of amide bonds. Molecules 2018, 23, 2615 4 of 43 Molecules 2018, 23, x 4 of 43

H O Asn H O Asn H O Asn N N N H H H

O O H O H2N N N H HO O N NH Asp O Asp O HO Asp O OH N NH HN + NH O O Ser His Ser Ser His His Oxyanion hole Tetrahedral TS

Figure 3. GeneralGeneral pathway pathway of of serine serine protease proteasess directed directed amide amide bond bond hydrolysis. hydrolysis.

2.2.2.2. CysteineCysteine Proteases CysteineCysteine proteasesproteases (CPs)(CPs) hydrolyzehydrolyze the the peptide peptide bonds bonds with with maximum maximum efficiency efficiency at pHat pH 4–6.5 4–6.5 [14 ]. The[14]. thiol The thiol group group of cysteine of cysteine protease protease is susceptible is susceptible to oxidationto oxidation so so the the environment environment of of the the enzyme enzyme is reducingis reducing in nature.in nature. Till Till now now 21 21 families families of of CPs CPs have have been been discovered discovered [34 [34–37].–37]. CPs CPs form form a triada triad at at the activethe active site site during during the the hydrolysis hydrolys ofis of the the peptide peptide bond bond made madeup up ofof Cys-His-Asn residues. residues. First, First, Asn Asn formsforms thethe hydrogenhydrogen bond with His, His, then then His His abstracts abstracts the the proton proton from from Cys Cys to to generate generate a anucleophilic nucleophilic − thiolatethiolate ionion (S(S−)) similar similar to to enolate enolate ion ion generated generated by by serine serine proteases proteases (Figure (Figure 3).3). Next, Next, the the thiolate thiolate ion ion − (S(S−)) attacks attacks the the carbonyl carbonyl group group of of th thee peptide peptide resulting resulting in in the the formation formation of of a atetrahedral tetrahedral intermediate intermediate TSTS followedfollowed byby thethe hydrolysis of the amide bond [34–37]. [34–37].

2.3.2.3. MetalloproteasesMetalloproteases MetalloproteasesMetalloproteases are are members members of of a aclass class of ofproteases proteases that that require require a metal a metal ion cofactor ion cofactor at the at theactive active site for site the for hydrolysis the hydrolysis of peptide of peptide bonds [ bonds38]. The [38 most]. Thecommon most metal common ion cofactor metal ion present cofactor in 2+ 2+ presentmetalloproteases in metalloproteases is the zinc ion is the(Zn2+ zinc) [39]. ion Other (Zn transition)[39]. Other metals transition such as Co metals2+ and suchMn2+ are as Cocapableand 2+ 2+ Mnof restoringare capable the functions of restoring in zinc-metalloproteases the functions in zinc-metalloproteases where the Zn2+ core where has the been Zn removedcore has [39]. been removedMetalloproteases [39]. Metalloproteases are divided areinto divided two into ma twojor majorfamilies: families: metalloendopeptidases metalloendopeptidases and and metalloexopeptidases.metalloexopeptidases. TheThe names names of of these these families families are are based based on theon sitethe ofsite the of hydrolysis the hydrolysis of the peptideof the bondspeptide [40 ,bonds41]. Metalloendopeptidases [40,41]. Metalloendopeptidase cleave the internals cleave amide the bondsinternal whereas amide metalloexopeptidases bonds whereas cleavemetalloexopeptidases the amide bonds cleave present the atamide the C- bonds or N-terminus present at ofthe peptides. C- or N-terminus of peptides.

2.3.1.2.3.1. Metalloendopeptidase: Thermolysin Thermolysin ThermolysinThermolysin (TLN) catalyzes the cleavage of of the the in internalternal peptide peptide bond bond at at the the amino-side amino-side of of large large hydrophobichydrophobic amino acids, such as leucine, isoleucine, isoleucine, or or phenylalanine. phenylalanine. TLN TLN and and TLN-like TLN-like proteins proteins 2+ requirerequire ZnZn2+ asas a a metal metal ion ion cofactor cofactor for for the the cleavage cleavage of of amide amide bonds bonds [42–47]. [42–47 ]. TLN-mediatedTLN-mediated hydrolysis of the pe peptideptide bond bond is is a a two-step two-step process process (Figure (Figure 4)4)[ [48–51].48–51]. The The active active sitesite ofof TLN TLN contains contains three three residues—His142, residues—His142, His146, His146, Glu166—and Glu166—and a water a water molecule, molecule, which which are bound are tobound the Znto 2+theion. Zn2+ First, ion. First, the carbonyl the carbonyl group group of the of peptidethe peptide coordinates coordinates with with Zn2+ Znand2+ and displaces displaces the hydrogenthe hydrogen of a waterof a water molecule molecule to form to anform H-bond an H-bond with Glu143 with Glu143 and the and oxygen the oxygen of the water of the molecule water remainsmolecule associated remains associated to the Zn2+ toion, the resulting Zn2+ ion, in resulting the formation in the of formation the enzyme-substrate of the enzyme-substrate complex (ES). Second,complex the (ES). oxygen Second, of the the water oxygen attached of the towater Zn2+ attachedattacks theto Zn carbonyl2+ attacks carbon the carbonyl of the peptide, carbon resulting of the inpeptide, the formation resulting of in transitionthe formation state of 1 transition (TS1). TS1 stat ise stabilized1 (TS1). TS1 by is the stabilized formation by the of formation the H-bond of withthe Asp226H-bond andwith His231 Asp226 at and the His231 carbonyl at oxygenthe carbonyl of the ox peptideygen of followed the peptide by followed the formation by the of formation intermediate of gem-diolateintermediate (INT) gem-diolate by the breakage(INT) by ofthe hydroxyl breakage OH of bondhydroxyl of water. OH bond The amideof water. of theThe peptide amide formsof the a hydrogenpeptide forms bond a with hydrogen the H ofbond H2O. with Third, the the H of carbonyl H2O. Third, bond the rearrangement carbonyl bond in TS2 rearrangement leads to the breakage in TS2 ofleads the to amide the breakage bond (CONH) of the amide of the peptidebond (CONH) and releases of the thepeptide N-terminal and releases peptide. the TheN-terminal rate-determining peptide. The rate-determining studies showed that the collapse of a zwitterionic tetrahedral intermediate (INT) is a rate-limiting step (Figure 4).

Molecules 2018, 23, 2615 5 of 43 studies showed that the collapse of a zwitterionic tetrahedral intermediate (INT) is a rate-limiting step

(FigureMolecules4 ).2018, 23, x 5 of 43

Glu143 Glu143 O O O H O H H N H N + H H O H H H O N H O N O N - N Asp His Zn 226 His Zn Asp226 142 142 Glu Glu166 O 166 O His146 His231 His146 His231 ES O TS1 O

Glu143 O O H H H N NH2 FA N OH O H N H + H O N N H O Asp226 O His142 Zn H Glu166 O - His231 Zwitterionic tetrahedral intermediate His146 O INT Glu H 143 H Glu143 N O O H H O O N H H O H H O N H O N H O N N Asp Asp226 His Zn 226 His142 Zn 142 Glu Glu166 O 166 O His231 His His His146 231 146 O O Product TS2

Figure 4. ThermolysinThermolysin Mechanistic Mechanistic pathway. pathway.

2.3.2.2.3.2. Metalloexopeptidase:Metalloexopeptidase: Carboxypeptidase A A 2+ CarboxypeptidaseCarboxypeptidase A (CPA) is a 35 kDakDa metalloenzyme and and contains contains a a Zn Zn2+ ionion cofactor cofactor in in its its activeactive sitesite [[52–54].52–54]. CPA CPA is is an an exopeptidase, exopeptidase, which which catalyzes catalyzes the the hydrolysis hydrolysis of amide of amide bonds bonds present present at atthe the C-terminus C-terminus comprising comprising large large hydrophobic hydrophobic side side chains. chains. Two Two different different mechanisms mechanisms have have been been proposedproposed forfor the the cleavage cleavage by by these these metalloproteases metalloproteases (Figure (Figure5) which 5) which showed showed the importance the importance of Lewis of acidLewis catalysis acid catalysis for the for activation the activation of amide of amide bonds bonds [53,54 ].[53,54]. One involves One involves the Lewis-acid the Lewis-acid activation activation of the 2+ carbonylof the carbonyl group ofgroup the amideof the bondamide by bond Zn by, followed Zn2+, followed by the by attack the attack of water of (Figurewater (Figure5). The 5). second The 2+ involvessecond involves the Lewis-acid the Lewis-acid activation activation of H2O of by H Zn2O byions Zn2+ followed ions followed by the by attack the attack of the of hydroxide the hydroxide ion of theion waterof the onwater the on carbonyl the carbonyl group group of the amideof the amide bond (Figurebond (Figure5). 5).

Zn Zn

O H O O NH H NH

Glu O Glu O

O O

Figure 5. Mechanisms of carboxypeptidase A.

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Glu143 Glu143 O O O H O H H N H N + H H O H H H O N H O N O N - N Asp His Zn 226 His Zn Asp226 142 142 Glu Glu166 O 166 O His146 His231 His146 His231 ES O TS1 O

Glu143 O O H H H N NH2 FA N OH O H N H + H O N N H O Asp226 O His142 Zn H Glu166 O - His231 Zwitterionic tetrahedral intermediate His146 O INT Glu H 143 H Glu143 N O O H H O O N H H O H H O N H O N H O N N Asp Asp226 His Zn 226 His142 Zn 142 Glu Glu166 O 166 O His231 His His His146 231 146 O O Product TS2

Figure 4. Thermolysin Mechanistic pathway.

2.3.2. Metalloexopeptidase: Carboxypeptidase A

Carboxypeptidase A (CPA) is a 35 kDa metalloenzyme and contains a Zn2+ ion cofactor in its active site [52–54]. CPA is an exopeptidase, which catalyzes the hydrolysis of amide bonds present at the C-terminus comprising large hydrophobic side chains. Two different mechanisms have been proposed for the cleavage by these metalloproteases (Figure 5) which showed the importance of Lewis acid catalysis for the activation of amide bonds [53,54]. One involves the Lewis-acid activation of the carbonyl group of the amide bond by Zn2+, followed by the attack of water (Figure 5). The 2+ Moleculessecond involves2018, 23, 2615 the Lewis-acid activation of H2O by Zn ions followed by the attack of the hydroxide6 of 43 ion of the water on the carbonyl group of the amide bond (Figure 5).

Zn Zn

O H O O NH H NH

Glu O Glu O

O O

Molecules 2018, 23, x Figure 5. 5. MechanismsMechanisms of of carboxypeptidase carboxypeptidase A. A. 6 of 43 2.3.3. Glutamate Glycosylation for Amide Bond Cleavage 2.3.3. Glutamate Glycosylation for Amide Bond Cleavage Human enzyme O-GlcNAc transferase (OGT) is essential for the cleavage of amide bonds Human enzyme O-GlcNAc transferase (OGT) is essential for the cleavage of amide bonds in host incell host factor-1 cell factor-1(HCF-1). (HCF-1).HCF-1 cleavage HCF-1 takes cleavage place takesat the placeN-terminal at the glutamic N-terminal acid glutamicby the glycosylation acid by the glycosylationwhich is catalyzed which isby catalyzed enzyme byOGT. enzyme Mechanistic OGT. Mechanistic studies showed studies that showed the glycosylation that the glycosylation of the ofglutamate the glutamate side chain side (intermediate chain (intermediate 1, Figure 1, 6) Figure leads6 to) leads the formation to the formation of an enzyme-catalyzed of an enzyme-catalyzed internal internalpyroglutamate pyroglutamate formation formation (intermediate (intermediate 2, Figure 6) 2, with Figure the6) amidic with the nitrogen amidic of nitrogen the peptide of the backbone peptide backbonechain, which chain, then which undergoes then undergoes spontaneous spontaneous hydrolysis hydrolysis (Figure 6) (Figure [55]. Detailed6)[ 55]. Detailedmechanistic mechanistic studies studiesshowed showed that the that rate the of rate conversion of conversion of glycopep of glycopeptidetide to internal to internal pyroglutamate pyroglutamate was was an anorder order of of magnitudemagnitude slowerslower thanthan observed observed in in the the presence presence of of OGT, OGT, thus, thus, it wasit was concluded concluded that that both both the the first first and secondand second steps steps occurred occurred while while the peptidethe peptide is bound is bound to OGT to OGT (Figure (Figure6). Hydrolysis 6). Hydrolysis likely likely occurs occurs after dissociationafter dissociation from thefrom enzyme. the enzyme. It has alsoIt has been also reported been reported that glycosylation that glycosylation on Thr nexton Thr to glutamate next to alsoglutamate prevents also the prevents cleavage the at cleavage the glutamate at the (Glu)glutam becauseate (Glu) of because the steric of hindrance the steric andhindrance thus the and enzyme thus isthe unable enzyme to carryis unable out theto carry glycosylation out the glycosylation of glutamate of [ 56glutamate]. [56].

OH OH H O O N O O N O O HN O HN H NH SH NH SH OH O O N O O O O HN O O HO O N SH NH HO HO O HO O HO NHAc N O HO AcHN Intermediate 1 Intermediate 2 O O OH O P O OH O O P OH O O O O N OH O HN + NH SH O

FigureFigure 6. 6. GlycosylationGlycosylation pathway. pathway.

2.3.4.2.3.4. NicotinamidaseNicotinamidase (Pnc1) for the Hydrolysis of of the the Amide Amide Bond Bond of of Nicotinamide Nicotinamide NicotinamidasesNicotinamidases catalyze the the cleavage cleavage of of nicotinamide, nicotinamide, which which is is a acritically critically important important part part of of + NADNAD+ andand NADH, NADH, to to nicotinic nicotinic acid acid and and ammonia ammonia (Figure (Figure 7).7). A A detailed detailed study study showed showed that that both both the the carbonylcarbonyl oxygenoxygen and the ring nitrogen of of nicotinami nicotinamidede are are critical critical for for binding binding to to the the nicotinamidases nicotinamidases andand reactivityreactivity [[57–60].57–60].

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O O

NH2 Pnc1 OH + H2O + NH3 N N Nicotinamide hydrolysis H53 H53 Mechanism D51 H94 D51 H94 H53 Zn Zn K122 NH3 N NH D51 H94 K122 3 K122 NH3 + D8 COO Zn + D8 COO N N O H H H2N O C167 SH D8 C167 S O O H H H N O H N O 2 (INT) 2 S

C167 -NH3

H53 H53 D51 H94 NH H53 D51 H94 K122 3 Zn Zn N D51 H94 Zn NH K122 NH3 + D8 COO + K122 3 N N C167 SH H HO O O H O O O H H D8 HO D8 H O O S O S O

C167 C167

Figure 7. Mechanistic pathway pathway of of Pnc1 Pnc1 for for hydrolysis hydrolysis of of nicotinamide. nicotinamide.

2+ ThreeThree residues—Asp51,residues—Asp51, His53, His53, and and His94—in His94—in nicotinamidase nicotinamidase (Pnc1) (Pnc1) directly directly coordinate coordinate with with Zn atZn the2+ at active the active site and site three and otherthreeresidues other residues act as a ac catalytict as a catalytic triad (Cys167, triad (Cys167, Asp8, and Asp8, Lys122) and (FigureLys122)7 ). In(Figure the first 7). step,In the the first substrate step, the binds substrate to the binds Zn2+ toby the nitrogen Zn2+ by ofnitrogen pyridine of ringpyridi andne ring displaces and displaces the water moleculesthe water ligatedmolecules to the ligated Zn2+ .to Next, the Asp8Zn2+. removesNext, Asp8 the removes proton from the Cys167,proton from forming Cys167, a thiolate, forming which, a inthiolate, turn, react which, with in the turn, amide react carbonyl with carbonthe amide of nicotinamide, carbonyl carbon leading of tonicotinamide, the formation leading of a tetrahedral to the intermediateformation of (INT).a tetrahedral The tetrahedral intermediate intermediate (INT). The collapsed, tetrahedral resulting intermediate in the collapsed, breakage resulting of the amide in bondthe breakage and release of the of theamide ammonia. bond and This release is followed of the byammonia. the release This of is the followed nicotinic by acid the fromrelease the of active the sitenicotinic of the acid enzyme from by the acid-base active site catalysis. of the enzyme by acid-base catalysis.

2.3.5.2.3.5. Flavoenzyme-MediatedFlavoenzyme-Mediated Hydrolysis of of the the Amide Amide Bond Bond BegleyBegley etet al.al. demonstrated demonstrated the the role role of of flavoenzyme flavoenzyme in thein the cleavage cleavage of theof the unactivated unactivated amide amide bond inbond uracil, in uracil, a building a building block block for RNA for RNA (Figure (Figure8)[ 61 8)– 63[61–63].]. The The detailed detailed mechanistic mechanistic analysis analysis showed showed that thethat reaction the reaction takes takes place place through through the oxidative the oxidative mechanism mechanism that isthat initiated is initiated by the by addition the addition of a flavinof a hydroperoxideflavin hydroperoxide to the C* to carbonylthe C* carbonyl of uracil, of forming uracil, forming a tetrahedral a tetrahedral intermediate intermediate (INT) (Figure (INT)8 ).(Figure This is followed8). This is by followed the collapsing by the collapsing of the tetrahedral of the tetrahed intermediateral intermediate (INT), leading (INT), to leading the cleavage to the cleavage of an amide of bondan amide in uracil. bond This in uracil. was the This first was example the first where example such chemistry where such was chemistry shown by was flavin shown hydroperoxides. by flavin hydroperoxides.

Molecules 2018, 23, 2615 8 of 43 Molecules 2018,, 23,, xx 8 of 43

O O RutA + RutF(or Fre) HN RutA + RutF(or Fre) HO O2 + NADH + FMN O N 2 HN H H N O H2N O Mechanism

O O O O O O O O O Flv Flv O O HN HN O HO HN * HN * Flv O HO O N O N HN HN H H INTINT H N O H N O H2N O H2N O

Figure 8. Flavoenzyme-mediatedFlavoenzyme-mediated hydrolysis hydrolysis of of amide amide bond. bond.

2.3.6.2.3.6. PrimaryPrimary Amide Bond Hydrolysis by by Antibodies Antibodies

TheThe antibodyantibody Fab-BL125 catalyzes the the hydrolysis hydrolysis of of the the unactivated unactivated primary primary amide amide bond bond of of the the LL-isomer-isomer ofof peptidespeptides to generate free free peptide peptide acid acid (Figure (Figure 9).9). The The antibody antibody showed showed high high regio- regio- and and diastereoselectivitydiastereoselectivity sincesince the theD-proline D-proline primary primary amide amide diastereoisomer diastereoisomer did not did undergo not undergo any hydrolysis. any Thehydrolysis. antibody The Fab-BL125 antibody decreasesFab-BL125 the decreases half-life the of half-life the peptide of the from pept 17.5ide yearsfrom 17.5 to only years 3.9 to h. only Such 3.9 an antibodyh. Such an was antibody obtained was by obtained using the byα -aminousing the boronic α-amino acid boronic Hapten acid by a Hapten direct selection byby aa directdirect strategy selectionselection from thestrategy antibody from combinatorial the antibody combinatorial libraries [64,65 libraries]. [64,65].

O O H O H O H +H O H N N +H2O N N N N H H O H O H AcHN O O NH AcHN O O NH AcHN O NH2 AcHN O NH2 OH

-NH -NH3 O H N N O R N H O H H H B(OH) N N O B(OH)2 N H O O -amino boronic AcHN O OH O -amino boronic O acid hapten HO C acid hapten R = HO2C

R = Boc

Figure 9. AntibodyAntibody Fab Fab catalyzed catalyzed primary primary amide amide bond bond hydrolysis. hydrolysis. 2.3.7. RNA-Assisted Cleavage of Amide Bonds 2.3.7. RNA-Assisted Cleavage of Amide Bonds A group I RNA obtained by in vitro evolution catalyzes the cleavage of unactivated alkyl amides A group I RNA obtained by in vitro evolution catalyzes the cleavage of unactivated alkyl amides of DNA analog. This includes substrates with an amide bond that joins either two DNAs, or a DNA of DNA analog. This includes substrates with an amide bond that joins either two DNAs, or a DNA with a short peptide. The RNA increases the rate of hydrolysis by more than 103 in comparison with a short peptide. The RNA increases the rate of hydrolysis by more than 103 inin comparisoncomparison toto thethe touncatalyzed the uncatalyzed reaction. reaction. The RNA-catalyzed The RNA-catalyzed amide amidebond cleavage bond cleavage was entirely was entirely dependent dependent on Mg2+ on uncatalyzed2+ reaction.2+ The RNA-catalyzed amide bond cleavage was entirely dependent on Mg Mg where2+ Mg acts as a Lewis acid thus activating the carbonyl group of the amide bond for where Mg2+ acts as a Lewis acid thus activating the carbonyl group of the amide bond for the

Molecules 2018, 23, 2615 9 of 43 Molecules 2018, 23, x 9 of 43 thenucleophilic nucleophilic attack attack by the by hydrox the hydroxyy group group of RNA of (Figure RNA (Figure10). No 10amide). No bond amide cleavage bond was cleavage detected was 2+ 2+ 2+ detectedin the presence in the presence of other metal of other ions metal such ions as Zn such2+, Ca as2+ Zn, or Sr, Ca2+. A, trace or Sr amount. A trace of amountamide bond of amide cleavage bond cleavagewas observed was observed in the presence in the presenceof MnCl2 ofwhich MnCl is2 inwhich contrast is in to contrast the RNA to and the DNA RNA cleavage and DNA reactions cleavage reactions[66]. [66].

DNA1 DNA1 5' O 5' O 5' O T IGS T T HN H N 2 H2N O HO C T Mg2+ Mg2+ 2 O H2O O H T O DNA2 Mg2+ DNA2 O 3' O 3' HO OH H T O Ribozyme G OH OH HO O 3' G O G O

Figure 10. RNARNA catalyzed catalyzed amide amide bond bond cleavage. cleavage.

ToTo determinedetermine thethe generalitygenerality of the amide bond bond cl cleavageeavage by by RNA, RNA, a a series series of of substrates substrates were were explored.explored. If If a ashort short DNA DNA is isattached attached to toa peptide a peptide or an or amino an amino acid acidby using by using an amide an amide bond, bond, the theimmediate immediate cleavage cleavage at the at theamide amide bond bond was wasobserved observed in the in presence the presence of the of RNA. the RNA. The cleavage The cleavage of the of theamide amide bond bond between between the theamino amino acid acid residues residues was wasnot observed. not observed.

3.3. MetalMetal ComplexesComplexes for the Activation of Amide Amide Bonds Bonds WeWe havehave summarizedsummarized different kinds of metal-base metal-basedd complexes, complexes, their their mechanisms mechanisms of of hydrolysis hydrolysis ofof unactivatedunactivated peptidepeptide bonds and point of cleavages in in Table Table 22..

Table 2. Metal catalyzed hydrolysis hydrolysis of of peptide peptide bonds. bonds.

Entry MetalMetal Complex Method of Hydrolysis Point of Cleavage Ref. Entry Method of Hydrolysis Point of Cleavage Ref. 1Complex Simple metal ions Lewis acidity of metal ion C-terminal of peptide [67–72] SimpleZr metal POMs, 1 2 LewisLewis acidity acidity of ofmetal metal ion ion C-terminal C-terminal of of peptide [67–72] [73–82 ] ionsZr MOF-808 Zr POMs, Zr Lewis acidity of metal ion and formation of 2 3 Mo(VI) Lewis acidity of metal ion C-terminalC-terminal sideof peptide of Asp [73–82] [81,82 ] MOF-808 5 membered ring Lewis acidity of metal ion and formation of 5 3 4Mo(VI) Co(III) N-terminal amine intiated tertiary complexC-terminal C-terminal side of peptide of Asp [81,82] [83–88 ] membered ring 5 Mo(II) Favorable six-membered chelate ring C-terminal side of Cys [89] 4 Co(III) N-terminal amine intiated tertiary complex C-terminal of peptide [83–88] Carboxylic group of amino acid and side chain of C-terminal side of Met, His, Cys 5 6 Mo(II) Pd(II), Pt(II) Favorable six-membered chelate ring C-terminal side of Cys [ [89]90–98 ] amino acid anchoring metal complex and S-MeCys Carboxylic group of amino acid and side chain of C-terminal side of Met, 6 Pd(II), Pt(II) Second amide bond upstream [90–98] 7 Pd(0) Methionineamino acid side anchoring chain anchoring metal complex metal complex His, Cys and S-MeCys [99–102] from Met Second amide bond 7 Pd(0) Methionine side chain anchoring metal complex Mb = Leu 89 − Ala 90 [99–102] Lewis acidity activation and PNA for selectivity upstream from Met 8 Co(III) and Cu(II) PDF = Gln 152 − Arg 153 [103–111] towards a particular protein BSAMb = Solvent = Leu 89 exposed − Ala portion90 Co(III) and Lewis acidity activation and PNA for selectivity PDF = Gln 152 − Arg 153 8 9 Ni(II) Non Lewis acid based N,O acyl rearrangement N-terminal side of Ser/Thr[103–111] [112,113] Cu(II) towards a particular protein BSA = Solvent exposed 10 Sc(III) Lewis acid based N,O acyl rearrangement N-terminalportion side of Ser/Thr [114] 9 Ni(II) Non Lewis acid based N,O acyl rearrangement N-terminal side of Ser/Thr [112,113] 10Another Sc(III) approach for Lewis the acid cleavage based N,O of acyl peptide rearrangement bonds involves N-terminal the side use of ofSer/Thr metal complexes. [114] This metal complex has potential applications in the field of chemical biology, biochemistry, Another approach for the cleavage of peptide bonds involves the use of metal complexes. This and bioengineering. A variety of metal ion complexes has been utilized for testing the reactivity metal complex has potential applications in the field of chemical biology, biochemistry, and with substrates such as peptides, and proteins [67]. Most of the metal catalyzed reactions reported so bioengineering. A variety of metal ion complexes has been utilized for testing the reactivity with far are based on the activation of amide carbonyl or water by the Lewis acid mechanism of the metal substrates such as peptides, and proteins [67]. Most of the metal catalyzed reactions reported so far are based on the activation of amide carbonyl or water by the Lewis acid mechanism of the metal ion.

Molecules 2018, 23, 2615 10 of 43 Molecules 2018, 23, x 10 of 43 ion.Another Another metal metal ion hydrolysis ion hydrolysis mechanism mechanism involves involves the formation the formation of a square of a square planar planar complex complex of the of themetal metal ions ions Cu(II), Cu(II), Ni(II), Ni(II), or Pd(II) or Pd(II) with with the theSer/Thr-His Ser/Thr-His or Ser/Thr-Xaa-His or Ser/Thr-Xaa-His sequence sequence leading leading to the to theN NO→ rearrangementO rearrangement of the of theacyl acyl moiety moiety resulting resulting in th ine cleavage the cleavage of the of peptide the peptide bond bond (Figure (Figure 11). In11 ). Inthis this section, section, we we will will provide provide few few examples examples of of both both Lewis Lewis acid acid and and the the N N→OO acyl acyl rearrangement rearrangement for for thethe cleavagecleavage ofof peptidepeptide bonds.bonds.

M HO O O O H R'' R'' N R' N R' N R' N R'' H H H O O H H O H H M M (a) Activation of a water (b) polarization of carbonyl group (c) N-O acyl rearrangement

Figure 11. Metal-assistedMetal-assisted peptide peptide bond bond hydrolysis. hydrolysis.

3.1.3.1. LewisLewis AcidAcid Mediated Hydrolysis 3.1.1. Simple Metal Ions 3.1.1. Simple Metal Ions Yashiro et al. reported that the rate of hydrolysis of peptide bonds increases by almost all the Yashiro et al. reported that the rate of hydrolysis of peptide bonds increases by almost all the metal ions and the highest conversion was observed for Zn(II) [68–72]. The active intermediate was metal ions and the highest conversion was observed for Zn(II) [68–72]. The active intermediate was metal complexes where metal binds to both the carbonyl group and the N-terminal amino group of metal complexes where metal binds to both the carbonyl group and the N-terminal amino group of thethe peptidepeptide bond.bond. Cleavage of the the peptide peptide bond bond in in the the presence presence of of the the metal metal takes takes place place through through the the mechanismmechanism inin FigureFigure 1111b.b. 3.1.2. Oxo Metal Ions 3.1.2. Oxo Metal Ions −4 −4 Parac-Vogt et al. proved that polyoxometalates oxo-metal compounds such as MoO2 −4 , WO2−4 , Parac-Vogt−4 et− al.4 proved that polyoxometalates oxo-metal compounds such as MoO2 , WO2 , CrO2 , and VO2 cleave the peptide bonds in various dipeptides [73–77]. They have shown CrO2−4, and VO2−4 cleave the peptide bonds in various dipeptides [73–77]. They have shown that these that these oxo-metal compounds also hydrolyze the amide bonds in their regular oligomeric forms, oxo-metal compounds also hydrolyze the amide bonds in their regular oligomeric forms, polyoxometalates (POMs). A POM is a unit where one or more atoms can be replaced by a metal center polyoxometalates (POMs). A POM is a unit where one or more atoms can be replaced by a metal leadingcenter leading to the change to the inchange the coordination in the coordination properties properties of POM. POMsof POM. were POMs utilized were for utilized the Zr(IV)- for the and Ce(IV)-assistedZr(IV)- and Ce(IV)-assisted peptide bond peptide hydrolysis bond becausehydrolysis they because are homogenous they are homogenous in nature [78 in, 79nature]. [78,79]. Zirconium Complex Mediated Hydrolysis of Peptide Bonds Zirconium Complex Mediated Hydrolysis of Peptide Bonds Parac-Vogt et al. reported the hydrolysis of peptide bonds catalyzed by a polyoxometalate Parac-Vogt et al. reported the hydrolysis of peptide bonds catalyzed by a polyoxometalate complex for the first time. They demonstrated the role of metal-substituted Wells-Dawson type complex for the first time. They demonstrated the role of metal-substituted Wells-Dawson type polyoxometalates K15H[Zr(α2-P2W17O61)2]·25H2O for the hydrolysis of peptide bonds in diglycine, polyoxometalates K15H[Zr(α2-P2W17O61)2]·25H2O for the hydrolysis of peptide bonds in diglycine, triglycine, tetraglycine, and pentaglycine (GG), yielding glycine as a final product (Figure 12)[80]. triglycine, tetraglycine, and pentaglycine (GG), yielding glycine as a final product (Figure 12) [80]. A Adetailed detailed mechanistic mechanistic investigation investigation by by NMR NMR showed showed that that the the free free amino amino terminus terminus and and both both carbonyl carbonyl functionalities of GG interact with polyoxometalates K H[Zr(α -P W O ) ]·25H O either by the functionalities of GG interact with polyoxometalates K1515H[Zr(α22-P2W1717O6161)2]·25H2 2O2 either by the formationformation ofof metalmetal ionion coordinationcoordination complex or or by by the the non-covalent non-covalent interactions interactions of of the the protonated protonated aminoamino groupgroup with with the the negatively negatively charged charged surface surface of POMs,of POMs, thus thus responsible responsible for thefor activationthe activation of amide of bondsamide towardsbonds towards hydrolysis. hydrolysis. These POMs These selectively POMs selectively cleave the cleave C-terminal the C-terminal amide bond amide of glycylglycyl bond of amideglycylglycyl (GGNH amide2), resulting (GGNH in2), the resulting formation in the of GG. formation No free of glycine GG. No amide free (GNHglycine2) amide was detected (GNH2) during was thedetected course during of the the reaction. course of the reaction.

Molecules 2018, 23, 2615 11 of 43 Molecules 2018,, 23,, xx 11 of 43

O

NH

HN

X O X cGG

O O K15H[Zr( 2-P2W17O61)2]] 25H2O H3N O H3N 3 N 2 3 O H GG O G

Figure 12. PeptidePeptide hydrolysis hydrolysis catalyzed catalyzed by by a a polyoxometalate polyoxometalate complex. complex.

Recently,Recently, VogtVogt etet al.al. utilized the MOF-808, a a Zr(IV)-based metal metal−−organicorganic complex complex for for the the hydrolysishydrolysis ofof thethe peptide bond in in a a wide wide range range of of peptides peptides and and proteins proteins such such as as hen hen egg egg white white lysozymelysozyme (HEWL)(HEWL) under under physiological physiological conditions conditions [81 [81,82].,82]. The The MOF-88 MOF-88 is is heterogeneous heterogeneous in naturein nature and isand thus is athus reusable a reusable catalyst. catalyst. The experimental The experimental studies andstudies calculations and calculations showed that showed MOF-808 that hydrolyzedMOF-808 thehydrolyzed Gly-Gly bondthe Gly-Gly by the bond formation by the of formation the active of complexes the active with complexes two adjacent with two Zr(IV) adjacent centers Zr(IV) of the {Zrcenters6O8} coreof the by {Zr coordination6O8} core by with coordination amide oxygen with and amide the amineoxygen nitrogen and the atoms. amine Thenitrogen catalytic atoms. efficiency The ofcatalytic MOF-808 efficiency towards of theMOF-808 hydrolysis towards of peptides the hydrolys is dependentis of peptides on the is dependent bulkiness andon the nature bulkiness of the and side chainnature amino of the acidside residues.chain amino Dipeptides acid residues. with smallDipeptides or hydrophilic with small residues or hydrophilic undergo residues cleavage undergo faster as comparedcleavage faster to those as compared with bulky to andthose hydrophobic with bulky and residues. hydrophobic residues.

Asp-XaaAsp-Xaa SelectiveSelective HydrolysisHydrolysis of the Peptide Bond by by Oxo-Metal Oxo-Metal Ions Ions ItIt hashas beenbeen reported that that oxomol oxomolybdate(VI)ybdate(VI) catalyzes catalyzes the the cleavage cleavage of of various various peptides peptides containing containing asparticaspartic acidacid (Asp)(Asp) with cleavage at at the the C-terminal C-terminal side side of of the the Asp Asp residue. residue. This This is is due due to to the the attack attack ofof thethe sideside chainchain ofof thethe AspAsp on the amidic carbon carbonylyl which which is is activated activated by by the the coordination coordination with with oxomolybdate(VI),oxomolybdate(VI), resultingresulting inin thethe formationformation ofof the the five-membered five-membered ring ring and and simultaneous simultaneous cleavage cleavage of theof the amide amide bonds bonds (Figure (Figure 13 )[13)81 [81,82].,82].

O O Mo O O O H O H H N N N R R1 R R OH N R2 1 R2 R1 OH + R2 R1 N O + H2N H2N H OH 2 OH O O O O O O

Figure 13. Oxomolybdate(VI)Oxomolybdate(VI) catalyzed catalyzed cleavage cleavage of of peptide peptide bonds. bonds.

VariousVarious OtherOther MetalMetal ComplexesComplexes TheThe WestheimerWestheimer and Trapmann groups showed showed th thatat the the metal metal complexes complexes containing containing Co(II), Co(II), Cu(II),Cu(II), andand Ni(II)Ni(II) ionsions cleavecleave various dipeptides dipeptides [83,84]. [83,84]. Out Out of of variousvarious various metalmetal metal complexes,complexes, complexes, thethe the Co(III)Co(III) Co(III) 2+2+ complexcomplex [Co(trien)OH(H[Co(trien)OH(H22O)]2+ isisis oneone one ofof of thethe the widelywidely widely studiedstudied studied metalmetal metal ionion ion complexescomplexes complexes andand and carriedcarried carried outout out thethe the rapidrapid hydrolysishydrolysis of of the the peptide peptide bond bond [85 [85–88].–88]. The The detailed detailed mechanistic mechanistic investigation investigation showed showed that that first, 2+ 2+ thefirst, metal the complexmetal complex [Co(trien)OH(H [Co(trien)OH(H2O)] forms2O)]2+ aforms tertiary a complextertiary complex with a dipeptide with a dipeptide by the replacement by the ofreplacement an equatorially of an coordinatedequatorially c wateroordinated molecule water in molecule the octahedral in the Co(III)octahedral complex Co(III) by complex the N-terminal by the amineN-terminal of the amine peptide. of the This peptide. mode This of coordination mode of coordination brings the brings axially the bound axially hydroxylbound hydroxyl group ingroup close proximityin close proximity to the peptide to the peptide carbonyl carbonyl resulting resulting in the hydrolysis in the hydrolysis of thepeptide of the peptide bond (Figure bond (Figure 14). 14).

Molecules 2018, 23, 2615 12 of 43 Molecules 2018,, 23,, xx 12 of 43

NH NH2 H HN N Co X NH Co X1 NH2 X2 HN NH 2 HN OH /OH HN NH2 HN OH2/OH R1 NH O N 1 NH2 Co OH H H OH H O HN N HN NH O HN N HN NH2 Co X OH /OH HN NH X1 OH2/OH HN NH2 NH2 + 2 H HN N X1 O HN N O 1 H N Co O H N N R H2N R1 X1 X2 HN NH2 X1 2 O X 2 H N O X2 H2N X R1 O X2 R1 HN O O R H O R1 H O

Figure 14. 14. Co(III)Co(III) Complex Complex catalyzed catalyzed peptide peptide hydrolysis. hydrolysis.

AnchoringAnchoring atat CysCys Side Chains: Molybdocene Molybdocene Erxleben et al. showed that molybdocene dichloride, Cp MoCl , cleaves the amide bond at Erxleben et al. showed that molybdocene dichloride, Cp2MoCl2 2,, cleavescleaves2 thethe amideamide bondbond atat thethe theC-terminal C-terminal side side of cysteine of cysteine to togenerate generate Cys-Gl Cys-Glyy from from Gly-Cys-Gly Gly-Cys-Gly [89]. [89 ].The The cleavage cleavage is ishighly highly selectiveselective forfor thethe C-side of Cys because because it it leads leads to to thethe the formationformation formation ofof of thethe the favorablefavorable favorable six-memberedsix-membered six-membered ring.ring. ring. TheThe mechanismmechanism ofof thisthis reactionreaction is illustrated in Figure 1515 [[89].89].

NH2 O 2+ NH2 O 2 H 2 H H O O N O O N O H2O Cp O N O N + pH 7.4 N H + Mo H O O O O O O O O O H O Cp room temp. O O S SH H2O Cp room temp. S OH Mo Cp Cp

NH2 O 2 H O N NH CH COO O NH2CH2COO + O O Cp S Mo Cp

FigureFigure 15. 15. MoMo catalyzed catalyzed peptide peptide hydrolysis. hydrolysis. 3.1.3. Anchoring at Met, His, and Cys Side Chains: Palladium(II) Complexes for the Cleavage of 3.1.3. Anchoring at Met, His, and Cys Side Chains: Palladium(II) Complexes for the Cleavage of Amide Bonds Amide Bonds Figure 16 shows the examples of platinum(II) and palladium(II) complexes which are known for Figure 16 shows the examples of platinum(II) and palladium(II) complexes which are known for the cleavage of amide bonds under mild conditions. These platinum(II) and palladium(II) complexes the cleavage of amide bonds under mild conditions. These platinum(II) and palladium(II) complexes attach to the sulfur atom of cysteine, S-methylcysteine, and methionine in peptides, thus, promoting the attach to the sulfur atom of cysteine, S-methylcysteine, and methionine in peptides, thus, promoting selective cleavage of the unactivated amide bonds at the C-side of the amino acid (Figure 16)[90–92]. the selective cleavage of the unactivated amide bonds at the C-side of the amino acid (Figure 16) [90– 92].

MoleculesMolecules 20182018,, 2323,, 2615x 1313 of of43 43 Molecules 2018, 23, x 13 of 43 2+ 2+ Cl N N 2+ 2+ S Cl N Pd N N PdN N N N N Pd N S N PdN N N Pd N S Pd OH2 Cl N PdN NCl PdCl N S Pd OH N Cl 2 N PdN Cl Cl OH2 N OH2 2+ [{Pd(en)Cl}2(1-pz)](NO3)2 [{Pd(en)Cl}2(1-pydz)](NO3)2 Cis[Pd(dtco)(H2O)2] 2+ [{Pd(en)Cl}2(1-pz)](NO3)2 [{Pd(en)Cl} (1-pydz)](NO ) Cis[Pd(dtco)(H O) ] 2 3 2 OH 2 2 H2O OH H2O OH H O 2 2 2 OH2 HO O OHO H O Pd H O Pd Pd -CD OHO 2 OH2 2 OH2 H2O OH HO H O N MeS 2 HO O O 2 Pd OH2 Pd N Pd S O OH -CD OH HO O H2O OH N N MeS HO OH 2 S O HO OH OHHO O 2+ 2+ O Pd(en)(H O) 2+ O HO OH Pd(H2O)4 2 2 [Pd(dtco)( -CD)(H2O)2] OH -CD O 2+ 2+ Pd(H O) Pd(en)(H O) 2+ O -CD OH 2 4 2 2 [Pd(dtco)( -CD)(H2O)2] OH O O O N OHO OHO OH O O Pt N OH O O OH O Pt N OOH OHO OH OH O O O N O OHO OH OH O[Pt(dach)(CBDCA-O,O')] HO O [Pt(dach)(CBDCA-O,O')] Cl O Cl O HO O O Cl O O N Cl Cl Pt N Cl Cl Pt N O O O O Pt N O Pt N Cl Pt N O Pt N Cl Cl Pt N O N CH3 O N Cl Pt N O Pt N O Pt N N N CH3 N CH3 N Cl N N N CH3 [Pt(en)Cl2] [Pt(en)(CBDCA-O,O')] [Pt(1,2-pn)Cl2] [Pt(1,2-pn)(CBDCA O,O')] [Pt(dach)Cl2]

[Pt(en)Cl2] [Pt(en)(CBDCA-O,O')] [Pt(1,2-pn)Cl2] [Pt(1,2-pn)(CBDCA O,O')] [Pt(dach)Cl2]

Figure 16. Pd and Pt complexes for activation of amide bonds. Figure 16. Pd and Pt Pt complexes complexes for for activation activation of of amide amide bonds. bonds. Pyrazine and Pyridazine Palladium(II)-Aqua Dimers Pyrazine and Pyridazine Palladium(II)-Aqua Dimers PyrazineThe completeand Pyridazine hydrolysis Palladium(II)-Aqua of the amide bonds Dimers of peptides N-acetylated-L-histidylglycine (Ac-L- His-Gly)TheThe completeand complete -L-methionylglycine hydrolysis hydrolysis of ofthe (Ac- theamideL-Met-Gly) amide bonds bonds atof thepeptides of C-terminal peptides N-acetylated- sideN-acetylated- of LMet-histidylglycine andL -histidylglycineHis in the (Ac- pHL- (Ac-rangeHis-Gly)L-His-Gly) 2.0 and < pH - andL-methionylglycine < -L2.5-methionylglycine was catalyzed (Ac- L (Ac-by-Met-Gly) twoL-Met-Gly) dinuclear at the at C-terminal the palladium(II) C-terminal side side complexes,of Met of Met and and His[{Pd(en)Cl} His in inthe the pH2(l- pH rangepz)](NOrange 2.0 2.03 <) 2 pH

Molecules 2018, 23, 2615 14 of 43 Molecules 2018, 23, x 14 of 43

PlatinumPlatinum ComplexesComplexes for the Cleavage of the the Amide Amide Bond Bond 1 1H-NMR investigation of of the the cleavage cleavage reactions reactions between between various various Pt(II) Pt(II) complexes complexes of of the the type type [Pt(L)Cl ] and [Pt(L)(CBDCA-O,O0] (L = ethylenediamine-en; (±)-trans-1,2-diaminocyclohexane-dach; [Pt(L)Cl22] and [Pt(L)(CBDCA-O,O′] (L = ethylenediamine-en; (±)-trans-1,2-diaminocyclohexane-dach; ((±)-1,2-propylenediamine-1,2-pn±)-1,2-propylenediamine-1,2-pn and and CBDCA CBDCA is the is 1,1-cyclobutanedicarboxylic the 1,1-cyclobutanedicarboxylic anion) and anion) the N and- theacetylated-N-acetylated-L-methionylglycineL-methionylglycine dipeptide dipeptide (MeCOMet-Gly) (MeCOMet-Gly) were reported were by reported Djuran and by co-workers Djuran and co-workers[93–96]. The [comparison93–96]. The of comparison the rate studies of theof these rate Pt studies complexes of these for the Pt complexescleavage of forN-acetylated- the cleavageL- ofmethionylglycineN-acetylated-L -methionylglycinedipeptide (MeCOMet-Gly) dipeptide showed (MeCOMet-Gly) that the rate showed of hydrolysis that the decreases rate of hydrolysiswith the decreasesincrease in with the thesteric increase bulk of in the the CBDCA steric bulk and of chlorido the CBDCA Pt(II) and complexes chlorido (en Pt(II) > 1,2-pn complexes > dach) (en (Figure > 1,2-pn >18). dach) (Figure 18).

O

H C NH O 3 H H C N 3 S O O MeCOMet-Gly pH 7.40 in phosphate buffer o [Pt(L)Cl2]+ t = 37 C + [Pt(L)(CBDCA-O,O)] (L = en, 1,2-pn or dach) O O O O H C NH O 3 H H C N H3C 3 S O O O O HN O N N Pt Cl H Pt N N O S N O CH3 [Pt(L)(CBDCA-O)(MeCOMet-Gly-S)]

- -Cl ; +H2O -CBCDA; +H2O O + HN CH3 O O + H N O H3C NH O H3C H N H3C O S O S OH2 O N Pt OH2 N Pt OH2 internal delivery external attack N N

+ hydrolytically active [Pt(L)(H 2O)(MeCOMet-Gly-S)] complex

O + N OH HN CH3 2 O Pt O + H2N S O N O CH3 + Gly [Pt(L)(H2O)(MeCOMetS)]

Figure 18. 18. HydrolyticHydrolytic reactions reactions of of Pt Pt complexes. complexes.

Later, these [Pd(en)(H2O)2]2+ and [Pt(en)(H2O)2]2+ complexes were used for the cleavage of tetrapeptide (MeCOMet-Gly-His-GlyNH2) in the pH range of 1.5–2.0 and at 60 °C. The study showed

Molecules 2018, 23, 2615 15 of 43

2+ 2+ Later, these [Pd(en)(H2O)2] and [Pt(en)(H2O)2] complexes were used for the cleavage of Molecules 2018, 23, x ◦ 15 of 43 tetrapeptide (MeCOMet-Gly-His-GlyNH2) in the pH range of 1.5–2.0 and at 60 C. The study showed thatthat thesethese complexescomplexes are highly selective selective for for the the cleavage cleavage of of the the amide amide bond bond at at the the C-terminal C-terminal side side of of methioninemethionine (Figure 1919))[ [97].97]. The The high high selectivity selectivity for for the the Met-Gly Met-Gly amide amide bond bond compared compared to to the the other other amidesamides isis due to the high affinity affinity of of the the Pt(II) Pt(II) and and Pd(II) Pd(II) ions ions for for the the sulfur sulfur atom atom on on Met. Met. Two Two different different mechanismsmechanisms for the cleavage of tetrapeptide tetrapeptide at at the the C-terminus C-terminus of of Met Met residue residue by by Pd Pd complexes complexes have have beenbeen proposed.proposed. One involves involves the the direct direct coordinati coordinationon of of the the Pd Pd complex complex with with Met Met followed followed by by the the cleavage.cleavage. The second involves the the formation formation of of ma macrochelatecrochelate with with both both His His and and Met Met followed followed by by the the hydrolysishydrolysis ofof the amide bond.

NH S N O O O H H N N H3C N N NH2 H H O O 2+ 2+ Pt(en)(H2O)2 Pd(en)(H2O)2 1.5

O O 2+ H H N N H C N N NH CH 3 N N 2 Pd 3 H H HN O O O S OH N O 2 NH HN N O S Pt N NH HN N O HN O O hydrolytically active form H2N macrochelate

2+ after 60min H2en 2+ H2O OH2 O CH H Pd 3 H C N S HN 3 OH N HN O O OH2 O NH HN S Pt N N H2N H O N O O hydrolytically active form

+ OH O H O 2+ N NH2 H2N N O NH S N H O O Pd NH H3C N N HN O N O O NH

H2N

Figure 19. 19. HydrolyticHydrolytic reactions reactions of of Pd Pd and and Pt Pt complexes. complexes.

2+ Cis[Pd(dtco)(HCis[Pd(dtco)(H22O)2]]2+ leadsleads to to the the selective selective cleavage cleavage of of the the amide amide bond bond at at the the C-terminal C-terminal of of Met Met (Figure(Figure 2020))[98 [98].]. Coordination Coordination of theof metalthe metal complex complex promotes promotes hydrolysis hydrolysis by two different by two mechanisms. different Themechanisms. first involves The first the formationinvolves the of formation a six-membered of a six-membered complex by complex the chelation by the ofchelation a metal of atom a metal with atom with both the sulfur of Met and the carbonyl of the amide bond, thus, activating the amide bond toward cleavage by an external attack from water. This mechanism is favorable in the case of platinum(II) promoters and substrates with smaller anchoring side chains. Second, the mechanism

Molecules 2018, 23, 2615 16 of 43 both the sulfur of Met and the carbonyl of the amide bond, thus, activating the amide bond toward cleavageMolecules 2018 by, an23, x external attack from water. This mechanism is favorable in the case of platinum(II)16 of 43 promoters and substrates with smaller anchoring side chains. Second, the mechanism involves the chelationinvolves the ofmetal chelation with of sulfur metal only, withfollowed sulfur only, by thefollowed internal by attackthe internal of water attack molecules of water to molecules the amide bond.to the Thisamide mechanism bond. This is favorablemechanism with is favorable palladium(II) with promoters palladium(II) and substratespromoters withand longersubstrates anchoring with sidelonger chains anchoring (Figure side 20)[ chains98]. (Figure 20) [98].

2+ H3C AcMet-Gly H3C AcMet-Gly 2+ S S S S S S +2 H O Pd Pd 2 Pd Pd -AcMet-Gly S S S S O O S H C H2 H2 3 AcMet-Gly

Mechanism HN CO2H O OH2

O S M CO2H O CO2H N NH external attack x S M Activation M S x S x O M H2O HN amide N-coordination CO2H inhibition internal delivery

2+2+ FigureFigure 20.20. [Pd(dtco)(H[Pd(dtco)(H22O)2]] mediatedmediated hydrolysis hydrolysis of of amide amide bond. bond.

KosticKostic etet al. utilizedal. utilized the Pd(H the 2O)Pd(H4 complex2O)4 complex for the cleavage for the of decapeptidecleavage of (Ac-AKYGGMAARA) decapeptide (Ac- underAKYGGMAARA) acidic conditions under (pH acidic 2.3) conditions at 60 ◦C[99 (pH]. The 2.3) cleavage at 60 °C of[99]. the The peptide cleavage bond of took the placepeptide at thebond Gly residuetook place next at tothe Met Gly on residue the N-terminal next to Met side on andthe N-terminal generated twoside fragmentsand generated after two 24 h.fragments The Pd(H after2O) 4 complex24 h. The bindsPd(H2 toO) the4 complex S of the binds Met to residue the S of leading the Met to residue the formation leading ofto twothe formation different complexesof two different under thecomplexes reaction under conditions the reaction (active conditions and inactive), (active which and areinactive), in equilibrium which are with in equilibrium each other (Figurewith each 21 ). Theother active (Figure complex 21). The led active to the complex formation led of to two the fragments formation through of two hydrolysisfragments through but theinactive hydrolysis complex but didthe notinactive undergo complex any hydrolysis. did not undergo The coordination any hydrolysis of two. The amide coordination nitrogen of atoms two amide in the inactivenitrogen complex atoms quenchin the inactive the Lewis complex acidity quench of Pd(II), the thusLewis no acidity hydrolysis of Pd(II), was thus observed. no hydrolysis was observed. Next, Kostic et al. used this metal complex Pd(H2O)4 for the cleavage of the amide bond O O underO neutral conditionsO in different peptideO substrates suchO as Gly-Met, Sar-Met,O and Pro-Met. H + H S This study showedN that the rate of hydrolysis-H at a neutralN pH is slowhydrolysis compared to that at a low N N S N N pH [44H]. Interestingly,H the complete cleavage+H+ ofH Sar-Met and Pro-Met was observed butN noPd cleavageOH2 O Pd O was observed for Gly-Met at a neutral pH. This is due to the differencePd S in the equilibriumO position of active NH2 the Gly-Met compared to that of Pro-Met. In Gly-Met, equilibrium is more shifted towards the+ inactive form at neutral pH due to the ability of Gly to form a strong coordinate bond with Pd.O In contrast, H for the peptide containing Sar-Met or Pro-Met,+H the+ -H equilibrium+ is shifted towards the hydrolyticallyN HO active form because they are unable to form a strong coordination with Pd due to the tertiary amide of O the Pro or Sar residue (Figure 22). O O S O S O S +H+ hydrolysis X + N Pd N Pd H HO N Pd N O -H+ N 2 O N O O O N O N

inactive N O H O

Figure 21. [Pd(H2O)4]2+ mediated hydrolysis of amide bond.

Molecules 2018, 23, x 16 of 43

involves the chelation of metal with sulfur only, followed by the internal attack of water molecules to the amide bond. This mechanism is favorable with palladium(II) promoters and substrates with longer anchoring side chains (Figure 20) [98].

2+ H3C AcMet-Gly H3C AcMet-Gly 2+ S S S S S S +2 H O Pd Pd 2 Pd Pd -AcMet-Gly S S S S O O S H C H2 H2 3 AcMet-Gly

Mechanism HN CO2H O OH2

O S M CO2H O CO2H N NH external attack x S M Activation M S x S x O M H2O HN amide N-coordination CO2H inhibition internal delivery

Figure 20. [Pd(dtco)(H2O)2]2+ mediated hydrolysis of amide bond.

Kostic et al. utilized the Pd(H2O)4 complex for the cleavage of decapeptide (Ac- AKYGGMAARA) under acidic conditions (pH 2.3) at 60 °C [99]. The cleavage of the peptide bond took place at the Gly residue next to Met on the N-terminal side and generated two fragments after 24 h. The Pd(H2O)4 complex binds to the S of the Met residue leading to the formation of two different complexes under the reaction conditions (active and inactive), which are in equilibrium with each other (Figure 21). The active complex led to the formation of two fragments through hydrolysis but

Moleculesthe inactive2018, 23complex, 2615 did not undergo any hydrolysis. The coordination of two amide nitrogen atoms17 of 43 in the inactive complex quench the Lewis acidity of Pd(II), thus no hydrolysis was observed.

O O O O O O O H + H S N -H N hydrolysis N N S N N H H +H+ H N Pd OH2 O Pd O Pd S O active NH2 + O Molecules 2018, 23, x H17 of 43 +H+ -H+ N HO Next, Kostic et al. used this metal complex Pd(H2O)4 for the cleavage of the amide bond underO neutral conditions in different peptide substratesO such as Gly-Met, Sar-Met, and Pro-Met. This study O S O S O showed thatS the rate of hydrolysis+H+ at a neutral pH is slowhydrolysis compared to that at a low pH [44]. X + Interestingly, the complete cleavage of Sar-MetN andPd Pro-Met was observedN butPd no cleavageH HO was N Pd N O -H+ N 2 observed for Gly-Met at a neutral pH. This isO due to the difference in the equilibrium position of the O N O O N Gly-Met comparedN to that of Pro-Met. In Gly-Met, equilibriumO is more shifted towards the inactive form at neutral pH due to the ability of Gly to forminactive a strong coordinate bond with Pd. In contrast, for N O the peptide containingO Sar-Met or Pro-Met, the equilibriumH is shifted towards the hydrolytically active form because they are unable to form a strong coordination with Pd due to the tertiary amide of the Pro or Sar residue (Figure 22). 2+ FigureFigure 21.21. [Pd(H[Pd(H22O)4]]2+ mediatedmediated hydrolysis hydrolysis of of amide amide bond. bond.

OH2 R O H2O OH2 H H O Pd S N Pd O H2O Pd S N N S S H H O O NH OH2 O O 2 R O Pd O H active (minor) OH2 H N H2O N N N N H H O O R R HN O NH OH2 N R 2 N Pd S OH O R N S H2O 2 O Pd N Pd N O O O N N O O S inactive (major) O O

Figure 22. PdPd triggered triggered pH pH dependent dependent hydrolysis hydrolysis of of amide amide bond. bond.

InIn thethe casecase ofof Gly-Pro-Met,Gly-Pro-Met, the hydrolysis of of the the amide amide bond bond ca cann take take place place either either through through the the externalexternal oror internalinternal attack of water molecules de dependingpending upon upon the the cis/trans cis/trans conformation conformation adopted adopted by by prolineproline (Figure(Figure 2323).). The ROESY NMR studies studies showed showed that that the the hydrolysis hydrolysis of of the the Gly-Pro-Met Gly-Pro-Met peptide peptide takestakes placeplace byby thethe externalexternal attack of water on on trans-Gly-Pro trans-Gly-Pro [100]. [100].

Figure 23. Cis/Trans conformations conformations of of Pd Pd complexes. complexes.

Overall, the Pd(H2O)4 complex is residue selective under acidic conditions and cleaves the second amide bond upstream from the Met residue. However, the same complex is sequence-specific under neutral conditions and cleaves the amide bond only at the Pro-Met or Sar-Met residue with no cleavage observed at Gly-Met peptide (Figure 24).

Molecules 2018, 23, 2615 18 of 43

Overall, the Pd(H2O)4 complex is residue selective under acidic conditions and cleaves the second amide bond upstream from the Met residue. However, the same complex is sequence-specific under neutral conditions and cleaves the amide bond only at the Pro-Met or Sar-Met residue with no cleavage observedMolecules 2018 at,Gly-Met 23, x peptide (Figure 24). 18 of 43

Ac Ac K K Y G Y G G M pH 2.0 Ac pH 7.0 A + K G M A A 2+ Y G A 2+ M A [Pd(H2O)4] G [Pd(H2O)4] G G A G G A P + + R A M residue-selective sequence-selective A R M A R M P M A G P A G

Figure 24. pHpH selective selective Pd Pd catalyst catalyst for for the the hydrolysis. hydrolysis.

Next,Next, thethe samesame groupgroup utilized the Pd(en)(H 2O)O)22 complexcomplex for for the the acidic acidic hydrolysis hydrolysis of of the the B-chain B-chain ofof bovinebovine insulininsulin containingcontaining two histidine residu residues.es. These These complexes complexes promoted promoted the the cleavage cleavage of of the the secondsecond amideamide bondbond upstream from histidine. The The detailed detailed mechanisti mechanisticc analysis analysis showed showed the the selective selective cleavagecleavage byby thethe coordinationcoordination of the Pd complexes with with the the histidine histidine side side chain chain and and amidic amidic nitrogen nitrogen followedfollowed byby thethe internalinternal attackattack of water on the the amide amide bond bond (Figure (Figure 25)25)[ [101].101].

O R'' O O R'' O H H H O N N N N N N 2+ O H [Pd(en)(H2O)2] R' O OH H N O 2 R' O NH NH N Pd N R'' N Pd N N Pd N NH N 2+ N [Pd(en)2] + N + 3H2O O R'

O R'' O O R'' H R'' N N H N N H N R' O O R' NH pH 2.0 H O Pd N H2O Pd N 2 O N H2O HN active inactive

Figure 25. AcidicAcidic hydrolysis hydrolysis of of amide amide bonds. bonds.

Next,Next, theythey usedused the β-cyclodextrin Pd Pd complex complex for for the the cleavage cleavage of of the the amide amide bond bond at at the the first first amideamide bondbond upstreamupstream from the Pro-Phe sequence. The The role role of of the the ββ-CD-CD complex complex was was to to bind bind to to the the hydrophobichydrophobic residueresidue (Phe) in an aq aqueousueous medium medium followed followed by by the the ac activationtivation of of the the amide amide carbonyl carbonyl groupgroup byby the the metal metal coordination, coordination, and and the the attack attack by the by externalthe external water water molecule molecule leading leading to the cleavageto the ofcleavage the amide of the bond amide (Figure bond 26 (Figure)[102]. 26) [102].

Molecules 2018, 23, 2615 19 of 43 Molecules 2018, 23, x 19 of 43

H2O

O R O H H N N N R R N N H N R O OH2 2 O H Pd cyclodextrin (CD) S S

Figure 26. ββ-Cyclodextrin-Cyclodextrin Pd-complex. Pd-complex.

3.1.4.3.1.4. ArtificialArtificial MetalMetal Proteases SuhSuh etet al.al. designed metal metal complexes complexes for for the the clea cleavagevage of of amide amide bonds bonds in in proteins proteins at at specific specific locationslocations [[103–107].103–107]. These These metal metal complexes complexes are are highly highly selective selective with with their their protein protein partners partners similar similar to tosome some natural natural proteases. proteases. They They also also demonstrated demonstrated thatthat the catalytic the catalytic rate rateof hy ofdrolysis hydrolysis increased increased by the by theformation formation of the of the complex complex between between the the substrate substrate and and catalyst. catalyst. This This is isachieved achieved by by increasing increasing the the multinuclearmultinuclear metalmetal centers, centers, which which provide provide extra extra metal metal centers centers as substrate as substrate binding binding sites. Theysites. showed They thatshowed the rate that of the hydrolysis rate of hydrolysis of the myoglobin of the myoglobi proteinn increasedprotein increased with the with increase the increase in the number in the number of metal centersof metal in thecenters mono in (half-lifethe mono for (half-life hydrolysis for 24 hydrolysis h, Figure 2724), dinuclearh, Figure (half-life27), dinuclear for hydrolysis (half-life 3.5 for h), andhydrolysis tetranuclear 3.5 h), metal and tetranuclear centers (half-life metal for centers hydrolysis (half-life 1.3 for h) [hydrolysis103]. 1.3 h) [103].

O O H H N N N N H H NH N N NH N N O O O Cu OH2 Cu OH2 O N N N N N N

mononuclear Cu OH2 dinuclear N N N N Cu H2O N N O HN O N HN O N O H Cu N N N H O H H2O N H H N N N O NH N N H O O O Cu OH2 tetranuclear tetranuclear N N N N

Cu OH2 N N

Figure 27. ArtificialArtificial metal metal complexes complexes with with different different numbers numbers of of metal metal centers. centers.

TheyThey showedshowed that the selectivity towards towards a a spec specificific protein protein was was achieved achieved by by the the addition addition of of organicorganic pendantspendants suchsuch asas peptidepeptide nucleicnucleic acidacid (PNA),(PNA), whichwhich areare responsibleresponsible forfor selectiveselective bindingbinding toto a particulara particular protein. protein. Overall, Overall, the goalthe goal of this of researchthis research was to was synthesize to synthesize peptide-cleavage peptide-cleavage agents selectiveagents forselective the hydrolysis for the hydrolysis of pathogenic of pathogenic proteins responsibleproteins responsible for Alzheimer’s for Alzheimer's disease, disease, type 2 diabetes type 2 diabetes mellitus, andmellitus, Parkinson’s and Parkinson’s disease. disease.

Aldehyde Pendant for the Cleavage of Proteins

Molecules 2018, 23, 2615 20 of 43

AldehydeMolecules 2018 Pendant, 23, x for the Cleavage of Proteins 20 of 43

AmmoniumAmmonium groupsgroups are abundant on the the surface surface of of proteins proteins and and form form imine imine with with aldehyde aldehyde faster faster thanthan thethe peptidepeptide bondbond cleavage and, and, through through this this pr process,ocess, brings brings the the reactive reactive metal metal catalytic catalytic center center inin closeclose proximity to to the the protein/peptide protein/peptide chain chain (Figure (Figure 28). 28 The). The rapid rapid formation formation of imine of imine makes makes the theattack attack of the of metal the metal center center on the on peptide the peptide bond an bond intramolecular an intramolecular process, process, thus, increases thus, increases the rate of the ratehydrolysis. of hydrolysis. Based Basedon this on concep this concept,t, several several artificial artificial metalloprot metalloproteaseseases have have been been developed developed by by incorporatingincorporating anan aldehydealdehyde handle handle close close to to the the metal metal center center and and has has been been successfully successfully been been applied applied for thefor fasterthe faster cleavage cleavage of peptide of peptide bonds bonds [104 [104].].

L L L L M O NH3 M N L L H3N H H protein substrate intermediate cleavage products

Figure 28. AldehydeAldehyde pendant pendant mediated mediated cleavage cleavage of of peptide peptide bonds. bonds.

Mb-SelectiveMb-Selective ArtificialArtificial Protease TheThe catalystcatalyst for the cleavage of myoglobin (Mb) (Mb) was was designed designed by by attaching attaching the the cyclen cyclen metal metal complexcomplex containingcontaining Cu(II) Cu(II) or or Co(III) Co(III) to theto the peptide peptide nucleic nucleic acid acid (PNA) (PNA) monomers, monomers, which which act as bindingact as sitesbinding for sites the Mb for (Figurethe Mb (Figure 29)[105 29)]. Varieties[105]. Varietie of linkerss of linkers were were inserted inserted between between the the PNA PNA binding binding site andsite and the catalyticthe catalytic cyclen cyclen site site for for the the formation formation of of Mb-cleaving Mb-cleaving catalysts.catalysts. MALDI-TOF MALDI-TOF MS MS showed showed thatthat thethe cleavagecleavage ofof the peptide backbone chain chain of of Mb Mb takes takes place place at at Leu89-Ala90. Leu89-Ala90. Various Various chelating chelating ligandsligands werewere testedtested forfor determining the the activity activity of of the the Mb-cleaving Mb-cleaving catalyst catalyst but but only only cyclen cyclen and and its its triaza-monooxotriaza-monooxo analoganalog showed efficient efficient catalytic activity. activity.

Cyc-metal complex peptide nucleic acid (PNA) catalyst site binding site nucleobase NH2 O NHNH O O NHN N NH2 N N H H n O

NH2 O O NH2 N N N N NH NH N N N N NH N S N N NH2 N O H 2 H H 2 H A* T* GC

Figure 29. 29. CatalystCatalyst design design for for protein protein cleavage. cleavage.

Mechanism:Mechanism: The first first step involves involves the the binding binding of of the the carbonyl carbonyl group group of of the the amide amide to to the the Co(III) Co(III) metalmetal centercenter toto formform a CS complex (Figure 30).30). The The selectivity selectivity of of one one amide amide carbonyl carbonyl over over the the rest rest of of thethe amidicamidic carbonylscarbonyls is based on the the other other half half of of the the catalyst, catalyst, which which is is responsible responsible for for the the recognition recognition ofof aa particularparticular proteinprotein andand a particularparticular site. This This Co(III) Co(III) carbonyl carbonyl coordinati coordinationon activates activates the the amidic amidic carbonylcarbonyl forfor aa nucleophilic attack by by the the hydroxide hydroxide ion ion on on the the metal metal center, center, resulting resulting in in the the formation formation ofof tetrahedraltetrahedral intermediate T. T. This is is followed followed by by the the collapse collapse of of the the tetrahedral tetrahedral intermediate intermediate (T), (T), resulting in the breakage of the amide bond to generate a peptide amine and corresponding peptide acid [106].

Molecules 2018, 23, 2615 21 of 43 resultingMolecules 2018 in, the 23, x breakage of the amide bond to generate a peptide amine and corresponding peptide21 of 43 Molecules 2018, 23, x 21 of 43 acid [106]. Suh and co-workers showed that the cleaving capability of the Co(III)/Cu(II) complexes of cyclen SuhSuh andand co-workers co-workers showed showed that that the cleaving the cleaving capability capability of the Co(III)/Cu(II) of the Co(III)/Cu(II) complexes of complexes cyclen increased by the replacement of one nitrogen atom of cyclen with an oxygen atom. The Co(III)- ofincreased cyclen increased by the replacement by the replacement of one nitrogen of one atom nitrogen of cyclen atom with of an cyclen oxygen with atom. an The oxygen Co(III)- atom. oxacyclen complexes (1-oxa-4,7,10-triazacyclododecane Co(III)) cleaved the proteins such as BSA, Theoxacyclen Co(III)-oxacyclen complexes complexes(1-oxa-4,7,10-triazacyclododeca (1-oxa-4,7,10-triazacyclododecanene Co(III)) cleaved Co(III)) the cleaved proteins the such proteins as BSA, such HEWL, Mb, and bovine serum-globulin with a 4–14 times higher catalytic efficiency compared to the asHEWL, BSA, HEWL, Mb, and Mb, bovine and bovineserum-globulin serum-globulin with a 4–14 with ti ames 4–14 higher times catalytic higher catalyticefficiency efficiency compared compared to the Co(III)-cyclen complexes [106]. toCo(III)-cyclen the Co(III)-cyclen complexes complexes [106]. [106].

O NH H O NH H R' N R' R N R' IIIO N R' R N X X CoIIIO NH H X X Co O R NH H OH R III H IIIOH2 NH X XCo OH2 NH X XCo OH OH NH CS NH CS XX == NHNH or O

NH - NH HH RCH2OO- N RCH2OO IIIO N R' X IIIO R' X XCo OO RR HH NH

NHNH T IIIIII O X O R XX XCoCo R OO NHNH H NH H22 NH N N R'R' IIIIIIOO X XCo OO RR R'NH2 NH

FigureFigure 30.30. MechanismMechanism of of Co(III)-metal Co(III)-metal complexes.complexes. complexes.

PDF-SelectivePDF-SelectivePDF-Selective ArtificialArtificial Artificial Protease ProteaseProtease TheTheThe catalyst catalystcatalyst for forfor the thethe cleavage cleavagecleavage of theofof the protein,the protein, peptide peptide deformylase deformylase (PDF), (PDF),(PDF), was obtained waswas obtainedobtained in a selective inin a a mannerselectiveselective by manner manner screening by by screening screening the library thethe of librarylibrary catalysts of catalysts (Figure 31(Figure). The 31).31). catalyst TheThe catalystcatalyst cleaved cleavedcleaved the backbone the the backbone backbone peptide chainpeptidepeptide of the chainchain PDF ofof at thethe position PDFPDF atat Gln152-Arg153. positionposition Gln152-Arg153. Docking simulations Docking simulationssimulations showed that showedshowed multiple thatthat interactions multiplemultiple wereinteractionsinteractions responsible were were for responsibleresponsible the formation forfor thethe of formation aformation complex of between a complex the catalystbetweenbetweenand thethe PDF.cacatalysttalyst Fifteen and and PDF. otherPDF. Fifteen proteinsFifteen wereotherother examined, proteins proteins were were but none examined, examined, of them butbut underwent nonenone of them cleavage underwent by this cleavage Co(III) complex, byby thisthis Co(III)Co(III) which complex, furthercomplex, confirmed which which thefurtherfurther highly confirmed confirmed selective the the nature highly highly of selective theseselective metal naturenature complexes of these [ 107metal]. complexescomplexes [107].[107].

NH X O OH XX OH2 O OH X Co 2 OH2 OH2 N XX == NH N

O N N NH NH O H2N O H2N

Figure 31. Metal-assisted catalyst for PDF. Figure 31. Metal-assistedMetal-assisted catalyst catalyst for for PDF. PDF.

Molecules 2018, 23, x 22 of 43

AmPs-Selective Artificial Protease AmPs are amyloidogenic peptides or proteins which lack active sites and are related to diseases Moleculessuch as 2018Alzheimer’s, 23, 2615 and type 2 diabetes. Based on the above concept, Suh et al. synthesized various22 of 43 other metal complexes for the selective cleavage of amide bonds in these proteins. The main Molecules 2018, 23, x 22 of 43 AmPs-Selectiveadvantage of this Artificial work is Protease that such amyloidogenic proteins cannot be targeted by conventional approaches due to the lack of an active site (Figure 32) [108–110]. AmPs-Selective Artificial Protease AmPsSoares areet amyloidogenical. utilized mononucl peptidesear orcopper(II) proteins whichcomplexes lack active[Cu(HL sites1)Cl2 and] and are [Cu(L related1)Cl] to for diseases the suchcleavageAmPs as Alzheimer’s of are unactivated amyloidogenic and amide type 2peptides bonds diabetes. of or the Basedproteins proteins on which the bovine above lack concept,activeserum sitesal Suhbumin and et al.are(BSA) synthesizedrelated and toTaq diseases variousDNA othersuchpolymerase, as metal Alzheimer’s complexes under mild and for typepH the and selective2 diabetes. temperature cleavage Based conditions ofonamide the above bonds (Figure concept, in these33). SuhThe proteins. etcleavage al. synthesized The occurred main advantage various at the ofotherspecific this metal work site iscomplexeson that the such solvent- amyloidogenicfor the exposed selective portions proteins cleavage cannotof theof amide beprotein targeted bonds to generate by in conventional these particular proteins. approaches proteolyticThe main due toadvantagefragments the lack of[111].of an this active work site is (Figurethat such 32)[ amyloidogeni108–110]. c proteins cannot be targeted by conventional approaches due to the lack of an active site (Figure 32) [108–110]. Soares et al. utilized mononuclear copper(II) complexes [Cu(HL1)Cl2] and [Cu(L1)Cl] for the cleavage of unactivated amide bonds of the(R).(LCo proteins3+) bovine serum albumin (BSA) and Taq DNA polymerase, under mild pH and temperature conditions (Figure 33). The cleavage occurred at the specific site on the solvent- exposed portions of the protein to generateNH particularH proteolytic N fragments [111]. O R' X XCo OH R n NH (R).(LCo3+)

(AMP) (AMP) ol-m (AMP) ol-m (R).(LCo3+) NH H N O R' X XCo OH R n NH

(AMP) (AMP) ol-m (AMP) ol-m (R).(LCo3+)

+ + n-1

+ + Figure 32. MetalMetal complexes complexes for for the the cleavage cleavage of of amyloidgenic amyloidgenic peptides. peptides. n-1 1 1 Soares et al. utilized mononuclear copper(II) complexes [Cu(HL )Cl2] and [Cu(L )Cl] for the cleavage of unactivated amide bonds of the proteins bovine serum albumin (BSA) and Taq DNA polymerase, under mild pH and temperature conditions (Figure 33). The cleavage occurred at the specific site on the solvent- exposed portions of the protein to generate particular proteolytic fragments [111]. Figure 32. Metal complexes for the cleavage of amyloidgenic peptides.

Figure 33. Cu complexes for activation of amide bonds.

3.2. The Non-Lewis Acid Reaction Mechanisms Based on the NO Rearrangement In some cases, metal catalysts showed a high rate of hydrolysis of the peptide backbone chain at the N-terminus of the serine and threonine residues. Such a cleavage was catalyzed by the NO

Figure 33. CuCu complexes complexes for for activation activation of of amide amide bonds. bonds.

3.2. The Non-Lewis Acid Reaction Mechanisms Based on the NO Rearrangement In some cases, metal catalysts showed a high rate of hydrolysis of the peptide backbone chain at the N-terminus of the serine and threonine residues. Such a cleavage was catalyzed by the NO

Molecules 2018, 23, 2615 23 of 43

3.2. The Non-Lewis Acid Reaction Mechanisms Based on the N→O Rearrangement In some cases, metal catalysts showed a high rate of hydrolysis of the peptide backbone chain Molecules 2018,, 23,, xx 23 of 43 at the N-terminus of the serine and threonine residues. Such a cleavage was catalyzed by the N→O rearrangementrearrangement andand doesdoes not employ the Lewis acid acid propertiesproperties ofof of thethe the metalmetal metal atom.atom. atom. BasedBased Based onon on thethe the proposedproposed mechanism,mechanism, the first first step involves involves the the formation formation of of the the Ni(II) Ni(II) complex complex with with 4 4N N of of the the backbonebackbone amideamide chainchain andand thethe sideside chainchain of thethe HisHis residueresidue with the (Ser/Thr)-Xaa-His sequence sequence (Figure(Figure 3434).). TheThe secondsecond step involves the the N N→OO rearrangement rearrangement from from the the side side chain chain of of the the Ser Ser or or Thr Thr thatthat transferstransfers thethe N-terminalN-terminal R1R1 moiety from the the pe peptideptide bond bond to to form form an an ester ester bond. bond. This This is is followed followed byby thethe hydrolysishydrolysis ofof thethe resulting ester, leading to to the the formation formation of of two two reaction reaction products, products, the the R1 R1 peptidepeptide acidacid andand thethe Ni(II)Ni(II) complexcomplex with the peptide [112,113]. [112,113].

R O Xaa O R O Xaa O O HO N R1 O N O H3O O O N Ni N Zaa H2N Ni N Zaa N N N N O N N O R1 H H HN R2 HN R2

OH

R O Xaa O O HO N R O 1 O + O H2N Ni N Zaa N N O H R = H or CH3 HN R2

Figure 34. NonNon lewis lewis acid acid mediated mediated N, N, O O Acyl Acyl rearrangement. rearrangement.

Scandium(III)Scandium(III) Triflate-PromotedTriflate-Promoted Serine/ThreonineSerine/Threonine Selective Selective Pept Peptideide Bond Bond Cleavage Cleavage KanaiKanai etet al. reported reported the the hydrolysis hydrolysis of of the the peptid peptidee bond bond at atthe the N-terminus N-terminus of Ser/Thr of Ser/Thr residue residue by byusing using scandium scandium triflate. triflate. This This chemical chemical cleavage cleavage relies relies on onSthe Sthe c triflate c triflate mediated mediated N Nto to O O acyl acyl ◦ rearrangementrearrangement followed followed by by the the subsequent subsequent hydrolysis hydrolysis of of the the ester ester by by heating heating it it at at 80–100 80–100C °C (Figure (Figure 35 ). Complete35). Complete hydrolysis hydrolysis took took place place in 18–20 in 18–20 h. Theh. The authors authors have have used used this this approach approach for for the the cleavage cleavage of variousof various peptides peptides including including posttranslationally posttranslationally modified modified (PTM) (PTM) peptidespeptides and the the cleavage cleavage of of native native proteinprotein AAββ1-42,1-42, whichwhich is closely related to Alzheimer’s disease disease [114]. [114].

HO H O H O O N N O NH2 N O H R R O

Figure 35. 35. N,N, O O Acyl Acyl rearrangement. rearrangement. 4. Organic Molecules for Activation of Amide Bonds 4. Organic Molecules for Activation of Amide Bonds We have summarized different kinds of nonmetal-based methods, their mechanisms of hydrolysis We have summarized different kinds of nonmetal-based methods, their mechanisms of of unactivated peptide bonds and the point of cleavages in Table3. hydrolysis of unactivated peptide bonds and the point of cleavages in Table 3.

Molecules 2018, 23, 2615 24 of 43

Table 3. Organic molecules based hydrolysis of peptide bonds.

Entry Organic Molecules Method of Hydrolysis Point of Cleavage Ref. Phenyl Through 5 membered cyclic N-terminal side 1 [115] isothiocyanate phenylisothiocyanate intermediate of peptide 2 Cyanogen bromide Through 5 membered iminolactone C-terminal side of Met [116] 2-Nitro-5-thiocyano 3 Through 5 membered thialactone N-terminal side of Cys [117] benzoic acid 4 2-iodosobenzoic acid Through iminospirolactone C-terminal side of Trp [118,119] 5 TBC Through Oxindole C-terminal side of Trp [118,119] N-terminal side 6 N-amidination Through 5 membered cyclic amidine ring [120] of peptide Protecting goups C-terminal side 7 Lactonization strategy [121–127] (Table4) of peptide H O responsive C-terminal side 8 2 2 Lactonization strategy [128] protecting groups of peptide Glutamic acid selective activation through 9 PyBroP N-terminal side of Glu [129,130] pyroglutamyl imide Hofmann rearrangement mediated 10 DIB N-terminal side of Asn [131] N-acylurea intermediate N-terminal side of Ser, 11 DSC Cyclic urethane amide activation [132–136] Thr, Cys and Glu A photo responsive amide cleavage 12 o-NBnoc C-terminal side of Asn [137–142] through succinimide ring Cu-organo radical Aerobic chemoselective oxidation of Ser 13 N-terminal side of Ser [143] conjugate followed by oxalimide formation C-terminal side 14 TFA/H O N-acyl group mediated oxazolinium specie [144] 2 of peptide Hydrazinolysis accelarated by addition of 15 Hydrazine hydrate N-terminal of peptide [145] ammonium salts C-terminal side 16 N-Mecysteine Through oxazolinium ion [146] of N-MeCys C-terminal side 17 Acylated N-MeAib Through oxazolinium ion [147] of N-MeAib

4.1. N-Terminal Cleavage of Amide Bonds

4.1.1. Edman’s Degradation This approach utilizes phenyl isothiocyanate for the cleavage of the peptide bond at the N-terminus. Phenyl isothiocyanate reacted with an uncharged N-terminal amino group, under mildly alkaline conditions, to form a cyclic phenylisothiocyanate derivative, which undergoes cleavage as a thiazolinone derivative under acidic conditions (Figure 36). We proposed that the activation of an amide bond is due to the formation of the five-membered cyclic phenylisothiocyanate intermediate which creates a twist in the amide bond, thus preventing the amidic nitrogen from forming a resonating structure and making it susceptible towards hydrolysis [115]. Molecules 2018, 23, 2615 25 of 43 Molecules 2018, 23, x 25 of 43

R S R N H H C N pH 8.0 N HN N S H2N Peptide HN N Peptide H O O

heat, H+

H O Peptide NH S Peptide NH N O heat, H+ S S OH HN R R R N N N N H H thiazolinone Peptide NH2

Figure 36. Edman’s degradation degradation approach approach for for cleavage cleavage of of peptide peptide bonds. bonds.

4.1.2.4.1.2. CyanogenCyanogen BromideBromide for Cleavage at Met Residue Residue CyanogenCyanogen bromidebromide led to the cleavage of of the the pept peptideide bond bond at at the the C-terminus C-terminus of of the the methionine methionine residueresidue inin a selectivea selective manner. manner. The The first stepfirst involvesstep involves the nucleophilic the nucleophilic attack of attack the sulfur of the of methioninesulfur of onmethionine cyanogen on bromide cyanogen (Figure bromide 37)[116 (Figure]. This displaces37) [116]. theThis bromide displaces from the cyanogen bromide bromide, from cyanogen followed bybromide, the attack followed of the amideby the carbonylattack of onthe the amide cyano carbon group,yl on resulting the cyano in the group, formation resulting of the in five-memberedthe formation ring,of the iminolactone, five-membered comprising ring, iminolactone, a double bondcomprising in the a ring double between bond nitrogenin the ring and between carbon. nitrogen This double and bondcarbon. results This indouble a rigid bond ring results conformation, in a rigid thus ring activating conformation, the amide thus bond activating towards the cleavage amide bond at the C-terminustowards cleavage of Met, at resulting the C-terminus in the generation of Met, resulting of homoserine in the generation lactone. This of homoserine approach has lactone. widely This been utilizedapproach for has the widely sequencing been utilized of proteins for the [116 sequencing]. of proteins [116].

O R' O R' RHN RHN N COOH N COOH H H

SCH H CS 3 N Br 3 N

O R' R' + H O RHN + 2 HN O H N COOH HN H2N COOH RHN COOH homoserine O lactone iminolactone

Figure 37. Cyanogen bromide bromide for for selective selective cleavage cleavage at at Met. Met.

4.1.3.4.1.3. 2-Nitro-5-Thiocyano2-Nitro-5-Thiocyano Benzoic Acid for Cleavage at at Cys Cys 2-Nitro-5-thiocyano2-Nitro-5-thiocyano benzoic acid led to to the the hydrolysis hydrolysis of of the the amide amide bond bond at at the the N-terminal N-terminal side side ofof thethe cysteinecysteine residue.residue. The first first step is is the the cyanylation cyanylation of of the the side side chain chain of of cysteine cysteine on on a apeptide peptide by by 2-nitro-5-thiocyano2-nitro-5-thiocyano benzoicbenzoic acid, acid, which which is is followed followed by by the the attack attack of of the the cysteine cysteine amidic amidic nitrogen nitrogen to to the cyanothe cyano group group on the on side-chain the side-chain of cysteine, of cysteine, resulting resulting in the formation in the formation of the 5-membered of the 5-membered thiolactone ring.thiolactone This, in ring. turn, This, activates in turn, the activates amide bond the amide towards bond hydrolysis towards hydrolysis under basic under conditions basic conditions (Figure 38 ). This(Figure is again 38). This due is to again the inability due to the of cysteineinability of amidic cysteine nitrogen amidic in nitrogen a thiolactone in a thiolactone to form a resonatingto form a structureresonating with structure the carbonyl with the of carbonyl peptide bondof peptide [117]. bond [117].

Molecules 2018, 23, 2615 26 of 43 Molecules 2018,, 23,, xx 26 of 43

O NCS O H H R N R' R N R' N + N H H O O SH COOH S N NO NO2 cleavage

O R OH O S NH + S thialactone R' thialactone O R' N NH H

Figure 38. 2-nitro-5-thiocyano2-nitro-5-thiocyano benzoic benzoic acid acid selective selective cleavage cleavage at at Cys. Cys.

4.1.4.4.1.4. 2-Iodosobenzoic2-Iodosobenzoic Acid for Cleavage at at Trp Trp 2-Iodosobenzoic2-Iodosobenzoic acid has been used used for for the the hydrolysis hydrolysis of of the the amide amide bond bond at at the the C-terminal C-terminal side side ofof thethe Trp residue. The The mechanism mechanism ofof thethe the cleavagecleavage cleavage isis aa is two-steptwo-step a two-step procproc process.ess. The Thefirst firststep involves step involves the theoxidation oxidation of the of side-chain the side-chain of tryptophan of tryptophan by 2-iodosobenzoic by 2-iodosobenzoic acid followed acid followed by the nucleophilic by the nucleophilic attack attackfrom the from neighboring the neighboring carbonyl carbonyl group group of the of theamide amide bond, bond, leading leading to tothe the formation formation of ofan an iminospirolactoneiminospirolactone whichwhich hydrolyzes the the peptide peptide chain chain in in the the presence presence of of water water (Figure (Figure 39) 39 )[[118,119].118,119 ].

HN OH HN II O NH NH O HO HO HN O O HN O O N O O H N N H H H

O O O O HN HN HN H2N + N O O O HN O HN H O O iminospirolactoneiminospirolactone

Figure 39. 39. IodosobenzoicIodosobenzoic acid acid for for hydrolysis. hydrolysis. 4.1.5. TBC for Cleavage at Trp 4.1.5. TBC for Cleavage at Trp Tryptophanyl peptide bonds underwent selective cleavage by 2,4,6-tribromo-4- Tryptophanyl peptide bonds underwent selective cleavage by 2,4,6-tribromo-4- methylcyclohexadienonemethylcyclohexadienone (TBC) (TBC) at at the the C-terminus C-terminus (Figure (Figure 40). 40Tyrosyl). Tyrosyl and histidyl and histidylpeptide peptidebonds α bondswhich whichare usually are usually cleaved cleaved by other by otherbrominating brominating agents agents (such (suchas α-bromosuccinimide, as -bromosuccinimide, α- α bromoacetamide,-bromoacetamide, etc.) etc.) are are stable stable to tothis this reagent. reagent. Additionally, Additionally, other other amino amino acids, acids, which which are are sensitive sensitive toto oxidation,oxidation, reactreact withwith TBC but do not cleave the peptide bonds. bonds. This This method method was was successfully successfully appliedapplied toto a varietyvariety of peptides and proteins [118,119]. [118,119]. According to the reaction mechanism suggested by Patchornik et al. (1960), oxidative bromine participates in the modification-cleavage reaction [118,119]. Two equivalents of bromine first brominate the indole nucleus followed by a spontaneous debromination through a series of oxidation and hydrolysis reactions (Figure 40). These reactions led to the formation of an oxindole derivative, which cleaves the peptide bond.

Molecules 2018, 23, x 27 of 43

NHR1 NHR1

NHR2 NHR2 Br Br Molecules 2018, 23, 2615 O O 27 of 43 Molecules 2018, 23, x N N 27 of 43 H H

NHR1 NHR1 NHR NHR 2 2 NHR1Br NHR1 H2O Br O O NH2R2 +N O O N O NHR2 H HN H HN HBr OH Br

NHR1 NHR1 H2O Figure 40. TBC for selective cleavage at Trp residue. NH2R2 + O O O NHR2 HN HN According to the reaction mechanism suggestedHBr by Patchornik et al. (1960), oxidative bromine OH Br participates in the modification-cleavage reaction [118,119]. Two equivalents of bromine first brominate the indole nucleus followed by a spontaneous debromination through a series of oxidation and hydrolysis reactions (FigureFigureFigure 40. 40).40.TBC TBC These forfor reactions selective cleavage cleavageled to theat atTrp formation Trp residue. residue. of an oxindole derivative, which cleaves the peptide bond. 4.2. N-AmidinationAccording forto the Cleavage reaction of mechanism the N-Terminal suggested Amide by Bond Patchornik et al. (1960), oxidative bromine 4.2.participates N-Amidination in the for modification-cleavageCleavage of the N-Terminal reacti Amideon [118,119].Bond Two equivalents of bromine first Hamada et al. reported the cleavage of the amide bonds by the N-amidination of peptides. brominate the indole nucleus followed by a spontaneous debromination through a series of oxidation Hamada et al. reported the cleavage of the amide bonds by the N-amidination of peptides. The The Nand-amidination hydrolysis reactions of peptides (Figure leads 40). to These the formation reactions ofled a to cyclic the formation moiety which of an oxindole resulted derivative, in the cleavage N-amidination of peptides leads to the formation of a cyclic moiety which resulted in the cleavage of of thewhich amide cleaves bond the at roompeptide temperature bond. (Figure 41)[120]. The rate of cleavage was slow under ambient the amide bond at room temperature◦ (Figure 41) [120]. The rate of cleavage was slow under ambient conditions (PBS buffer, pH 7.4) at 37 C with t1/2 = 35.7 h, but a rapid cleavage was observed under conditions (PBS buffer, pH 7.4) at 37 °C with t1/2 = 35.7 h, but a rapid cleavage was observed under basic4.2. conditions N-Amidination (2% NaOH for Cleavage aq) with of the t 1/2N-Terminal= 1.5 min. Amide To Bond evaluate the broad applicability of this cleavage basic conditions (2% NaOH aq) with t1/2 = 1.5 min. To evaluate the broad applicability of this cleavage reaction,reaction,Hamada a series a series et of ofal. peptides peptidesreported with withthe cleavage differentdifferent of amino aminothe amide ac acidsids bonds at at the the by N-terminus N-terminusthe N-amidination such such as theof as peptides. Lys, the Lys,Glu, The Glu,Ser, Ser, Cys, Tyr, Val, and Pro residues were cleaved with t values from 1 min to 10 min. A slightly slow Cys,N-amidination Tyr, Val, and of peptidesPro residues leads were to the cleaved formation with of ta1/21/2 cyclic values moiety from which1 min resultedto 10 min. in theA slightly cleavage slow of cleavagecleavagethe amide was was observedbond observed at room with with temperature bulky bulky aminoamino (Figure acids 41) at at[120 th thee]. terminusThe terminus rate of such cleavage such as asVal was Val or Pro,slow or Pro, whichunder which mightambient might be be hinderinghinderingconditions the the path(PBS path ofbuffer, of cyclization. cyclization. pH 7.4) at 37 °C with t1/2 = 35.7 h, but a rapid cleavage was observed under basic conditions (2% NaOH aq) with t1/2 = 1.5 min. To evaluate the broad applicability of this cleavage

reaction, a series of peptides with different amino acids at the N-terminusR 1such as the Lys, Glu, Ser, Cys, Tyr, Val, and Pro residues were cleaved with t1/2 values from 1 min to 10 min. A slightly slow HN cleavage was observed with bulky amino acids at the terminus such as Val orO Pro, which might be O R HN N R hindering the path of2 cyclization. O O R2 H 2 H2N H2N N N H2N H amidination H novel N-terminalR1 R1 O NH R1 O O degradationHN reaction N-amidinopeptide O O R HN N R 2 O O R2 H 2 H2N H2N N N H2N H amidinationFigureFigure 41. N-amidinationN-amidination strategy. strategy.novel N-terminal R O H O 1 NH R1 O degradation reaction N-amidinatedN-amidinated peptide peptide with with a Cysa Cys residue residueN-amidinopeptide at at the th N-terminuse N-terminus also also generated generated a a five-membered five-membered ring, thiazolidinering, thiazolidine by path by b path (intermediate b (intermediate B) which B) whic didh notdid leadnot lead to anyto any cleavage, cleavage, therefore, therefore, the the tt1/21/2 ofof the peptidethe peptide withCys with at Cys the atN-terminus the N-terminus in 2%in 2% NaOH NaOH aq aq at at 3737 ◦°CC was was 3.4 3.4 min min (slower (slower than than with with other other Figure 41. N-amidination strategy. amino acids at the N-terminus) (Figure 42) [120]. aminoMolecules acids 2018 at, 23 the, x N-terminus) (Figure 42)[120]. 28 of 43 N-amidinated peptide with a Cys residue at the N-terminus also generated a five-membered ring, thiazolidine by path b (intermediate B) which did not lead to any cleavage, therefore, the t1/2 of the peptide with Cys at the N-terminus in 2% NaOH aq at 37 °C was 3.4 min (slower than with other amino acids at the N-terminus) (Figure 42) [120].

FigureFigure 42. 42.N-amidination N-amidination strategy strategy with with N-terminal N-terminal cysteine. cysteine.

4.3. Lactonization Mediated Cleavage of Amide Bonds Otaka et al. developed an auxiliary with special protecting groups (PGs), which is capable of forming a lactone with the carbonyl of an amide bond, resulting in the cleavage of the amide bond (Table 4) [121–128]. Depending on the nature of the protecting groups, an amide bond cleavage can be initiated in peptides by using different responsive reagents for the deprotection of PGs (Table 4). Table 4 showed various PGs and the corresponding responsive reagents for their deprotection. The thiol responsive reagent was applied for the cleavage of the PNA/DNA complex using thiol- responsive protecting groups [121–128].

Table 4. Lactonization mediated cleavage of amide bonds.

Reagent/Condition PG R1 R2 R3 NO2 H H NO2 OMe OMe H OTBDPS H Ultraviolet Near-infrared Fluoride hypoxia

H NO2 H

Thiol 4-nitrobenzenesulfonyl - - - Phosohatase phosphate - - -

4.4. Hydrogen Peroxide-Induced Amide Bond Cleavage

Later, the hydrogen peroxide (H2O2)-responsive protecting group was introduced to the amino acid. This protecting group contains a boronate or boronic acid moiety which underwent deprotection in oxidative stress because of the release of hydrogen peroxide followed by the formation of lactone and the cleavage of the peptide bond (Figure 43) [128].

Molecules 2018Molecules, 23, x 2018, 23, x 28 of 43 28 of 43

Molecules 2018, 23, 2615 28 of 43 Figure 42. N-amidinationFigure 42. N-amidination strategy with strategy N-terminal with cysteine. N-terminal cysteine.

4.3. Lactonization4.3.4.3. LactonizationLactonization Mediated Cleavage MediatedMediated of Amide Cleavage Bonds of Amide Bonds Otaka et al.Otaka developed et al. developedan auxiliary an with auxiliary special with protecting special groupsprotecting (PGs), groups which (PGs), is capable which ofis capable of Otaka et al. developed an auxiliary with special protecting groups (PGs), which is capable of forming a lactoneforming with a lactone the carbonyl with the of carbonyl an amide of bo annd, amide resulting bond, in resulting the cleavage in the of cleavage the amide of bondthe amide bond forming a lactone with the carbonyl of an amide bond, resulting in the cleavage of the amide bond (Table 4) [121–128].(Table 4) Depending [121–128]. Dependingon the nature on ofthe the nature protecting of the groups,protecting an groups,amide bond an amide cleavage bond can cleavage can (Table4)[ 121–128]. Depending on the nature of the protecting groups, an amide bond cleavage can be be initiatedbe in initiatedpeptides inby peptides using different by using responsi differentve responsireagentsve for reagents the deprotection for the deprotection of PGs (Table of PGs4). (Table 4). initiated in peptides by using different responsive reagents for the deprotection of PGs (Table4). Table4 Table 4 showedTable various 4 showed PGs various and the PGs corresponding and the corresponding responsive rereagentssponsive for reagents their deprotection. for their deprotection. The The showed various PGs and the corresponding responsive reagents for their deprotection. The thiol thiol responsivethiol responsivereagent was reagent applied was for applied the cleavage for the of cleavage the PNA/DNA of the PNA/DNAcomplex using complex thiol- using thiol- responsive reagent was applied for the cleavage of the PNA/DNA complex using thiol-responsive responsive responsiveprotecting groupsprotecting [121–128]. groups [121–128]. protecting groups [121–128].

Table 4. LactonizationTable 4. Lactonization mediated cleavage mediated of amide cleavage bonds. of amide bonds. Table 4. Lactonization mediated cleavage of amide bonds.

Reagent/Condition PG R1 R2 R3 Reagent/ConditionReagent/Condition PGPG R1 R1R2 R3 R2 R3 NO2 H NO2 H H H NO2 NOOMe2 NO 2 OMeHHOMe OMe Ultraviolet Near-infrared H NOOTBDPS2 H HOMeOTBDPS H OMe Ultraviolet Near-infraredUltraviolet Near-infraredFluoride hypoxia Fluoride hypoxia Fluoride hypoxia H OTBDPS H

H HNO2 H H NO NO2 2 H H

Thiol 4-nitrobenzenesulfonyl - - - Thiol Thiol 4-nitrobenzenesulfonyl4-nitrobenzenesulfonyl ------PhosohatasePhosohatase Phosohatase phosphatephosphate phosphate------

4.4. Hydrogen4.4.4.4. Peroxide-Induced HydrogenHydrogen Peroxide-InducedPeroxide-Induced Amide Bond AmideCleavage Bond Cleavage

Later, the hydrogenLater, the peroxide hydrogen (H peroxide2O2)-responsive (H2O2)-responsive protecting groupprotecting was groupintroduced was introducedto the amino to the amino Later, the hydrogen peroxide (H2O2)-responsive protecting group was introduced to the amino acid. This acid.acid.protecting ThisThis protecting protectinggroup contains group group contains a containsborona a boronatete a orborona boronic or boronicte oracid acidboronic moiety moiety acid which which moiety underwentunderwent which deprotectionunderwent deprotectionindeprotection oxidativein oxidative stress in stressoxidative because because ofstress the of release becausethe release of of hydrogen theof hydrogenrelease peroxide of peroxidehydrogen followed followed peroxide by the formationby followed the of by lactone the formation offormation lactone and of lactone the cleavage and the of cleavagethe peptide of the bond peptide (Figure bond 43) (Figure[128]. 43) [128]. andMolecules the cleavage 2018, 23, x of the peptide bond (Figure 43)[128]. 29 of 43

O FmocHN OH R = B(OH)2, B(MIDA) O N B(MIDA) = B R O O O O

O O O H H H H RYDR N H RYDR N H RYDR N O GAQSGY NH2 GAQSGY NH2 O OH

OH +

H GAQSGY NH2

FigureFigure 43. Hydrogen peroxide peroxide responsive responsive probes. probes.

4.5. Glutamic Acid Specific Activation of Amide Bonds We have also reported a new method for the site-specific cleavage of peptide bonds at glutamic acid under physiological conditions [129,130]. The method involves the activation of the backbone peptide chain at the N-terminal side of glutamic acid by the formation of a pyroglutamyl imide (pGlu) moiety using bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP) (Figure 44). This activation increases the susceptibility of the peptide bond toward various nucleophiles including thiol and water (Figure 44). We showed that this pyroglutamyl imide activated peptide chain underwent the complete cleavage of the peptide bond under neutral buffer conditions (pH 7.5). It was observed that the rate of hydrolysis increase under basic pH conditions (pH = 10.5). Although the Asp has a carboxylic group on the side chain, no cleavage was observed under the reaction conditions. Jensen et al. exposed the pGlu activated peptide bond towards thiol, resulting in the formation of peptide thioesters [129,130]. A noted feature about this approach is that it leads to the formation of epimerization free peptide acids and peptide thioesters. This method is highly specific and exhibits a broad substrate scope including the cleavage of bioactive peptides with unnatural amino acids, which are unsuitable substrates for enzymes.

Molecules 2018, 23, 2615 29 of 43

4.5. Glutamic Acid Specific Activation of Amide Bonds We have also reported a new method for the site-specific cleavage of peptide bonds at glutamic acid under physiological conditions [129,130]. The method involves the activation of the backbone peptide chain at the N-terminal side of glutamic acid by the formation of a pyroglutamyl imide (pGlu) moiety using bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP) (Figure 44). This activation increases the susceptibility of the peptide bond toward various nucleophiles including thiol and water (Figure 44). We showed that this pyroglutamyl imide activated peptide chain underwent the complete cleavage of the peptide bond under neutral buffer conditions (pH 7.5). It was observed that the rate of hydrolysis increase under basic pH conditions (pH = 10.5). Although the Asp has a carboxylic group on the side chain, no cleavage was observed under the reaction conditions. Jensen et al. exposed the pGlu activated peptide bond towards thiol, resulting in the formation of peptide thioesters [129,130]. A noted feature about this approach is that it leads to the formation of epimerization free peptide acids and peptide thioesters. This method is highly specific and exhibits a broad substrate scope including the cleavage of bioactive peptides with unnatural amino acids,Molecules which 2018, 23 are, x unsuitable substrates for enzymes. 30 of 43

Figure 44. GlutamicGlutamic acid acid selective selective activation activation of of peptide peptide bonds. bonds.

4.6.4.6. AsparagineAsparagine Selective Cleavage of Amide Bonds Bonds KanaiKanai etet al.al. described the method for for the the site-sel site-selectiveective chemical chemical activation activation of of peptide peptide bonds bonds for for hydrolysishydrolysis atat thethe asparagineasparagine residue using diacet diacetoxyiodobenzeneoxyiodobenzene (DIB) (DIB) [131]. [131]. The The reaction reaction of of the the side-chainside-chain ofof AsnAsn with DIB leads to the formation of of isocyanate isocyanate by by Hofmann Hofmann rearrangement. rearrangement. This This is is followedfollowed byby the attack of of the the N N-terminal-terminal amidic amidic nitrogen nitrogen of of the the peptide peptide backbone backbone chain, chain, affording affording a afive-membered five-membered N-acylureaN-acylurea intermediate, intermediate, thus thusactivating activating the amide the bond amide towards bond towardshydrolysis hydrolysis (Figure (Figure45). Asn-selective 45). Asn-selective peptide bond peptide cleavage bond was cleavage proceeded was in proceeded an aqueous in neutral an aqueous solution neutral at 37 solution°C and ◦ atexhibited 37 C and a broad exhibited substrate a broad scope. substrate The Gln-site scope. was The Gln-sitenot cleaved was under not cleaved the reaction under conditions. the reaction conditions.Specifically, Specifically, this method this methodis applicable is applicable to peptides to peptides containing containing unnatural unnatural amino amino acids acids and/or and/or posttranslationalposttranslational modificationsmodifications where enzymaticenzymatic cleavage is is not not very very efficient. efficient.

O R O O R O H H N Peptide B N Peptide B Peptide A N N N N H H Peptide A H H O O O N NH2 C Hofmann rearrangement O O R cyclization OH Peptide A N H HN Peptide B O O O O + R H hydrolysis N Peptide B Peptide A N N NH O N H H HN O O

Figure 45. Asparagine selective cleavage of peptide bonds.

4.7. Cyclic Urethane Mediated Activation of Amide Bonds We have developed a method for the cleavage of the amide backbone chain at the N-terminal side of Ser, Thr, and Cys by the formation of a five-membered cyclic urethane moiety [132]. The

Molecules 2018, 23, x 30 of 43

Figure 44. Glutamic acid selective activation of peptide bonds.

4.6. Asparagine Selective Cleavage of Amide Bonds Kanai et al. described the method for the site-selective chemical activation of peptide bonds for hydrolysis at the asparagine residue using diacetoxyiodobenzene (DIB) [131]. The reaction of the side-chain of Asn with DIB leads to the formation of isocyanate by Hofmann rearrangement. This is followed by the attack of the N-terminal amidic nitrogen of the peptide backbone chain, affording a five-membered N-acylurea intermediate, thus activating the amide bond towards hydrolysis (Figure 45). Asn-selective peptide bond cleavage was proceeded in an aqueous neutral solution at 37 °C and exhibited a broad substrate scope. The Gln-site was not cleaved under the reaction conditions. MoleculesSpecifically,2018, 23this, 2615 method is applicable to peptides containing unnatural amino acids and/or30 of 43 posttranslational modifications where enzymatic cleavage is not very efficient.

O R O O R O H H N Peptide B N Peptide B Peptide A N N N N H H Peptide A H H O O O N NH2 C Hofmann rearrangement O O R cyclization OH Peptide A N H HN Peptide B O O O O + R H hydrolysis N Peptide B Peptide A N N NH O N H H HN O O

Figure 45. AsparagineAsparagine selective selective cleavage cleavage of of peptide peptide bonds. bonds.

4.7.4.7. CyclicCyclic Urethane Mediated Activation of of Amide Amide Bonds Bonds WeWe havehave developeddeveloped a a method method for for the the cleavage cleavage of of the the amide amide backbone backbone chain chain at theat the N-terminal N-terminal side ofsideMolecules Ser, of Thr, Ser, 2018 and Thr,, 23,Cys x and by Cys the formationby the formation of a five-membered of a five-membered cyclic urethane cyclic urethane moiety [ 132moiety]. The [132]. formation31 ofThe 43 of the cyclic urethane moiety with an amide backbone makes the amidic carbonyl group susceptible toformation nucleophilic of the attack. cyclic Thisurethane is presumably moiety with due an toamide the twist backbone in the makes backbone the amidic amide carbonyl chain caused group by thesusceptible cyclic urethane to nucleophilic moiety. attack. Thus, itThis was is nopresum longerably able due to to form the twist a resonating in the backbone structure. amide To achievechain caused by the cyclic urethane moiety. Thus, it was no longer able to form a resonating structure. To this goal, we screened various carbonylating reagents and a maximum conversion to cyclic urethane achieve this goal, we screened various carbonylating reagents and a maximum conversion to cyclic moiety was achieved with N,N-disuccinimidyl carbonate (DSC). We proposed that the hydroxymethyl urethane moiety was achieved with N,N-disuccinimidyl carbonate (DSC). We proposed that the group of the side chain of Ser reacted with DSC to generate an activated intermediate, A, which then hydroxymethyl group of the side chain of Ser reacted with DSC to generate an activated intermediate, undergoes nucleophilic displacement by the amidic nitrogen on the N-side of serine through the A, which then undergoes nucleophilic displacement by the amidic nitrogen on the N-side of serine path to generate a five-membered cyclic urethane intermediate B (Figure 46). The formation of the through the path to generate a five-membered cyclic urethane intermediate B (Figure 46). The cyclicformation urethane of the intermediate cyclic urethane B makes intermediate the amide B makes bond susceptiblethe amide bond to nucleophilic susceptible attackto nucleophilic and led to theattack cleavage and led of theto the amide cleavage bond of in the neutral amide aqueousbond in neutral conditions aqueous (room conditions temp, pH (room 7.5, 12temp, h). TherepH 7.5, is a possibility12 h). There of is nucleophilic a possibility displacement of nucleophilic of displace the intermediatement of the A intermediate by the amidic A nitrogenby the amidic through nitrogen path b. 0 Thisthrough could path lead b. to This the formationcould lead ofto six-memberedthe formation of ring six-membered B , but we did ring not B′, observebut we did the not formation observe of the any six-memberedformation of any ring six-membered as analyzed by ring NMR as analyzed studies. by NMR studies.

O O H backbone H N activation N N N H H O O OH O path a path b path b O O A O N H O O N N O B’ O O path a

O O

H O hydrolysis O N O N O O + HO N O B H HN

FigureFigure 46.46. CyclicCyclic urethane mediated activation activation of of peptide peptide bond. bond.

The side chain of Glu on reaction with DSC also led to the formation of a pyroglutamyl imide moiety with an amide backbone chain, thus making it susceptible to nucleophilic attack (Figure 47) [132]. We have used this approach for the selective hydrolysis of peptides/proteins at the N-terminus of Ser, Thr, Cys, and Glu. This method cleaved various bioactive peptides containing posttranslational modifications (e.g., N-acetylation and -methylation) and mutations (D-and β-amino acids), which are not suitable substrates for enzymes, thus exhibited a broad substrate scope. We have also used this approach for the synthesis of a variety of functionalized C-terminal peptides such as esters, amides, alcohols, and thioesters (Figure 48) by exposing the cyclic urethane activated peptide towards various nucleophiles such as alcohols, amines, reducing agents, and thiols [133,134]. The attractive feature of this approach is that it leads to the formation of epimerization free C- functionalized peptides.

Molecules 2018, 23, 2615 31 of 43

The side chain of Glu on reaction with DSC also led to the formation of a pyroglutamyl imide moiety with an amide backbone chain, thus making it susceptible to nucleophilic attack (Figure 47)[132]. We have used this approach for the selective hydrolysis of peptides/proteins at the N-terminus of Ser, Thr, Cys, and Glu. This method cleaved various bioactive peptides containing posttranslational modifications (e.g., N-acetylation and -methylation) and mutations (D-and β-amino acids), which are not suitable substrates for enzymes, thus exhibited a broad substrate scope. We have also used this approach for the synthesis of a variety of functionalized C-terminal peptides such as esters, amides, alcohols, and thioesters (Figure 48) by exposing the cyclic urethane activated peptide towards various nucleophiles such as alcohols, amines, reducing agents, and thiols [133,134]. The attractive feature of this approach is that it leads to the formation of epimerizationMolecules 2018, 23, free x C-functionalized peptides. 32 of 43

O backbone O H backbone H N activation N N N H H O 2 O 2 O O HO O O O N O

O

O O

N O H O hydrolysis O N O O + HO N H HN

Figure 47. Pyroglutamyl imide imide mediated mediated activation activation of of peptide peptide bond. bond.

O CH3OH 3 OCH OCH3 O O H NH2CH2Ph 2 2 NHCH2Ph N O 2 N O NaBH O NaBH4 OH O OH

RSH O

= peptide SR

Figure 48. Cyclic urethane urethane mediated mediated synt synthesishesis of of C-terminal C-terminal peptides. peptides.

Later,Later, thisthis cycliccyclic urethaneurethane amide-activationamide-activation approachapproach waswas appliedapplied forfor the the cleavage cleavage of of a a variety variety of cyclicof cyclic and and lasso lasso peptides peptides obtained obtained from from nature nature to todetermine determine theirtheir sequence, which which is is difficult difficult to to be be determineddetermined byby conventional approaches (Figure (Figure 49)49)[ [135,136].135,136]. We We have have also also applied applied this this method method for for thethe synthesissynthesis ofof a peptide-based molecular molecular machine machine (rotaxanes) (rotaxanes) for for the the first first time time (Figure (Figure 50) 50 [135,136].)[135,136 ].

X X X X X X O X X X DIEA, DMAP, O O X X X DMF, DSC X O HN X N O HN N O X H O OH O O OH O

O X O X X O X X NH O phosphate buffer X X X X OH N O X X O O H O X H2O X X O

Molecules 2018, 23, x 32 of 43

O O H backbone H N activation N N N H H O 2 O 2 O O HO O O O N O

O

O O

H O hydrolysis N O N O O + HO N H HN

Figure 47. Pyroglutamyl imide mediated activation of peptide bond.

O CH3OH OCH3 O O H NH2CH2Ph N NHCH2Ph N O O NaBH4 O OH

RSH O

= peptide SR

Figure 48. Cyclic urethane mediated synthesis of C-terminal peptides.

Later, this cyclic urethane amide-activation approach was applied for the cleavage of a variety of cyclic and lasso peptides obtained from nature to determine their sequence, which is difficult to be Moleculesdetermined2018, 23by, 2615conventional approaches (Figure 49) [135,136]. We have also applied this method32 for of 43 the synthesis of a peptide-based molecular machine (rotaxanes) for the first time (Figure 50) [135,136].

X X X X X X X DIEA, DMAP, O O X X DMF, DSC X O X N HN N O X H O O OH O

O X O X X X X NH O phosphate buffer X X X OH N X O O X H2O X X O Molecules 2018, 23, x 33 of 43

Figure 49. 49. CyclicCyclic urethane urethane for for cleavage cleavage of of cyclic cyclic peptides. peptides.

H N O S9 I10 O L11 L11 I10 Q12 OH Q12 S9 P7Q13R6 P7Q13R6 O 1 D8 D8 G5 DSC,H2O G5 E14 G1 E14 G1 F4 F4 V2 G3 V2 G3 Q15 Q15

A17 A17 K17 K17

P18 P18

M19 M19

Figure 50. 50. SynthesisSynthesis of of rotaxane rotaxane from from lasso lasso peptide. peptide. 4.8. Intein-Inspired Amide Cleavage Chemical Device 4.8. Intein-Inspired Amide Cleavage Chemical Device AA photoresponsivephotoresponsive devicedevice waswas developeddeveloped forfor thethe cleavagecleavage ofof thethe amideamidebond bond at at the the C-terminus C-terminus of theof the Asn Asn residue residue [137 ].[137]. This This approach approach was inspiredwas inspired by intein-mediated by intein-mediated protein protein splicing splicing and its chemicaland its environmentchemical environment was mimicked was bymimicked the incorporation by the incorporation of geminal dimethyl of geminal groups dimethyl and a secondarygroups and amine a onsecondary the asparagine amine on scaffold. the asparagine scaffold. TheThe secondary amine amine acts acts as as an an intramolecular intramolecular base, base, which which enhances enhances the nucleophilicity the nucleophilicity of the of theamide amide nitrogen nitrogen (Figure (Figure 51). The51). geminal The geminal dimethyl dimethyl groups groupsled to a led Thorpe-Ingold to a Thorpe-Ingold effect, which effect, whichenhances enhances the intramolecular the intramolecular attack, thus attack, assisting thus in assistingthe formation in the of the formation succinimide of thering succinimide [138–140]. ringThe o [138-nitrobenzyloxycarbonyl–140]. The o-nitrobenzyloxycarbonyl (o-NBnoc) masks ( othe-NBnoc) basic character masks the of basicthe secondary character amine of the [141,142], secondary aminethus leading [141,142 to], the thus photo leading triggered to the cleavage photo triggered of an amide cleavage bond of by an the amide deprotection bond by the of the deprotection secondary of theamine secondary unit containing amine unit the containingo-nitrobenzyloxycarbonyl the o-nitrobenzyloxycarbonyl group. group.

O O H UV H N irradiation N N N H NH H N O N +

O O H2N

O2N Mechanism O H N N H H N N R

Figure 51. Intein-inspired amide bond cleavage.

Molecules 2018, 23, x 33 of 43

Figure 49. Cyclic urethane for cleavage of cyclic peptides.

H N O S9 I10 O L11 L11 I10 Q12 OH Q12 S9 P7Q13R6 P7Q13R6 O 1 D8 D8 G5 DSC,H2O G5 E14 G1 E14 G1 F4 F4 V2 G3 V2 G3 Q15 Q15

A17 A17 K17 K17

P18 P18

M19 M19

Figure 50. Synthesis of rotaxane from lasso peptide.

4.8. Intein-Inspired Amide Cleavage Chemical Device A photoresponsive device was developed for the cleavage of the amide bond at the C-terminus of the Asn residue [137]. This approach was inspired by intein-mediated protein splicing and its chemical environment was mimicked by the incorporation of geminal dimethyl groups and a secondary amine on the asparagine scaffold. The secondary amine acts as an intramolecular base, which enhances the nucleophilicity of the amide nitrogen (Figure 51). The geminal dimethyl groups led to a Thorpe-Ingold effect, which enhances the intramolecular attack, thus assisting in the formation of the succinimide ring [138–140]. The o-nitrobenzyloxycarbonyl (o-NBnoc) masks the basic character of the secondary amine [141,142], Moleculesthus leading2018, 23 to, 2615the photo triggered cleavage of an amide bond by the deprotection of the secondary33 of 43 amine unit containing the o-nitrobenzyloxycarbonyl group.

O O H UV H N irradiation N N N H NH H N O N +

O O H2N

O2N Mechanism O H N N H H N N R

Figure 51. 51. Intein-inspiredIntein-inspired amide amide bond bond cleavage. cleavage.

4.9. Serine-Selective Aerobic Cleavage of Peptides Kanai et al. reported a use of the water-soluble copper-organoradical conjugate for the selective cleavage of the peptide bond at the N-terminus of a serine residue under mild conditions and at room temperature [143]. They used this approach for the selective cleavage of a variety of different peptides/proteins containing D-amino acids or sensitive disulfide pairs. Ser-selective cleavage of the peptide bond was initiated by the aerobic chemoselective oxidation of the hydroxymethyl moiety of Ser to a formyl group (A) (Figure 52). This produced a β-formyl glycineamide intermediate B which, on further oxidation, led to the formation of oxalamide C by undergoing oxidative deformylation. Oxalamide C then underwent hydrolysis under mild conditions because the carbonyl groups of the oxalamide are more electrophilic than those of simple amides, resulting in the formation of the cleaved fragments D and D0. By using molecular oxygen as a terminal oxidant, water and a C1 molecule (possibly HCO2H) become stoichiometric side products. This strategy is widely distinct from Lewis acid, promoted by the Ser-selective peptide hydrolysis through N-to-O rearrangement.

4.10. Hydrolysis of Amide Bonds by the Formation of Oxazolinium Specie: Function of Acyl Protecting Group Peptides containing a simple N-acyl group activates the amide bond four bonds away from an acyl group for cleavage under acidic conditions [144]. First, TFA leads to the protonation of amide carbonyl followed by the nucleophilic attack from the oxygen of the acyl carbonyl to generate a five-membered oxazolinium specie A in the peptide chain. Second, the collapse of the oxazolinium intermediate A leads to the cleavage of an unactivated amide bond (Figure 53). The nature of the aromatic group, G, was responsible for the rate of hydrolysis of the peptide bond. Electron-donating groups increase the rate of hydrolysis whereas electron withdrawing substituents decrease it (Figure 53). Molecules 2018, 23, x 34 of 43

4.9. Serine-Selective Aerobic Cleavage of Peptides Kanai et al. reported a use of the water-soluble copper-organoradical conjugate for the selective cleavage of the peptide bond at the N-terminus of a serine residue under mild conditions and at room temperature [143]. They used this approach for the selective cleavage of a variety of different peptides/proteins containing D-amino acids or sensitive disulfide pairs. Ser-selective cleavage of the peptide bond was initiated by the aerobic chemoselective oxidation of the hydroxymethyl moiety of Ser to a formyl group (A) (Figure 52). This produced a β-formyl glycineamide intermediate B which, on further oxidation, led to the formation of oxalamide C by undergoing oxidative deformylation. Oxalamide C then underwent hydrolysis under mild conditions because the carbonyl groups of the oxalamide are more electrophilic than those of simple amides, resulting in the formation of the cleaved fragments D and D′. By using molecular oxygen as a terminal oxidant, water and a C1 molecule (possibly HCO2H) become stoichiometric side products. MoleculesThis strategy2018, 23 is, 2615 widely distinct from Lewis acid, promoted by the Ser-selective peptide hydrolysis34 of 43 through N-to-O rearrangement.

CuI + keto-ABNO-H H2O NO

H NO2 2 N N HO O CuII O H O N N Peptide A N Peptide B O H H O (1e + 1e) oxidation

O O O O H O O (Cu) O H N 2 N Peptide A N Peptide B Peptide A N Peptide B H H ABO O

HCO2H O O O O H H + N hydrolysis N X = NH or OH X X N 2 D’ D H C O O

Molecules 2018, 23, x 35 of 43 Figure 52. SerineSerine selective selective aerobic aerobic cleavage cleavage of of peptide peptide bonds. bonds.

O O H H 4.10. Hydrolysis of Amide Bonds by the FormationTFA/H of Oxazolinium2O Specie: Function of Acyl Protecting R2 N R3 R2 N Group N OH + R3NH2 H O R O R Peptides containing a simple1 N-acyl group activates the amide1 bond four bonds away from an R = H, Ala-NH acyl group for cleavage under acidic conditions [144]. First,3 TFA leads2 to the protonation of amide carbonyl followed by the nucleophilic attack from the oxygenR = alkyl, of theG acyl carbonyl to generate a five- Mechanism 2 membered oxazolinium specie A in the peptide chain. Second, the collapse of the oxazolinium R NH intermediate A leads toO the cleavageR1 of an unactivatedO R amide1 bond (Figure 53).3 OH The nature of the aromatic group,NHR3 G, was responsibleNHR for the3 rate ofO hydrolysis of the peptide R2 N R2 N R1 bond. Electron-donating Hgroups increase the rateH of hydrolysis whereas electron withdrawing O O R N substituents decrease it (Figure 53). H 2 A H

O O R1 OH O R2 N R NH + R H 3 2 1 O N R2 H

Figure 53. OxazoliniumOxazolinium species species formation. formation. 4.11. Hydrazinolysis for the Cleavage of Amide Bonds 4.11. Hydrazinolysis for the Cleavage of Amide Bonds TheThe hydrazinolysishydrazinolysis of unactivated amide amide bonds bonds was was accelerated accelerated by by the the addition addition of of ammonium ammonium ◦ salts.salts. The reaction proceeds proceeds at at 50–70 50–70 °CC to to give give peptide peptide cleavage cleavage products products and and exhibits exhibits a abroad broad substratesubstrate scopescope thatthat out-performsout-performs existi existingng amide amide bond bond cleavage cleavage reactions reactions [145]. [145]. This This approach approach was was appliedapplied forfor thethe cleavagecleavage of the peptide bonds without without racemization racemization at at the the αα-position-position of of the the amino amino acidsacids (Figure(Figure 5454).). It was also applied to the cleavage cleavage of of the the NN-acetyl-acetyl group group from from the the amino amino sugar. sugar.

O O NH I H H 4 H N N hydrazine hydrate N N N H2N H H cleavage O O O

OH OH O NH4I O HO hydrazine hydrate HO HO HO HN O cleavage H2N O O

Figure 54. Hydrazinolysis for the cleavage of peptide bonds.

4.12. Amide Bond Cleavage of the N-Methylcysteinyl Peptide Tam et al. developed a selective bi-directional peptide bond cleavage approach utilizing N- methylcysteine (MeCys) in the Xaa-MeCys-Yaa peptides (Xaa and Yaa, non-cysteine residues) [146]. Under strong acidic conditions, peptide Xaa-MeCys-Yaa led to the formation of an oxazolinium intermediate, resulting in the cleavage of the Xaa-MeCys bond. The oxazolinium intermediate was later trapped by thiocresol (TC) to form a Xaa-MeCys-TC thioester (Figure 55). The replacement of MeCys by Cys residue did not result in the peptide bond cleavage, suggesting the important role of N-methylation in MeCys residue for the formation of oxazolone.

Molecules 2018, 23, x 35 of 43

O O H H TFA/H2O R2 N R3 R2 N N OH + R3NH2 H O R1 O R1 R3 = H, Ala-NH2

R = alkyl, G Mechanism 2

O R1 O R1 R3NH OH NHR3 NHR3 O R2 N R2 N R H H 1 O O R N H 2 A H

O O R1 OH O R2 N R NH + R H 3 2 1 O N R2 H

Figure 53. Oxazolinium species formation.

4.11. Hydrazinolysis for the Cleavage of Amide Bonds The hydrazinolysis of unactivated amide bonds was accelerated by the addition of ammonium salts. The reaction proceeds at 50–70 °C to give peptide cleavage products and exhibits a broad substrate scope that out-performs existing amide bond cleavage reactions [145]. This approach was Moleculesapplied2018 for, 23the, 2615 cleavage of the peptide bonds without racemization at the α-position of the amino35 of 43 acids (Figure 54). It was also applied to the cleavage of the N-acetyl group from the amino sugar.

O O NH I H H 4 H N N hydrazine hydrate N N N H2N H H cleavage O O O

OH OH O NH4I O HO hydrazine hydrate HO HO HO HN O cleavage H2N O O

Figure 54. Hydrazinolysis for for the the cleavage cleavage of of peptide peptide bonds. bonds.

4.12.4.12. AmideAmide BondBond Cleavage of the N-Methylcysteinyl Peptide Peptide TamTam etet al. developed developed a aselective selective bi-directional bi-directional peptide peptide bond bond cleavage cleavage approach approach utilizing utilizing N- Nmethylcysteine-methylcysteine (MeCys) (MeCys) in inthe the Xaa-MeCys-Yaa Xaa-MeCys-Yaa peptid peptideses (Xaa (Xaa and and Yaa, Yaa, non-cysteine non-cysteine residues) residues) [146]. [146 ]. UnderUnder strongstrong acidicacidic conditions, peptide Xaa-MeCys-Yaa Xaa-MeCys-Yaa led led to to the the formation formation of of an an oxazolinium oxazolinium intermediate,intermediate, resultingresulting in the cleavage of the Xaa-MeCys bond. bond. The The oxazolinium oxazolinium intermediate intermediate was was laterlater trappedtrapped byby thiocresolthiocresol (TC) to form a Xaa-MeCys-TCXaa-MeCys-TC thioester thioester (Figure (Figure 55). 55). The The replacement replacement of of MeCysMeCys byby CysCys residueresidue did not result in the peptide bond bond cleavage, cleavage, suggesting suggesting the the important important role role of of NMoleculesN-methylation-methylation 2018, 23, inxin MeCysMeCys residue for the formation of of oxazolone. oxazolone. 36 of 43

O R O O R O 1 H 1 H N protonated N Peptide N NH2 Peptide N NH2 O R O R 2 H 2 O R1 O R1 S OH Peptide N ptol Peptide N O O 4-Mercaptotoluene H2O O O HO O O + O Peptide H N NH2 N NH 2 R1 H 2 R N R 2 Peptide N R1 2

Figure 55. NN-methylcysteinyl-methylcysteinyl peptide peptide cleavage. cleavage.

4.13.4.13. N-MeAibN-MeAib Induced Unusual Cleavage of of Amide Amide Bonds Bonds PeptidesPeptides containingcontaining acylated N-methyl-aminoisobutyryl (NMeAib) (NMeAib) residues residues showed showed unusual unusual cleavagecleavage of amide bonds bonds under under acidic acidic conditions. conditions. The The cleavage cleavage takes takes place place at the at theC-terminal C-terminal side sideof ofthe the NMeAib NMeAib residue residue (Figure (Figure 56) [147]. 56)[ X-ray147]. diffraction X-ray diffraction studies of studies the NMeAib of the containing NMeAib molecules containing moleculesshowed that showed the oxygen that atom the oxygen of the carbonyl atom of group the carbonyl of the preceding group of residue the preceding is close to residue the carbonyl is close tocarbon the carbonyl of the NMeAib carbon of residue, the NMeAib thus acting residue, as thusan internal acting asnucleophile an internal forming nucleophile a tetrahedral forming a tetrahedralintermediate. intermediate. Once this tetrahedra Once thisl intermediate tetrahedral intermediate was formed, waslone formed,pair electrons lone pair on the electrons nitrogen on of the nitrogenthe phenylalanine of the phenylalanine were no longer were be no able longer to form be able resonating to form resonatingstructures with structures the carbonyl with the group carbonyl of groupNMeAib. of NMeAib.In fact, the In phenylalanine fact, the phenylalanine nitrogen nitrogenbecomes becomesa proton aacceptor proton acceptorlike the amines, like the amines,which whichresulted resulted in the incleavage the cleavage of the of amide the amide bond bond followed followed by bythethe removal removal of ofphenylalanine phenylalanine and and the the formationformation ofof anan oxazoliniumoxazolinium ion intermediate, which which further further reacts reacts with with water water to to form form a acarboxylic carboxylic acidacid product.product.

O O O O H H N protonated N N OCH3 N OCH3 O O H

O O O O OH N work up O NH O N OH PheOMe O oxazolinium ion N tetrahedral intermediate

Figure 56. N-Me Aib mediated amide bond cleavage.

5. Conclusions This mini-review highlights the methods for the activation of unactivated amide bonds in biomolecules. This review further highlights the application of these methods in the sequencing of proteins and the synthesis of peptide acids, thioesters, alcohols, and amides. These studies showed that there is still a lot to learn from enzymes catalyzed pathways and how we can develop enzyme mimetics for catalyzing the cleavage of unactivated and highly stable amide bonds at particular

Molecules 2018, 23, x 36 of 43

O R O O R O 1 H 1 H N protonated N Peptide N NH2 Peptide N NH2 O R O R 2 H 2 O R1 O R1 S OH Peptide N ptol Peptide N O O 4-Mercaptotoluene H2O O O HO O O + O Peptide H N NH2 N NH 2 R1 H 2 R N R 2 Peptide N R1 2

Figure 55. N-methylcysteinyl peptide cleavage.

4.13. N-MeAib Induced Unusual Cleavage of Amide Bonds Peptides containing acylated N-methyl-aminoisobutyryl (NMeAib) residues showed unusual cleavage of amide bonds under acidic conditions. The cleavage takes place at the C-terminal side of the NMeAib residue (Figure 56) [147]. X-ray diffraction studies of the NMeAib containing molecules showed that the oxygen atom of the carbonyl group of the preceding residue is close to the carbonyl carbon of the NMeAib residue, thus acting as an internal nucleophile forming a tetrahedral intermediate. Once this tetrahedral intermediate was formed, lone pair electrons on the nitrogen of the phenylalanine were no longer be able to form resonating structures with the carbonyl group of NMeAib. In fact, the phenylalanine nitrogen becomes a proton acceptor like the amines, which resulted in the cleavage of the amide bond followed by the removal of phenylalanine and the

Moleculesformation2018 of, 23 an, 2615 oxazolinium ion intermediate, which further reacts with water to form a carboxylic36 of 43 acid product.

O O O O H H N protonated N N OCH3 N OCH3 O O H

O O O O OH N work up O NH O N OH PheOMe O oxazolinium ion N tetrahedral intermediate

Figure 56. N-MeN-Me Aib Aib mediated mediated amide amide bond bond cleavage. cleavage.

5.5. ConclusionsConclusions ThisThis mini-reviewmini-review highlights the the methods methods for for the the activation activation of of unactivated unactivated amide amide bonds bonds in in biomolecules.biomolecules. This This review review further further highlights highlights the the application application of ofthese these methods methods in the in thesequencing sequencing of ofproteins proteins and and the the synthesis synthesis of ofpeptide peptide acids, acids, thio thioesters,esters, alcohols, alcohols, and and amides. amides. These These studies studies showed showed thatthat therethere isis stillstill aa lotlot toto learnlearn from enzymes catalyzed catalyzed pathways pathways and and how how we we can can develop develop enzyme enzyme mimeticsmimetics for catalyzing the cleavage of of unactivated unactivated and and highly highly stable stable amide amide bonds bonds at at particular particular residues in a selective manner. We assume that these enzyme mimetics can have potential applications in various fields.

Author Contributions: M.R., S.M. and K.-C.T. collected the literature references. M.R., S.M. and K.-C.T. contributed to the manuscript writing. Funding: This research was supported by start up funds granted to M.R. by Auburn University. Conflicts of Interest: The authors declare no conflicts of interest.

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