molecules

Review
Review
Amide Bond
Amide Bond Activation
Activation of
of Biological
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;
Department of Chemistry
mzr0068@auburn.edu andkzt0026@tigermail.auburn.edu
(S.M.); Biochemistry, Auburn University, Auburn, AL 36849, USA;
(K.-C.T.)
mzs0154@auburn.edu (S.M.); kzt0026@tigermail.auburn.edu
* Correspondence: mzr0068@auburn.edu; Tel.: +1-334-844-6986 (K.-C.T.)
* Correspondence: mzr0068@auburn.edu; Tel.: +1-334-844-6986
Academic Editor: Michal Szostak
Academic
Received: Editor: Michal
7 September Szostak
2018; 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-dimensional
review article is to structures, which,
report the in turn, arefor
methodologies responsible for their
the activation of functions. The main
the unactivated focusbonds
amide of this
review article is to report the methodologies for the activation of the unactivated amide
present in biomolecules, which includes the enzymatic approach, metal complexes, and non-metal bonds present
in biomolecules,
based methods. which includes
This article alsothe enzymatic
discusses some approach, metal complexes,
of the applications and bond
of amide non-metal based
activation
methods.
approachesThis
in article also discusses
the sequencing some ofand
of proteins the the
applications
synthesisofofamide
peptide bond activation
acids, esters, approaches
amides, andin
the sequencing
thioesters. of proteins and the synthesis of peptide acids, esters, amides, and thioesters.

Keywords: peptide bond

Keywords: peptide bond cleavage; amide bond
cleavage; amide bond resonance;
resonance; twisted
twisted amides;
amides; enzymes;
enzymes; metal
metal
complexes;
complexes; catalysts
catalysts

1.1. Introduction
Introduction
The
The amide
amide bond
bond is
is one of the
one of the most
most abundant
abundant chemical
chemicalbonds
bondsandandwidely
widelyexists
existsininmany
manyorganic
organic
molecules
moleculesand andbiomolecules
biomolecules[1–6]. Nature
[1–6]. has used
Nature has amide
used bonds
amidetobonds
make these important
to make thesebiomolecules
important
because of the high stability of amide bonds towards various reaction conditions
biomolecules because of the high stability of amide bonds towards various reaction conditions (acidic and basic
(acidic
conditions), high temperature,
and basic conditions), and the presence
high temperature, and the of other chemicals
presence [7]. The high
of other chemicals [7]. Thestability of amide
high stability
bonds is attributed
of amide to its tendency
bonds is attributed to formto
to its tendency a form
resonating structure,
a resonating whichwhich
structure, provides a double
provides bond
a double
character to the amide CO-N bond (Figure 1) [8–10]. The resonance of these
bond character to the amide CO-N bond (Figure 1) [8–10]. The resonance of these amide bonds formsamide bonds forms
aa planar
planar structure andhinders
structure and hindersthe thefree
freerotation
rotation around
around thethe CO-N
CO-N bond,
bond, thus,thus,
it is it is responsible
responsible for 3Dfor
3D structures
structures adopted
adopted by by proteins
proteins andand other
other biomolecules.
biomolecules. These
These 3D3D structuresofofbiomolecules
structures biomoleculesareare
responsible
responsible for
for various
various important
important biological functions.
functions.

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

Classicalamide
Figure 1. Classical amidebond
bondresonance.
resonance.

Hansen
Hansen et et al. carried out
al. carried out the
the rate
rate studies
studies on
on the
the hydrolysis
hydrolysisofofamide
amidebondsbondsatatvarious
variouspH pH
conditions [11]. The study concluded that at pH 7, the rate of hydrolysis is due to the
conditions [11]. The study concluded that at pH 7, the rate of hydrolysis is due to the direct attack of direct attack
of water
water onon peptide
peptide and
and measured
measured asaskH kH 2 O.
2O. The
The rate
rate constant
constant showed
showed thatthe
that thehalf-life
half-lifeofofthe
theamide
amide
bonds
bonds is
is 267
267 years,
years, similar to the value determined
determined by byRadzicka
RadzickaandandWolfenden
Wolfenden[12]. [12].This
Thisstudy
studyalso
also
showed that the rates of acid (kH O + ) and base hydrolysis (kOH − ) are identical, therefore, the rate of
showed that the rates of acid (kH33 ) and base hydrolysis (kOH ) are identical, therefore, the rate of
+ −

the
thehydrolysis
hydrolysis ofof the
the peptide
peptide bond is dominated by kH kH22O
Othroughout
throughoutthe thepHpHrange
rangefrom
frompH pH5–9.
5–9.

Molecules 2018, 23, x; doi: www.mdpi.com/journal/molecules

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,various
variousmethods
methodshave havebeen
beenreported in the
reported in literature to activate
the literature the amide
to activate bonds towards
the amide bonds
atowards
variety aofvariety
nucleophiles or electrophiles for the synthesis of other organic compounds.
of nucleophiles or electrophiles for the synthesis of other organic compounds. This includes
This
the use of enzymes, metal complexes, and non-metal based methods [13–15].
includes the use of enzymes, metal complexes, and non-metal based methods [13–15]. One widely One widely reported
approach for the activation
reported approach of amideofbonds
for the activation amideinvolves the distortion
bonds involves of amide
the distortion bonds,bonds,
of amide thus, the
thus,amide
the
bond is no longer able to form a resonating structure, loses its double bond
amide bond is no longer able to form a resonating structure, loses its double bond character, andcharacter, and becomes
more susceptible
becomes to nucleophilic
more susceptible or electrophilic
to nucleophilic attack. A attack.
or electrophilic higher A distortion of the amide
higher distortion bond
of the from
amide
the
bond from the planar structure makes it more reactive, as evidenced by various twisted amide bondsin
planar structure makes it more reactive, as evidenced by various twisted amide bonds present
cyclic nonplanar
present in cyclic bridged
nonplanar lactams,
bridgedas demonstrated by Stoltz [16,17],
lactams, as demonstrated Kirby[16,17],
by Stoltz [18–20], and others
Kirby [18–20],[21–23]
and
(Figure 2). One (Figure
others [21–23] of the special
2). One cases to achieve
of the special maximum rotational
cases to achieve inversion
maximum of the amide
rotational bondofsothe
inversion that
itamide
remains in the twisted conformation is the use of N-acyl-glutarimides [24–29]
bond so that it remains in the twisted conformation is the use of N-acyl-glutarimides [24–29]and N,N-substituted
amide bonds [30,31] (Figure
and N,N-substituted 2). It is[30,31]
amide bonds this strong distortion
(Figure of amide
2). It is this strongbonds that provides
distortion of amideamide
bondsbonds
that
with a high
provides reactivity
amide bondstoward
with a ahighvariety of nucleophiles
reactivity and electrophiles.
toward a variety 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' O
[M]n n+2 N R-Y
R'
R N or N R [M]
R'' R 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)

Figure
Figure 2.
2. Twisted
Twisted amides
amidesfor
foractivation
activationof
ofamide
amidebonds.
bonds.

There
There are already some excellent
excellent review
review articles
articles in
in the
the literature
literaturecovering
coveringthe
thereactivity
reactivityofof
twisted/activated amidebonds
twisted/activated amide bondsfor
forthe
thesynthesis
synthesisofofthe
thevariety
varietyofofdifferent
differentorganic
organicmolecules
moleculessuch
suchasas
ketones, esters,acids,
ketones, esters, acids,and
and alcohols,
alcohols, by cross-coupling
by cross-coupling reactions
reactions [24–31].
[24–31]. The mainThefocus
mainoffocus of this
this review
review is to summarize
is to summarize the for
the methods methods for the ofactivation
the activation of less
less reactive amide reactive amide bonds
bonds present present in
in biomolecules
biomolecules suchproteins,
such as peptides, as peptides, proteins, glycopeptides,
glycopeptides, nucleotides in DNAnucleotides
and RNA in DNA and RNA
and various andpeptide
other various
bioconjugates,
other toward attack toward
peptide bioconjugates, by various
attacknucleophiles. This task wasThis
by various nucleophiles. accomplished by various
task was accomplished
methods
by varioussuch as by using
methods such biological
as by usingmolecules,
biologicalmetal complexes,
molecules, metal and non-metaland
complexes, based methodsbased
non-metal and
is discussed
methods andbelow.
is discussed below.

2.
2. Biomolecules Activation of
Biomolecules for the Activation of Amide
AmideBonds—The
Bonds—TheEnzyme-Directed
Enzyme-DirectedHydrolysis
Hydrolysisof
of Amides
Amides
We
We have
have summarized different kinds
summarized different kinds of
of enzymes,
enzymes,their
theirmechanisms
mechanismsofofhydrolysis
hydrolysisofofunactivated
unactivated
peptide
peptide bonds,
bonds, and
and the
the point of cleavages
point of cleavages in
in Table
Table 1.
1.
Molecules 2018, 23, 2615 3 of 43

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 (Figure 3). 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 H 2N
O
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

General pathway
Figure 3. General pathwayof
ofserine
serineproteases
proteasesdirected
directedamide
amidebond
bondhydrolysis.
hydrolysis.

2.2.
2.2. Cysteine
Cysteine Proteases
Proteases
Cysteine
Cysteine proteases
proteases(CPs) (CPs)hydrolyze
hydrolyzethe thepeptide
peptidebonds
bondswith
withmaximum
maximum efficiency at pH
efficiency at pH4–6.5 [14].
4–6.5
The
[14]. The thiol group of cysteine protease is susceptible to oxidation so the environment of the enzymeis
thiol group of cysteine protease is susceptible to oxidation so the environment of the enzyme
reducing
is reducing in nature.
in nature. TillTill
now now21 21
families
families of of
CPs have
CPs been
have discovered
been discovered[34–37].
[34–37].CPs form
CPs forma triad at at
a triad the
active site during the hydrolysis of the peptide bond made up of Cys-His-Asn
the active site during the hydrolysis of the peptide bond made up of Cys-His-Asn residues. First, Asnresidues. First, Asn
forms
forms thethe hydrogen
hydrogen bondbond withwith His,
His, then
then His
His abstracts
abstractsthe
theproton
protonfrom
fromCys
Cystotogenerate
generatea anucleophilic
nucleophilic
thiolate −
thiolate ion
ion (S
(S−))similar
similarto toenolate
enolateion
iongenerated
generatedby byserine
serineproteases
proteases(Figure
(Figure3).
3).Next,
Next,the
thethiolate
thiolateion
ion
(S − ) attacks the carbonyl group of the peptide resulting in the formation of a tetrahedral intermediate
(S ) attacks the carbonyl group of the peptide resulting in the formation of a tetrahedral intermediate
−

TS
TS followed
followed by by the
the hydrolysis of the amide bond bond [34–37].
[34–37].

2.3.
2.3. Metalloproteases
Metalloproteases
Metalloproteases
Metalloproteases are are members
membersofofa aclass
class
of of proteases
proteases thatthat require
require a metal
a metal ion cofactor
ion cofactor at theat
the active
active sitethe
site for forhydrolysis
the hydrolysis of peptide
of peptide bonds
bonds [38]. The[38].
mostThe
commonmostmetal
common metal ion
ion cofactor cofactor
present in
present in metalloproteases is the zinc ion (Zn 2+ ) [39]. Other transition metals such as Co2+ and
metalloproteases is the zinc ion (Zn ) [39]. Other transition metals such as Co and Mn are capable
2+ 2+ 2+

Mn 2+ are capable of restoring the functions in zinc-metalloproteases where Zn2+removed

of restoring the functions in zinc-metalloproteases where the Zn2+ core hasthe
been core has[39].
been
removed [39]. Metalloproteases
Metalloproteases are dividedareinto divided
twointomajor
two major families:
families: metalloendopeptidasesand
metalloendopeptidases and
metalloexopeptidases. The names of these families are based on the site of the hydrolysis
metalloexopeptidases. The names of these families are based on the site of the hydrolysis of the of the peptide
bonds
peptide[40,41].
bondsMetalloendopeptidases cleave the internal
[40,41]. Metalloendopeptidases cleaveamide
the bonds whereas
internal amidemetalloexopeptidases
bonds whereas
cleave the amide bondscleave
metalloexopeptidases present
theat the C-bonds
amide or N-terminus
present at ofthepeptides.
C- or N-terminus of peptides.

2.3.1.
2.3.1. Metalloendopeptidase: Thermolysin
Metalloendopeptidase: Thermolysin
Thermolysin
Thermolysin (TLN)(TLN) catalyzes the cleavage
catalyzes the cleavage ofof the
theinternal
internalpeptide
peptidebond
bondatatthetheamino-side
amino-sideofoflarge
large
hydrophobic amino acids, such as leucine, isoleucine, or phenylalanine.
hydrophobic amino acids, such as leucine, isoleucine, or phenylalanine. TLN and TLN-like proteinsTLN and TLN-like proteins
require 2+ as a metal ion cofactor for the cleavage of amide bonds [42–47].
require ZnZn2+ as a metal ion cofactor for the cleavage of amide bonds [42–47].
TLN-mediated
TLN-mediated hydrolysis
hydrolysis of the peptide bond bond is isaatwo-step
two-stepprocess
process(Figure
(Figure4)4)[48–51].
[48–51].TheTheactive
active
site
site ofofTLN
TLNcontains
containsthree residues—His142,
three residues—His142, His146,
His146, Glu166—and
Glu166—and a water molecule,
a water whichwhich
molecule, are bound
are
to the Zn 2+ ion. 2+First, the carbonyl group of the peptide coordinates with Zn2+ and displaces the
bound to the Zn ion. First, the carbonyl group of the peptide coordinates with Zn and displaces 2+

hydrogen
the hydrogen of a water molecule
of a water to form
molecule to an H-bond
form with Glu143
an H-bond and theand
with Glu143 oxygen of the water
the oxygen of themolecule
water
remains
moleculeassociated to the Zn2+toion,
remains associated theresulting
Zn2+ ion,inresulting
the formationin theofformation
the enzyme-substrate complex (ES).
of the enzyme-substrate
Second,
complexthe oxygen
(ES). Second,of the
thewater
oxygenattached
of the to Zn2+attached
water attacks the carbonyl
to Zn carbon
2+ attacks of the peptide,
the carbonyl carbonresulting
of the
in the formation
peptide, resultingof intransition stateof1transition
the formation (TS1). TS1 is stabilized
state 1 (TS1). TS1 byisthe formation
stabilized by theof formation
the H-bond of with
the
H-bondand
Asp226 withHis231
Asp226 atand
the His231
carbonyl at oxygen
the carbonyl
of theoxygen
peptide offollowed
the peptideby followed
the formationby theofformation of
intermediate
intermediate(INT)
gem-diolate gem-diolate (INT) by of
by the breakage thehydroxyl
breakageOH of bond
hydroxyl OH bond
of water. of water.
The amide Thepeptide
of the amide forms
of the a
peptide forms
hydrogen bondawith
hydrogen
the H of bond
H2 O.with
Third, thethe
H of H2O. Third,
carbonyl bond the carbonyl bond
rearrangement rearrangement
in TS2 in TS2
leads to the breakage
leads
of the to the breakage
amide bond (CONH) of the amide bond (CONH)
of the peptide of the the
and releases peptide and releases
N-terminal the The
peptide. N-terminal peptide.
rate-determining
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
(Figure
Molecules4).
2018, 23, x 5 of 43

Glu143 Glu143
O O
O H O H
H N H N
H + H N H
H N H O
H O O N
O N
-
Asp226 His142 Zn Asp226
His142 Zn
Glu166 O Glu166
O
His146 His231 His146 His231
ES O TS1 O

Glu143
O
O H H
H N
NH2
H N H
FA N OH + H O O N
N H O Asp226
O His142 Zn
- H Glu166 O
His231
Zwitterionic tetrahedral intermediate His146
O
INT
Glu143 H Glu143
H
O N O
O H H
O
N
H N H
H O O
H N H H O O N
N Asp226
Asp226 His142 Zn
His142 Zn Glu166 O
Glu166 O His231
His146 His231 His146
O O
Product TS2

ThermolysinMechanistic
4. Thermolysin
Figure 4. Mechanisticpathway.
pathway.

2.3.2.
2.3.2. Metalloexopeptidase: Carboxypeptidase A
Metalloexopeptidase: Carboxypeptidase A
Carboxypeptidase
Carboxypeptidase A A (CPA)
(CPA) isis aa 35
35 kDa
kDa metalloenzyme
metalloenzyme and and contains Zn2+2+ion
contains aa Zn ioncofactor
cofactorininitsits
active site [52–54]. CPA is an exopeptidase, which catalyzes the hydrolysis of amide
active site [52–54]. CPA is an exopeptidase, which catalyzes the hydrolysis of amide bonds present bonds present
at
at the C-terminus comprising large hydrophobic side chains. Two different
the C-terminus comprising large hydrophobic side chains. Two different mechanisms have been mechanisms have been
proposed
proposedfor forthe
thecleavage
cleavage byby
these metalloproteases
these metalloproteases (Figure 5) which
(Figure showed
5) which the importance
showed of Lewis
the importance of
acid
Lewiscatalysis for the for
acid catalysis activation of amide
the activation bondsbonds
of amide [53,54]. One involves
[53,54]. the Lewis-acid
One involves activation
the Lewis-acid of the
activation
carbonyl group of
of the carbonyl the amide
group of the bond
amideby Zn2+by
bond , followed by the by
Zn2+, followed attack of water
the attack of (Figure 5). The5).
water (Figure second
The
involves the Lewis-acid activation of H O by Zn 2+ ions followed by the attack of the hydroxide ion of
second involves the Lewis-acid activation 2 of H2O by Zn ions followed by the attack of the hydroxide
2+

the
ionwater
of theon the on
water carbonyl group group
the carbonyl of the amide bond (Figure
of the amide 5).
bond (Figure 5).

Zn Zn

O
H
O O
NH
H
NH

Glu O Glu O

O O

Figure 5. Mechanisms of carboxypeptidase A.

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
second involves
Molecules the Lewis-acid activation of H2O by Zn2+ ions followed by the attack of the hydroxide
2018, 23, 2615 6 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.

Figure Mechanismsof
5. Mechanisms ofcarboxypeptidase
carboxypeptidaseA.
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
in
cellhost cell factor-1
factor-1 (HCF-1).(HCF-1). HCF-1takes
HCF-1 cleavage cleavage
place takes
at the place at theglutamic
N-terminal N-terminalacid glutamic acid by the
by the glycosylation
glycosylation which is catalyzed by enzyme OGT. Mechanistic studies
which is catalyzed by enzyme OGT. Mechanistic studies showed that the glycosylation of the showed that the glycosylation
of the glutamate
glutamate side(intermediate
side chain chain (intermediate
1, Figure1, 6)
Figure
leads 6)to leads to the formation
the formation of an enzyme-catalyzed
of an enzyme-catalyzed internal
internal
pyroglutamate formation (intermediate 2, Figure 6) with the amidic nitrogen ofnitrogen
pyroglutamate formation (intermediate 2, Figure 6) with the amidic the peptideof the peptide
backbone
backbone chain, which then undergoes spontaneous hydrolysis (Figure 6)
chain, which then undergoes spontaneous hydrolysis (Figure 6) [55]. Detailed mechanistic studies[55]. Detailed mechanistic
studies
showedshowed
that thethat theofrate
rate of conversion
conversion of glycopeptide
of glycopeptide to internal
to internal pyroglutamate
pyroglutamate was was
anan orderofof
order
magnitude slower than observed in the presence of OGT, thus, it was concluded
magnitude slower than observed in the presence of OGT, thus, it was concluded that both the first that both the first and
second steps occurred while the peptide is bound to OGT (Figure 6). Hydrolysis
and second steps occurred while the peptide is bound to OGT (Figure 6). Hydrolysis likely occurs likely occurs after
dissociation from the
after dissociation fromenzyme. It has also
the enzyme. been
It has alsoreported that glycosylation
been reported on Thr next
that glycosylation to glutamate
on Thr next to
also prevents the cleavage at the glutamate (Glu) because of the steric hindrance
glutamate also prevents the cleavage at the glutamate (Glu) because of the steric hindrance and and thus the enzyme
thus
isthe
unable
enzymeto carry out the
is unable glycosylation
to carry of glutamateof[56].
out the glycosylation glutamate [56].

OH OH H
O O N O O N O
O HN O HN H
NH SH NH SH
OH
O O O N
O
O O HN
O O O
HO N SH
NH HO
HO O O
HO HO
N O NHAc
AcHN HO
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

Figure 6.
Figure Glycosylationpathway.
6. Glycosylation pathway.

2.3.4.
2.3.4. Nicotinamidase
Nicotinamidase (Pnc1) for the
(Pnc1) for the Hydrolysis
Hydrolysis of
of the
the Amide
AmideBond
Bondof
ofNicotinamide
Nicotinamide
Nicotinamidases catalyze the
Nicotinamidases catalyze the cleavage
cleavage ofof nicotinamide,
nicotinamide,which
whichisisaacritically
criticallyimportant
importantpart
partofof
NAD + and NADH, to nicotinic acid and ammonia (Figure 7). A detailed study showed that both the
NAD and NADH, to nicotinic acid and ammonia (Figure 7). A detailed study showed that both the
+

carbonyl
carbonyl oxygen
oxygen and
and the
the ring nitrogen of
ring nitrogen of nicotinamide
nicotinamidearearecritical
criticalfor
forbinding
bindingtotothe
thenicotinamidases
nicotinamidases
and
and reactivity
reactivity [57–60].
[57–60].
Molecules 2018, 23, 2615 7 of 43
Molecules 2018, 23, x 7 of 43

O O

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

C167
-NH3

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

Mechanistic pathway
Figure 7. Mechanistic pathwayof
ofPnc1
Pnc1for
forhydrolysis
hydrolysisofofnicotinamide.
nicotinamide.

Three
Threeresidues—Asp51,
residues—Asp51,His53, His53,and
andHis94—in
His94—in nicotinamidase
nicotinamidase (Pnc1)
(Pnc1)directly coordinate
directly coordinate Zn2+
withwith
at
Znthe active
2+ at site and
the active three
site andother
threeresidues act as aact
other residues catalytic triad (Cys167,
as a catalytic Asp8, and
triad (Cys167, Lys122)
Asp8, and (Figure
Lys122)7).
In the first 2+
(Figure 7). step,
In thethe substrate
first step, thebinds to the
substrate Zn toby
binds nitrogen
the Zn by of
2+ pyridine
nitrogen of ring and ring
pyridine displaces the water
and displaces
molecules
the water ligated
moleculesto the Zn2+ .to
ligated Next,
the Asp8
Zn2+. removes
Next, Asp8 the removes
proton from the Cys167,
proton fromforming a thiolate,
Cys167, formingwhich,
a
in turn, react with the amide carbonyl carbon of nicotinamide, leading to the
thiolate, which, in turn, react with the amide carbonyl carbon of nicotinamide, leading to the formation of a tetrahedral
intermediate
formation of (INT). The tetrahedral
a tetrahedral intermediate
intermediate (INT). Thecollapsed,
tetrahedral resulting in thecollapsed,
intermediate breakage resulting
of the amide
in
bond and release
the breakage of the
of the amideammonia.
bond andThis is followed
release of the by the release
ammonia. Thisofisthe nicotinic
followed byacid
the from
releasetheofactive
the
nicotinic
site of theacid
enzymefromby theacid-base
active site of the enzyme by acid-base catalysis.
catalysis.

2.3.5.
2.3.5. Flavoenzyme-Mediated Hydrolysis of
Flavoenzyme-Mediated Hydrolysis of the
the Amide
Amide Bond
Bond
Begley
Begley etetal.
al.demonstrated
demonstratedthe therole ofof
role flavoenzyme
flavoenzyme in the cleavage
in the cleavageof the unactivated
of the amide
unactivated bond
amide
in uracil,
bond a building
in uracil, blockblock
a building for RNA (Figure
for RNA 8) [61–63].
(Figure The The
8) [61–63]. detailed mechanistic
detailed analysis
mechanistic showed
analysis showedthat
the
thatreaction takestakes
the reaction placeplace
through the oxidative
through mechanism
the oxidative that is
mechanism initiated
that by the
is initiated byaddition of a flavin
the addition of a
hydroperoxide to the C*
flavin hydroperoxide tocarbonyl of uracil,offorming
the C* carbonyl a tetrahedral
uracil, forming intermediate
a tetrahedral (INT) (Figure
intermediate (INT)8). This is
(Figure
followed
8). This isby the collapsing
followed of the tetrahedral
by the collapsing intermediate
of the tetrahedral (INT), leading
intermediate (INT),toleading
the cleavage of an amide
to the cleavage of
an amide
bond bondThis
in uracil. in uracil.
was theThis
firstwas the first
example where example where such
such chemistry waschemistry
shown bywas shown
flavin by flavin
hydroperoxides.
hydroperoxides.
Molecules 2018, 23, 2615 8 of 43
Molecules 2018,
Molecules 2018, 23,
23, xx 88 of
of 43
43

O
O O
O
HN
HN RutA +
RutA RutF(or Fre)
+ RutF(or Fre) HO
HO
O22 +
O + NADH
NADH +
+ FMN
FMN
O
O N
N HN
HN
H
H
H22N
H N O
O
Mechanism
Mechanism

O
O O
O
Flv O
O O
O O O
O O
O O
O
Flv Flv
Flv O
O
O
HN
HN * HN
HN * Flv
Flv
O
O HO
HO
O
O N
N O
O N
N HN
HN HN
HN
H
H H
H
INT
INT H22N
N O H22N
N O
H O H O

Figure 8.
Figure 8. Flavoenzyme-mediated
Flavoenzyme-mediated hydrolysis
Flavoenzyme-mediatedhydrolysis of
hydrolysisof amide
ofamide bond.
amidebond.
bond.

2.3.6.
2.3.6. Primary
2.3.6. Primary Amide
Primary Bond Hydrolysis
Amide Bond Hydrolysis by
by Antibodies
Antibodies
Antibodies
The
The antibody
The antibody Fab-BL125
antibody catalyzes the
Fab-BL125 catalyzes the hydrolysis
the hydrolysisof
hydrolysis ofthe
of theunactivated
the unactivatedprimary
unactivated primaryamide
primary amidebond
amide bondof
bond ofofthe
the
the
L-isomer
LL -isomer of
-isomer of peptides
of to generate
peptides to
peptides generate free
free peptide
free peptide acid
peptide acid(Figure
acid (Figure9).
(Figure 9).The
9). Theantibody
The antibodyshowed
antibody showedhigh
showed highregio-
high regio-and
regio- andand
diastereoselectivity
diastereoselectivity since
sincethe
the
D -proline
D primary
-proline amide
primary diastereoisomer
amide did
diastereoisomer not
diastereoselectivity since the D-proline primary amide diastereoisomer did not undergo any undergo
did not any hydrolysis.
undergo any
The antibody
hydrolysis.
hydrolysis. The
TheFab-BL125
antibody decreases
antibody Fab-BL125the
Fab-BL125 half-lifethe
decreases
decreases of half-life
the the peptide
half-life from
of the
of the 17.5 years
peptide
peptide to only
from 17.5
from 17.5 3.9to
years
years toh.only
Such
only 3.9an
3.9
antibody
h. Such
h. Such an
anwas obtained
antibody
antibody wasbyobtained
was using theby
obtained α-amino
by boronic
using the
using the acidboronic
α-amino
α-amino Haptenacid
boronic by aHapten
acid direct selection
Hapten strategy
by aa direct
by direct from
selection
selection
the antibody
strategy from combinatorial
the antibody libraries [64,65].
combinatorial libraries
strategy from the antibody combinatorial libraries [64,65]. [64,65].

O
O O
O
H
H H
H
N
N N +H22O
+H O N
N N
N N N N
N N
H
H H
H
O
O O
O O
O O
O
AcHN
AcHN O NH22
NH AcHN
AcHN O NH2
NH
O O OH 2
OH

-NH33
-NH
O
O
H
H
N
N N
N O
R
R N
N H O
H H
H B(OH)22 N
N N
N
O
O B(OH) N
N
H
H
O
O O
O
-amino boronic
-amino boronic AcHN
AcHN O OH
OH
O
O O
acid hapten
acid hapten
R=
R = HO C
HO22C

R=
R = Boc
Boc

Figure 9.
Figure Antibody
9. Antibody Fab
Antibody Fab catalyzed
Fabcatalyzed primary
catalyzedprimary amide
primaryamide bond
amidebond hydrolysis.
bondhydrolysis.
hydrolysis.

2.3.7.
2.3.7. RNA-Assisted Cleavage
RNA-Assisted Cleavage
Cleavage ofof Amide
of Amide Bonds
Bonds
2.3.7. RNA-Assisted Amide Bonds
AA group
group III RNA obtained
RNA obtained
obtained byby in
by in vitro
vitro evolution
evolutioncatalyzes
catalyzesthe
thecleavage
cleavageofofunactivated
unactivatedalkylalkylamides
amides
A group RNA in vitro evolution catalyzes the cleavage of unactivated alkyl amides
of
ofDNA
DNA analog.
analog. This includes
This includes substrates with
includes substrates
substrates withananamide
amidebond
bondthat
thatjoins
joinseither
eithertwotwoDNAs,
DNAs,orora aDNADNA
of DNA analog. This with an amide bond that joins either two DNAs, or a DNA
3 in comparison
with a short
with aa short peptide.
short peptide.
peptide. TheThe
The RNARNA increases
RNA increases
increases the the
the rate rate
rate of of hydrolysis
of hydrolysis
hydrolysis by by
by more more
more than
than 10than
103 in
3 10
in comparison
comparison to to the
the
with
to the uncatalyzed
uncatalyzed reaction.
reaction. The The RNA-catalyzed
RNA-catalyzed amide amide
bond bond cleavage
cleavage was was entirely
entirely dependent
dependent on Mg on
2+
uncatalyzed
2+
reaction.
2+
The RNA-catalyzed amide bond cleavage was entirely dependent on Mg2+
Mg
wherewhereMg2+ Mg asacts
2+ acts as a Lewis acid activating
thus activating the carbonyl
groupgroup theofamide
the amide
bond bond for
where Mg acts as aa Lewis
Lewis acid thus
acid thus activating the the carbonyl
carbonyl group of the
of amide bond for the
for the
Molecules 2018, 23, 2615 9 of 43
Molecules 2018, 23, x 9 of 43

the nucleophilic
nucleophilic attack
attack byhydroxy
by the the hydroxy
groupgroup
of RNA of(Figure
RNA (Figure
10). No 10).
amide No amide
bond bond was
cleavage cleavage was
detected
detected in the presence of other metal ions such as Zn 2+ , Ca 2+ , or Sr2+ . A trace amount of amide bond
in the presence of other metal ions such as Zn , Ca , or Sr . A trace amount of amide bond cleavage
2+ 2+ 2+

cleavage was observed

was observed in the presence
in the presence of MnCl2 of MnCl
which is2inwhich
contrastis intocontrast
the RNAtoandthe DNA
RNA cleavage
and DNAreactions
cleavage
reactions
[66]. [66].

DNA1 DNA1
5' O 5' O 5' O
T T
IGS T
HN H2N H2N
O HO2C
T Mg2+ Mg2+ H 2O O
O
H T
DNA2 O O DNA2
Mg2+ 3' 3'
OH H O
HO T
O
Ribozyme G OH
OH HO
O 3'
G
O O
G

Figure 10. RNA

RNA catalyzed
catalyzedamide
amidebond
bondcleavage.
cleavage.

To
To determine
determine thethe generality amide bond
generality of the amide bond cleavage
cleavage by
byRNA,
RNA,aaseries
seriesofofsubstrates
substrateswere
were
explored.
explored. IfIfaashort
shortDNA
DNAis isattached
attachedto to a peptide
a peptide or amino
or an an amino
acidacid by using
by using an amide
an amide bond,bond,
the
the immediate
immediate cleavage
cleavage at the
at the amide
amide bondbond
waswas observed
observed inpresence
in the the presence ofRNA.
of the the RNA. The cleavage
The cleavage of theof
the amide
amide bondbond between
between the the amino
amino acidacid residues
residues waswas not observed.
not observed.

3.3. Metal
Metal Complexes
Complexes for
for the Activation of
the Activation of Amide
Amide Bonds
Bonds
We
Wehave
havesummarized
summarized different kinds of metal-based
metal-basedcomplexes,
complexes,their
theirmechanisms
mechanismsofofhydrolysis
hydrolysis
of
ofunactivated
unactivated peptide
peptide bonds and point of cleavages
cleavages in
in Table
Table 2.
2.

catalyzed hydrolysis
Table 2. Metal catalyzed hydrolysisof
ofpeptide
peptidebonds.
bonds.

Entry MetalComplex
Metal Method of Hydrolysis Point of Cleavage Ref.
Entry Method of Hydrolysis Point of Cleavage Ref.
1 Complex
Simple metal ions Lewis acidity of metal ion C-terminal of peptide [67–72]
SimpleZrmetal
POMs,
12 Lewis acidity
Lewis ofof
acidity metal
metalion
ion C-terminal
C-terminal of
of peptide
peptide [67–72]
[73–82]
ions
Zr MOF-808
Zr POMs, Zr LewisLewis
acidityacidity
of metal
23 Mo(VI) ofion andion
metal formation of C-terminal of peptide
C-terminal side of Asp [73–82]
[81,82]
MOF-808 5 membered ring
Lewis acidity of metal ion and formation of 5
34 Co(III)
Mo(VI) N-terminal amine intiated tertiary complex C-terminalside
C-terminal of peptide
of Asp [83–88]
[81,82]
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]
56 Mo(II) Carboxylic
Favorablegroup of amino acid
six-membered and side
chelate chain of
ring C-terminal side side
C-terminal of Met, His, Cys
of Cys [89]
Pd(II), Pt(II) [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) amino acid
Methionine anchoring
side metal complex
chain anchoring 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 = Leu 89
= Solvent − Ala portion
exposed 90
Co(III) and Lewis acidity activation and PNA for selectivity PDF = Gln 152 − Arg 153
89 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-terminal side of Ser/Thr [114]
portion
9 Ni(II) Non Lewis acid based N,O acyl rearrangement N-terminal side of Ser/Thr [112,113]
10 Sc(III) Lewis acid based N,O acyl rearrangement N-terminal side of Ser/Thr [114]
Another approach for the cleavage of peptide bonds involves the use of metal complexes.
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 metalmetal ion hydrolysis
ion hydrolysis mechanism
mechanism involves
involves the formation
the formation of a square
of a square planarplanar complex
complex of theof
the
metal ions Cu(II), Ni(II), or Pd(II) with the Ser/Thr-His or Ser/Thr-Xaa-His sequence leading to theto
metal ions Cu(II), Ni(II), or Pd(II) with the Ser/Thr-His or Ser/Thr-Xaa-His sequence leading
the
NON→rearrangement
O rearrangement of the
of the acylacyl moiety
moiety resulting
resulting in cleavage
in the the cleavage of peptide
of the the peptide
bondbond (Figure
(Figure 11).
11). In
In this section, we will provide few examples of both Lewis acid and the N → O acyl rearrangement
this section, we will provide few examples of both Lewis acid and the NO acyl rearrangement for for
the cleavage of peptide
the cleavage of peptide bonds.

M
O O HO
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

Metal-assistedpeptide
Figure 11. Metal-assisted peptidebond
bondhydrolysis.
hydrolysis.

3.1.
3.1. Lewis
Lewis Acid
Acid Mediated
Mediated Hydrolysis
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
the
thepeptide
peptide bond.
bond. Cleavage of the
Cleavage of the peptide
peptide bond
bondin
inthe
thepresence
presenceof
ofthe
themetal
metaltakes
takesplace
placethrough
throughthe
the
mechanism
mechanism in in Figure
Figure 11b.
11b.
3.1.2. Oxo Metal Ions
3.1.2. Oxo Metal Ions
Parac-Vogt et al. proved that polyoxometalates oxo-metal compounds such as MoO2 −−44 , WO2−4−4 ,
Parac-Vogt et al. proved that polyoxometalates oxo-metal compounds such as MoO2 , WO2 ,
CrO2 − 4 , and VO −4 cleave the peptide bonds in various dipeptides [73–77]. They have shown
CrO2−4, and VO2−4 2cleave 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
leading to the change
center leading to the in the coordination
change properties
in the coordination of POM. POMs
properties were
of POM. POMsutilized
wereforutilized
the Zr(IV)- and
for the
Ce(IV)-assisted peptide bond hydrolysis because they are homogenous in nature [78,79].
Zr(IV)- and Ce(IV)-assisted peptide bond hydrolysis because they are homogenous in nature [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 K15 H[Zr(α2 -P2 W17 O61 )2 ]·25H2 O 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
detailedmechanistic
mechanisticinvestigation
investigationbybyNMR NMRshowed
showedthat
thatthe
thefree
freeamino
aminoterminus
terminusand andboth
bothcarbonyl
carbonyl
functionalities
functionalities ofof GGGGinteract
interactwithwithpolyoxometalates
polyoxometalatesKK 1515H[Zr(α
H[Zr(α22-P W1717OO6161
-P22W 2 ]·25H
)2)]·25H O either by the
2O2 either by the
formation of metal ion coordination complex or by the non-covalent
formation of metal ion coordination complex or by the non-covalent interactions of the interactions of theprotonated
protonated
amino
aminogroup
groupwith
withthe thenegatively
negatively charged
charged surface of POMs,
surface of POMs, thusthus
responsible
responsiblefor the foractivation of amide
the activation of
bonds towards hydrolysis. These POMs selectively cleave the C-terminal
amide bonds towards hydrolysis. These POMs selectively cleave the C-terminal amide bond of amide bond of glycylglycyl
amide (GGNH
glycylglycyl 2 ), resulting
amide (GGNH in2),the formation
resulting of GG.
in the No freeofglycine
formation GG. No amide
free (GNH
glycine 2 ) amide
was detected
(GNH2)during
was
the courseduring
detected of the the
reaction.
course of the reaction.
Molecules 2018, 23, 2615 11 of 43
Molecules 2018, 23, x 11 of 43

NH
HN

X
cGG

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

Peptide hydrolysis
Figure 12. Peptide hydrolysiscatalyzed
catalyzedby
byaapolyoxometalate
polyoxometalatecomplex.
complex.

Recently,
Recently, VogtVogt et et al.
al. utilized the MOF-808,
utilized the MOF-808, aa Zr(IV)-based metal−organiccomplex
Zr(IV)-based metal−organic complexfor forthe
the
hydrolysis of the peptide bond in a wide range of peptides and proteins
hydrolysis of the peptide bond in a wide range of peptides and proteins such as hen egg white such as hen egg white
lysozyme
lysozyme(HEWL)(HEWL)underunderphysiological
physiologicalconditions
conditions[81,82].
[81,82].The
TheMOF-88
MOF-88 is is
heterogeneous
heterogeneous in nature
in natureand
isand
thusis athus
reusable catalyst.
a reusable The experimental
catalyst. studies and
The experimental calculations
studies showed that
and calculations MOF-808
showed that hydrolyzed
MOF-808
the Gly-Gly bond
hydrolyzed by the bond
the Gly-Gly formation of formation
by the the active of
complexes
the activewith two adjacent
complexes Zr(IV)
with two centers
adjacent of the
Zr(IV)
{Zr 6 O8 } core
centers by{Zr
of the coordination
6O8} core by with amide oxygen
coordination withand the amine
amide oxygennitrogen
and theatoms.
amine The catalytic
nitrogen efficiency
atoms. The
of MOF-808
catalytic towards
efficiency of the hydrolysis
MOF-808 of peptides
towards is dependent
the hydrolysis on the
of peptides bulkiness and
is dependent nature
on the of theand
bulkiness side
natureamino
chain of theacid
side residues.
chain amino acid residues.
Dipeptides Dipeptides
with small with small
or hydrophilic or hydrophilic
residues undergoresidues
cleavageundergo
faster as
cleavage faster
compared as compared
to those with bulky toand
those with bulky and
hydrophobic hydrophobic residues.
residues.

Asp-Xaa
Asp-Xaa Selective
Selective Hydrolysis
Hydrolysis of Bond by
of the Peptide Bond by Oxo-Metal
Oxo-MetalIons
Ions
ItIt has
has been reported that oxomolybdate(VI)
oxomolybdate(VI)catalyzes
catalyzesthe
thecleavage
cleavageofofvarious
variouspeptides
peptidescontaining
containing
aspartic
aspartic acid
acid (Asp)
(Asp) with cleavage
cleavage at at the
the C-terminal
C-terminalside
sideof
ofthe
theAsp
Aspresidue.
residue.This
Thisisisdue
duetotothe
theattack
attack
of the side chain of the
of the side chain of the Asp Asp on the amidic carbonyl which is activated by the coordination
amidic carbonyl which is activated by the coordination with with
oxomolybdate(VI),
oxomolybdate(VI), resulting
resultingininthe
theformation
formationofofthe
thefive-membered
five-memberedringringand
andsimultaneous
simultaneouscleavage
cleavageof
of the
the amideamide bonds
bonds (Figure
(Figure 13)13) [81,82].
[81,82].

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

Figure 13. Oxomolybdate(VI)

Oxomolybdate(VI)catalyzed
catalyzedcleavage
cleavageofofpeptide
peptidebonds.
bonds.

Various
VariousOther
Other Metal
Metal Complexes
Complexes
The
The Westheimer
Westheimer and and Trapmann
Trapmann groups groups showed
showed that that the
the metal
metalcomplexes
complexescontaining
containingCo(II),
Co(II),
Cu(II),
Cu(II), and
and Ni(II)
Ni(II) ions
ions cleave various dipeptides
cleave various dipeptides[83,84].
[83,84].Out
Outofofvarious
variousmetal
metalcomplexes,
complexes,the theCo(III)
Co(III)
complex
complex [Co(trien)OH(H
[Co(trien)OH(H22O)] O)]2+2+isisone
oneofofthe
thewidely
widelystudied
studiedmetal
metalion
ioncomplexes
complexesand
andcarried
carriedout
outthethe
rapid hydrolysis of the peptide bond [85–88]. The detailed mechanistic investigation
rapid hydrolysis of the peptide bond [85–88]. The detailed mechanistic investigation showed that showed that first,
the metal 2+
first, the complex [Co(trien)OH(H
metal complex 2 O)] forms
[Co(trien)OH(H 2O)]2+ aforms
tertiarya complex with a dipeptide
tertiary complex by the replacement
with a dipeptide by the
of an equatorially
replacement of an coordinated water molecule
equatorially coordinated water in molecule
the octahedral
in the Co(III) complex
octahedral Co(III)bycomplex
the N-terminal
by the
amine
N-terminal amine of the peptide. This mode of coordination brings the axially bound hydroxyl in
of the peptide. This mode of coordination brings the axially bound hydroxyl group close
group
in close proximity
proximity to the peptide
to the peptide carbonylcarbonyl
resultingresulting in the hydrolysis
in the hydrolysis of the peptide
of the peptide bond (Figure
bond (Figure 14). 14).
Molecules 2018, 23, 2615 12 of 43
Molecules
Molecules 2018,
2018, 23,
23, xx 12
12 of
of 43
43

NH
NH22
H
H
HN
HN N
N
NH Co
Co X
X11 X
NH22 X22
HN OH HN
HN NH
NH22
HN OH22/OH
/OH R
R11 NH
NH22
O
O N
N
Co
Co OH
OH H
H H
H
O
O HN
HN N
N
HN
HN NH
NH22
Co
Co
OH HN NH X
X11
OH22/OH
/OH HN NH22
NH
NH22 H
+ H
X
X11 O
O HN
HN N
N O
O
H
H
N
N Co
Co O
O
H
H22N
N R
R11 X
X11 X
X22
HN
HN NH
NH22
O
O X
X22 H
H22N
N
O
O X
X22 R
R11
HN
HN O
O
O R
R11
H O
H
H
H O
O

Figure
Figure 14.
14. Co(III)
Co(III)Complex
Co(III) Complexcatalyzed
Complex catalyzedpeptide
catalyzed peptidehydrolysis.
peptide hydrolysis.
hydrolysis.

Anchoring
Anchoring at
Anchoring at Cys
at Cys Side
Cys Side Chains:
Side Chains: Molybdocene
Chains: Molybdocene
Molybdocene
Erxleben
Erxleben
Erxleben et et al.
al. showed
al. showedthat
showed thatmolybdocene
that molybdocene
molybdocene dichloride,
dichloride,
dichloride, CpCp
Cp 2 MoCl
22MoCl
MoCl 2 , cleaves
22,, cleaves
cleaves the the amide
the amide
amide bondbond
bond at theat
at the
the C-terminal
C-terminal
C-terminal sideside
side of of cysteine
of cysteine
cysteine to to
to generate
generate
generate Cys-Gly
Cys-Gly
Cys-Gly from
from
from Gly-Cys-Gly
Gly-Cys-Gly
Gly-Cys-Gly [89].
[89].
[89]. The
TheThe cleavage
cleavage
cleavage is ishighly
is highly
highly
selective
selective for the C-side Cys because it leads to the formation of the favorable
selective for the C-side of Cys because it leads to the formation of the favorable six-membered ring.
of Cys because it leads to the formation of the favorable six-membered
six-membered ring.
ring.
The
The mechanism
The mechanism
mechanism of of this reaction is illustrated in Figure
of this reaction is illustrated in Figure
Figure 1515 [89].
15 [89].
[89].

NH
NH22 O
O 2+
2+ NH
NH22 O
O
H
H H
H
O
O N
N O
O H
H22O
O Cp
Cp O
O N
N O
O
N
N + pH
pH 7.4
7.4 N
N
H
H + Mo
Mo H
H
O
O O
O O
O O
O O
O O
O
SH
SH H
H22O
O Cp
Cp room
room temp.
temp. S
S OH
OH
Mo
Mo
Cp
Cp Cp
Cp

NH
NH22 O
O
H
H
O
O N
N
NH O
O
NH22CH
CH22COO
COO +
+
O
O O
O Cp
Cp
S
S Mo
Mo
Cp
Cp

Figure
Figure 15.
Figure Mocatalyzed
15. Mo
15. Mo catalyzedpeptide
catalyzed peptidehydrolysis.
peptide hydrolysis.
hydrolysis.

3.1.3.
3.1.3. Anchoring at Met, His,
His, and Cys
Cys Side Chains:
Chains: Palladium(II)Complexes
Complexes forthethe Cleavageofof
3.1.3. Anchoring
Anchoring at at Met,
Met, His, and
and Cys Side
Side Chains: Palladium(II)
Palladium(II) Complexes for for the Cleavage
Cleavage of
Amide Bonds
Amide
Amide Bonds
Bonds
Figure 16 shows the examples of platinum(II) and palladium(II) complexes which are known for
Figure
Figure 1616 shows
shows the
the examples
examples of
of platinum(II)
platinum(II) and
and palladium(II)
palladium(II) complexes
complexes which
which areare known
known forfor
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)
the cleavage of amide bonds under mild conditions. These platinum(II) and palladium(II) complexes complexes
attach
attach to the sulfur atom of cysteine, S-methylcysteine, and methionine ininpeptides, thus, promoting the
attach toto the
the sulfur
sulfur atom
atom ofof cysteine,
cysteine, S-methylcysteine,
S-methylcysteine, andand methionine
methionine in peptides,
peptides, thus,
thus, promoting
promoting
selective
the cleavage of the unactivated amide bonds at the C-side of the amino acid (Figure 16) [90–92].
the selective
selective cleavage
cleavage of
of the
the unactivated
unactivated amide
amide bonds
bonds atat the
the C-side
C-side of
of the
the amino
amino acid
acid (Figure
(Figure 16)
16) [90–
[90–
92].
92].
Molecules2018,
Molecules 2018, 23,
23, 2615
x 1313
ofof
4343
Molecules 2018, 23, x 13 of 43
2+ 2+
Cl N
2+ N 2+ S
N Cl
Pd N N N
Pd N N N N
Pd N S
N Pd
N N N Pd N S Pd OH2
Cl N N N Cl Pd
Pd Cl N
N S Pd OH2
Cl N Pd
N Cl Cl OH2

[{Pd(en)Cl}2(1-pz)](NO3)2 N 2(1-pydz)](NO3)2 OH
Cis[Pd(dtco)(H 2 2+
[{Pd(en)Cl} 2O)2]
[{Pd(en)Cl}2(1-pz)](NO3)2 [{Pd(en)Cl}2(1-pydz)](NO3)2 Cis[Pd(dtco)(H 2O)2]2+
OH
H2O OH2 H2O OH2 H 2O OH2 OH O
HO O
H2O Pd
OH2 H2O Pd
OH2 H 2O Pd
OH2 -CD OH OHO
H2O OH2 N N MeS HO O O
OH
Pd Pd Pd S -CD OH OHO
H2O N MeS HO O
OH2 N S O OH
HOO OH
OHHO
2+ O
Pd(H2O)4
2+
Pd(en)(H2O)2 2+ O -CD HO OH
[Pd(dtco)( -CD)(H2O)2] OH O
2+
2+
Pd(en)(H2O)2 O OH
Pd(H2O)4 [Pd(dtco)( -CD)(H2O)2]2+ OH -CD O
O O N O
O
OH OHOH
O O Pt N OH O
O OH
O Pt N OOH OHO OH OH
O O
O N O OHO OH OH
[Pt(dach)(CBDCA-O,O')]
O HO O
[Pt(dach)(CBDCA-O,O')] HO
Cl O Cl O
Cl O O Cl O O Cl N
Cl Pt N Cl Pt N
O O O
O Pt N Cl Pt N
Cl Pt N O
O Pt N Cl Pt
N
N CH3 O O
N Cl Pt N
O Pt N CH3 O Pt N CH3
N N N N
Cl N
N N CH3
[Pt(en)Cl2] [Pt(en)(CBDCA-O,O')] [Pt(1,2-pn)Cl 2] [Pt(1,2-pn)(CBDCA O,O')] [Pt(dach)Cl 2]
[Pt(en)Cl2] [Pt(en)(CBDCA-O,O')] [Pt(1,2-pn)Cl 2] [Pt(1,2-pn)(CBDCA O,O')] [Pt(dach)Cl 2]

Figure 16. Pd and Pt complexes for activation of amide bonds.

Pd and
Figure 16. Pd and Pt
Pt complexes
complexesfor
foractivation
activationof
ofamide
amidebonds.
bonds.
Pyrazine and Pyridazine Palladium(II)-Aqua Dimers
Pyrazine
Pyrazine and
and Pyridazine
Pyridazine Palladium(II)-Aqua
Palladium(II)-Aqua Dimers
Dimers
The complete hydrolysis of the amide bonds of peptides N-acetylated-L-histidylglycine (Ac-L-
The
His-Gly) complete
The complete hydrolysis
and -L-methionylglycine
hydrolysis of the of the
(Ac- amide
L-Met-Gly)
amide bonds
bonds atof theofC-terminal
peptides
peptides N-acetylated-
side of LMet
N-acetylated- andL-histidylglycine
His in the
-histidylglycine pH
(Ac- L-
(Ac- L-His-Gly)
range 2.0and
His-Gly) < pH-and< -L2.5
-methionylglycine
was catalyzed
L-methionylglycine (Ac-L(Ac-
by L-Met-Gly)
two
-Met-Gly) at C-terminal
dinuclear
at the the C-terminal
sideside
palladium(II) of Met
complexes,
of Met and and Hisin in
[{Pd(en)Cl}
His thepH
the pH
2(l-

range 2.0
pz)](NO <
) pH
and < 2.5 was catalyzed
[{Pd(en)Cl} by two
(l-pydz)](NO dinuclear
) at 37 palladium(II)
°C. The complexes,
hydrolysis is [{Pd(en)Cl}
assisted
range 2.0 < pH < 2.5 was catalyzed by two dinuclear palladium(II) complexes, [{Pd(en)Cl}2(l-
3 2 2 3 2 by the (l-pz)](NO
formation
2 3 )2
of
and [{Pd(en)Cl} (l-pydz)](NO ) at 37 ◦ C. The hydrolysis is assisted by the formation of complexes
complexes
pz)](NO3)2 between the side2(l-pydz)](NO
and 2[{Pd(en)Cl} chains
3 2 of methionine
3)2 at 37 °C. andThe
histidine and the
hydrolysis metal complexes
is assisted (Figure 17)
by the formation of
between
[93–95].
complexes thebetween
side chains of methionine
the side and histidine
chains of methionine andand the metal
histidine andcomplexes (Figure 17)(Figure
the metal complexes [93–95].17)
[93–95].
O O
HN O O O
HN H O HN O HN H
HN O
N O OH H N
HNN H N HN HN
N OH 2
HO O H
H
N
O OH N O O OH
N OH HO O
N Pd N ON Pd 2 N S S
O O OH
L = pz or pydz O
N Pd
N N N PdN N S N
H2O Pd S OH2
N Pd
L = pz or pydz
N N H2O PdOH2N N PdOH2 OH2
4+ 4+
[{Pd(en)(Ac-His-Gly-N3)}( -L){Pd(en)(H2O)}] [{Pd(Ac-L-Met-Gly-S)(H
OH2 2O)22} 2( -L)]
OH
4+
[{Pd(en)(Ac-His-Gly-N3)}( -L){Pd(en)(H2O)}] 4+ [{Pd(Ac-L-Met-Gly-S)(H 2O)2} 2( -L)]
Figure 17.
Figure Pd(II)aqua
17. Pd(II) aquadimers.
dimers.
Figure 17. Pd(II) aqua dimers.
Molecules 2018, 23, 2615 14 of 43
Molecules 2018, 23, x 14 of 43

Platinum
Platinum Complexes
Complexes forfor the Cleavage of
the Cleavage of the
the Amide
Amide BondBond
1 H-NMR investigation of the cleavage reactions between various Pt(II) complexes of the type
1H-NMR investigation of the cleavage reactions between various Pt(II) complexes of the type
[Pt(L)Cl
[Pt(L)Cl22]] and [Pt(L)(CBDCA-O,O0 ] (L
and [Pt(L)(CBDCA-O,O′] (L ==ethylenediamine-en; (±)-trans-1,2-diaminocyclohexane-dach;
ethylenediamine-en;(±)-trans-1,2-diaminocyclohexane-dach;
±)-1,2-propylenediamine-1,2-pnand
((±)-1,2-propylenediamine-1,2-pn andCBDCACBDCA is 1,1-cyclobutanedicarboxylic
is the the 1,1-cyclobutanedicarboxylic anion)
anion) and and
the N-
the N-acetylated- L -methionylglycine dipeptide (MeCOMet-Gly) were reported
acetylated-L-methionylglycine dipeptide (MeCOMet-Gly) were reported by Djuran and co-workers by Djuran and
co-workers
[93–96]. The[93–96].
comparison Theofcomparison of the
the rate studies rate Pt
of these studies of these
complexes Pt complexes
for the cleavage of for the cleavage
N-acetylated- L-
of N-acetylated-L-methionylglycine
methionylglycine dipeptide
dipeptide (MeCOMet-Gly) (MeCOMet-Gly)
showed that the rateshowed that the
of hydrolysis rate of hydrolysis
decreases with the
decreases
increase inwith
the the increase
steric inthe
bulk of theCBDCA
steric bulkandofchlorido
the CBDCA Pt(II)and chlorido(en
complexes Pt(II) complexes
> 1,2-pn (en(Figure
> dach) > 1,2-pn
>18).
dach) (Figure 18).

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

O O
O
O
H3C NH O
H
H 3C N H 3C
S O
O
O O HN O N
N Pt Cl H Pt
N S
N O N
O CH3
[Pt(L)(CBDCA-O)(MeCOMet-Gly-S)]

-Cl-; +H2O -CBCDA; +H 2O

O +
HN CH3 O
O + H
N
H 3C NH O O
H3C H H 3C O
S N S OH2
O
N Pt OH2 O N Pt OH2
internal delivery external attack
N N

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

complex

O +
N HN CH3
OH2
O
Pt O + H 2N
S O
N O
CH3
+ Gly
[Pt(L)(H2O)(MeCOMetS)]

Figure
Figure 18.
18. Hydrolytic
Hydrolyticreactions
reactionsof
ofPt
Ptcomplexes.
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

Later, these [Pd(en)(H2 O)2 ]2+ and [Pt(en)(H2 O)2 ]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
that
that these
these complexes
complexes are highly selective
are highly selective for
for the
thecleavage
cleavageof ofthe
theamide
amidebond
bondatatthe
theC-terminal
C-terminalsidesideofof
methionine (Figure 19) [97]. The high selectivity for the Met-Gly amide bond compared
methionine (Figure 19) [97]. The high selectivity for the Met-Gly amide bond compared to the other to the other
amides is due to the high affinity of the Pt(II) and Pd(II) ions for the sulfur atom on Met. Two
amides is due to the high affinity of the Pt(II) and Pd(II) ions for the sulfur atom on Met. Two different different
mechanisms
mechanisms for the cleavage of of tetrapeptide
tetrapeptideat atthe
theC-terminus
C-terminusofofMet Metresidue
residueby byPdPdcomplexes
complexeshave have
been proposed. One involves the direct coordination of the Pd complex with
been proposed. One involves the direct coordination of the Pd complex with Met followed by theMet followed by the
cleavage.
cleavage. The second involves
involves the
the formation
formationof ofmacrochelate
macrochelatewithwithboth
bothHis
Hisand
andMet Metfollowed
followedbybythe the
hydrolysis
hydrolysis of
of the amide bond.

NH
S
N
O O O
H H
N N
H 3C N N NH2
H H
O O
2+ 2+
Pt(en)(H2O)2 Pd(en)(H2O)2

1.5<pH<2.0; t = 60 oC

O O 2+
H H N N
H3C N N NH2 CH3
N N Pd
H H HN
O O O N S
OH2 HN O
NH
S Pt N N O
NH HN
N O HN
O O
hydrolytically active form H 2N macrochelate

H2en2+ after 60min

2+
H 2O OH2
O CH3
H Pd
H3C N S HN
OH N O
HN
O OH2 O
NH HN
S Pt N H2N N
H O O
N O hydrolytically active form

+ OH
O O 2+
H
N NH2
H 2N N O NH S N
H
O O Pd
NH H 3C N N
HN O
N O
O
NH
H2N

Figure
Figure 19.
19. Hydrolytic
Hydrolyticreactions
reactionsof
ofPd
Pdand
andPt
Ptcomplexes.
complexes.

Cis[Pd(dtco)(H 2+ leads to the selective cleavage of the amide bond at the C-terminal of Met
Cis[Pd(dtco)(H22 O)22]]2+ leads to the selective cleavage of the amide bond at the C-terminal of Met
(Figure
(Figure20)20)[98]. Coordination
[98]. Coordinationof the
of metal complex
the metal promotes
complex hydrolysis
promotes by two different
hydrolysis by twomechanisms.
different
The first involves
mechanisms. the formation
The first involves theofformation
a six-membered complex bycomplex
of a six-membered the chelation
by theof a metalof
chelation atom with
a metal
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
cleavage by an external attack from water. This mechanism is favorable in the case of platinum(II)
Molecules 2018, 23, x 16 of 43
promoters and substrates with smaller anchoring side chains. Second, the mechanism involves the
chelation
involves theof metal withofsulfur
chelation metalonly,
withfollowed by the
sulfur only, internal
followed by attack of water
the internal molecules
attack of watertomolecules
the amide
bond. This mechanism is favorable with palladium(II) promoters and substrates with
to the amide bond. This mechanism is favorable with palladium(II) promoters and substrates longer anchoring
with
side chains
longer (Figureside
anchoring 20) chains
[98]. (Figure 20) [98].

H3C AcMet-Gly 2+ H3C AcMet-Gly 2+

S S S S S S
+2 H2O Pd Pd
Pd Pd
S S -AcMet-Gly S O O S
S
H3C H2 H2
AcMet-Gly
Mechanism HN
CO2H

O OH2

O S M
CO2H O CO2H
N NH external attack
x
S M x Activation
M S S x O
M
H 2O HN
amide N-coordination CO2H
inhibition internal delivery

Figure20.
Figure [Pd(dtco)(H22O)22]]2+2+mediated
20. [Pd(dtco)(H mediatedhydrolysis
hydrolysisofofamide
amidebond.
bond.

Kostic
Kosticet et
al. utilized
al. utilizedthe Pd(Hthe2 O) 4 complex
Pd(H for the cleavage
2O)4 complex for theof decapeptide
cleavage of(Ac-AKYGGMAARA)
decapeptide (Ac-
under acidic conditions ◦
AKYGGMAARA) under(pH 2.3)conditions
acidic at 60 C [99]. (pHThe2.3)cleavage
at 60 °Cof[99].
the The
peptide bondof
cleavage took
theplace at the
peptide bondGly
residue next
took place atto Met
the Glyon the N-terminal
residue next to Met side
onand generated two
the N-terminal sidefragments
and generatedaftertwo
24 h. The Pd(H
fragments O)
after
2 4
complex
24 h. Thebinds
Pd(H2toO)the S of thebinds
4 complex Met toresidue leading
the S of the Met toresidue
the formation
leadingof totwo different complexes
the formation under
of two different
the reactionunder
complexes conditions (activeconditions
the reaction and inactive), which
(active and are in equilibrium
inactive), which arewith each other (Figure
in equilibrium with each 21).
other
The (Figure
active 21). The
complex led active
to the complex
formationled of to
twothefragments
formationthrough
of two hydrolysis
fragments through hydrolysis
but the inactive but
complex
the not
did inactive complex
undergo did not undergo
any hydrolysis. any hydrolysis.
The coordination of twoTheamide
coordination
nitrogenof two amide
atoms nitrogencomplex
in the inactive atoms
in the inactive
quench complex
the Lewis acidityquench the thus
of Pd(II), Lewis noacidity of Pd(II),
hydrolysis was thus no hydrolysis was observed.
observed.
Next, Kostic et al. used this metal complex Pd(H2 O)4 for the cleavage of the amide bond
under Oin different peptide substrates such as Gly-Met, O
O neutral conditions
O O O Sar-Met,
O
and Pro-Met.
H H
This study showed
N that the rate of -H +
hydrolysis at a neutral
N pH is slow compared
hydrolysis to thatSat a low
N N S N N
pH [44].
H Interestingly, H the complete cleavage+H+ of HSar-Met and Pro-Met was observed butNnoPd OH2
cleavage
O Pd O
was observed for Gly-Met at a neutral pH. This is due to the difference Pd S in the equilibrium
O position
NH2 of
active
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 +
H
for the peptide containing Sar-Met or Pro-Met,+H the equilibrium is shifted towards the hydrolytically
N
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 S O
O S S O
+H+ hydrolysis +
X

N Pd N Pd NH2 HO
N Pd N O -H+
O N O O N
O N O
inactive N
O H O

Figure 21. [Pd(H2O)4]2+ mediated hydrolysis of amide 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
the inactive
Molecules complex
2018, 23, 2615 did not undergo any hydrolysis. The coordination of two amide nitrogen atoms
17 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+ H S
N N hydrolysis
N N S N N
H H +H+ H N Pd OH2
O Pd O
Pd S O NH2
active
+
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 under O
neutral conditions in different peptide substrates
O
such as Gly-Met, Sar-Met, and Pro-Met. This study
O S O O
showed S the rate of hydrolysis
that +H+ at a neutral pH is slow compared to thatSat a low pH [44].
hydrolysis +

X
Interestingly, the complete cleavage of Sar-MetN andPd Pro-Met was observed N butPd no NH
cleavage was
N Pd N O -H+ 2 HO
observed for Gly-Met at a neutral pH. This isOdue to the difference
O
in the equilibrium position of the
O N O N
Gly-Met N
compared to that of Pro-Met. In Gly-Met, equilibrium O is more shifted towards the inactive
inactive
form at neutral pH due to the ability of Gly to form a strong
N coordinate bond with Pd. In contrast, for
O H O
the peptide containing Sar-Met or Pro-Met, the equilibrium 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
Figure 22). O) ]2+2+ mediated hydrolysis of amide bond.
21. [Pd(H
Figure 21. [Pd(H22O)44] mediated hydrolysis of amide bond.

OH2 R O H2O OH2

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

Pd triggered
Figure 22. Pd triggeredpH
pHdependent
dependenthydrolysis
hydrolysisofofamide
amidebond.
bond.

In
In the
the case
case of
of Gly-Pro-Met, the
the hydrolysis
hydrolysis of
of the
theamide
amidebond
bondcan
cantake
takeplace
placeeither
eitherthrough
throughthe
the
external
external or
or internal
internal attack of water molecules depending
depending upon
uponthethecis/trans
cis/transconformation
conformationadopted
adoptedbyby
proline
proline (Figure
(Figure 23).
23). The ROESY NMR
NMR studies
studiesshowed
showedthat
thatthe
thehydrolysis
hydrolysisofofthe
theGly-Pro-Met
Gly-Pro-Metpeptide
peptide
takes
takes place
place byby the external attack of water on
on trans-Gly-Pro
trans-Gly-Pro [100].
[100].

Cis/Transconformations
Figure 23. Cis/Trans conformationsofofPd
Pdcomplexes.
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(H2 O)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
Molecules 2018, 23, x peptide (Figure 24). 18 of 43

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

pH selective
Figure 24. pH selectivePd
Pdcatalyst
catalystfor
forthe
thehydrolysis.
hydrolysis.

Next,
Next, the
the same
same group
group utilized
utilized the Pd(en)(H22O)
the Pd(en)(H O)22complex
complexforforthe
theacidic
acidichydrolysis
hydrolysisofofthe
theB-chain
B-chain
of
of bovine
bovine insulin
insulin containing
containing two histidine residues.
two histidine residues. These
Thesecomplexes
complexespromoted
promotedthethecleavage
cleavageofofthe
the
second amide bond upstream from histidine. The detailed mechanistic analysis showed
second amide bond upstream from histidine. The detailed mechanistic analysis showed the selective the selective
cleavage
cleavage by
by the
the coordination complexes with
coordination of the Pd complexes withthethehistidine
histidineside
sidechain
chainand
andamidic
amidicnitrogen
nitrogen
followed
followed by
by the
the internal
internal attack of water on the amide
amide bond
bond (Figure
(Figure25)
25)[101].
[101].

O R'' O O R'' O O
H H H
N N N N
N N
H H [Pd(en)(H2O)2]2+ O
R' O OH2 R' O N
NH NH R''
N Pd N N Pd N N Pd N
NH
N
N [Pd(en)2]2+
N
+ 3H2O+ O
R'

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

Figure 25.
Figure Acidichydrolysis
25. Acidic hydrolysisof
ofamide
amidebonds.
bonds.

Next,
Next, they
they used
used the
the β-cyclodextrin
β-cyclodextrin Pd Pd complex
complexforforthe
thecleavage
cleavageofofthe
theamide
amidebond
bondatatthe
thefirst
first
amide
amide bond
bond upstream
upstream from the Pro-Phe
from the Pro-Phe sequence.
sequence. The
Therole
roleof
ofthe
theβ-CD complexwas
β-CDcomplex wastotobind
bindtotothe
the
hydrophobic
hydrophobic residue
residue (Phe) in an aqueous
aqueous medium
medium followed
followedby bythe
theactivation
activationofofthe
theamide
amidecarbonyl
carbonyl
group
groupby bythe
themetal
metalcoordination, andand
coordination, the the
attack by the
attack byexternal waterwater
the external molecule leadingleading
molecule to the cleavage
to the
of the amide
cleavage bond
of the (Figure
amide bond26) [102]. 26) [102].
(Figure
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 OH2
R O O H
Pd cyclodextrin (CD)
S S

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

3.1.4.
3.1.4. Artificial
Artificial Metal
Metal Proteases
Proteases
Suh
Suh etet al. designed metal
al. designed metal complexes
complexesfor forthe
thecleavage
cleavageofofamide
amidebondsbondsininproteins
proteinsatatspecific
specific
locations
locations [103–107]. Thesemetal
[103–107]. These metalcomplexes
complexesarearehighly
highly selective
selective with
with their
their protein
protein partners
partners similar
similar to
to
some natural proteases. They also demonstrated that the catalytic rate of hydrolysis increased by theby
some natural proteases. They also demonstrated that the catalytic rate of hydrolysis increased
the formation
formation of the
of the complex
complex between
between thethe substrate
substrate andand catalyst.
catalyst. This
This is is achievedbybyincreasing
achieved increasingthethe
multinuclear
multinuclearmetal metalcenters,
centers,which
which provide
provideextra metal
extra centers
metal as substrate
centers binding
as substrate sites. They
binding sites.showed
They
that the rate of hydrolysis of the myoglobin protein increased with the increase in the
showed that the rate of hydrolysis of the myoglobin protein increased with the increase in the number number of metal
centers
of metal centers in the mono (half-life for hydrolysis 24 h, Figure 27), dinuclear (half-life forh),
in the mono (half-life for hydrolysis 24 h, Figure 27), dinuclear (half-life for hydrolysis 3.5
and tetranuclear
hydrolysis 3.5 h),metal centers (half-life
and tetranuclear metalfor hydrolysis
centers 1.3for
(half-life h) [103].
hydrolysis 1.3 h) [103].

O O
H H
N N N N
H N N H N N
NH O NH
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 2O N H H
N N NH N N
H O O O
Cu OH2
tetranuclear N N N N
Cu OH2
N N

Artificial metal
Figure 27. Artificial metal complexes
complexeswith
withdifferent
differentnumbers
numbersofofmetal
metalcenters.
centers.

They
They showed
showed that
that the
the selectivity towards aa specific
selectivity towards specific protein
proteinwas
wasachieved
achievedby bythetheaddition
additionofof
organic
organic pendants
pendants such
such as
as peptide
peptide nucleic
nucleic acid
acid (PNA),
(PNA), which
which are
are responsible
responsible for
for selective
selective binding
binding to
to a
particular protein.
a particular Overall,
protein. the goal
Overall, of this
the goal of research was towas
this research synthesize peptide-cleavage
to synthesize agents selective
peptide-cleavage agents
for the hydrolysis
selective of pathogenic
for the hydrolysis proteins responsible
of pathogenic for Alzheimer’s
proteins responsible disease, disease,
for Alzheimer's type 2 diabetes mellitus,
type 2 diabetes
and Parkinson’s
mellitus, disease. disease.
and Parkinson’s

Aldehyde Pendant for the Cleavage of Proteins

Molecules 2018, 23, 2615 20 of 43

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

Ammonium
Ammonium groups
groups are abundant on
are abundant on the
the surface
surfaceofofproteins
proteinsandandform
formimine
iminewith
withaldehyde
aldehydefaster
faster
than the peptide bond cleavage and, through this process, brings the reactive metal
than the peptide bond cleavage and, through this process, brings the reactive metal catalytic center catalytic center
in
in close
close proximity to the
proximity to theprotein/peptide
protein/peptide chain
chain (Figure
(Figure 28).28).
TheThe
rapidrapid formation
formation of imine
of imine makes makes
the
the attack
attack of metal
of the the metal
center center onpeptide
on the the peptide
bond anbond an intramolecular
intramolecular process,process, thus, increases
thus, increases the
the rate of
rate
hydrolysis. Based on this concept, several artificial metalloproteases have been developed byby
of hydrolysis. Based on this concept, several artificial metalloproteases have been developed
incorporating
incorporating ananaldehyde
aldehydehandle
handleclose
closetotothe
themetal
metalcenter
centerandandhas
hasbeen
beensuccessfully
successfullybeen
beenapplied
applied for
the
for faster cleavage
the faster of peptide
cleavage bonds
of peptide [104].
bonds [104].

L L L L
M O NH3 M N
L L H 3N
H H
protein substrate intermediate cleavage products

Aldehyde pendant
Figure 28. Aldehyde pendantmediated
mediatedcleavage
cleavageofofpeptide
peptidebonds.
bonds.

Mb-Selective
Mb-Selective Artificial
Artificial Protease
Protease
The
The catalyst
catalyst for
for the
the cleavage
cleavage of of myoglobin
myoglobin (Mb)
(Mb) waswas designed
designedby byattaching
attachingthe
thecyclen
cyclenmetal
metal
complex
complexcontaining
containingCu(II)
Cu(II)oror
Co(III) to the
Co(III) peptide
to the nucleic
peptide acid acid
nucleic (PNA) monomers,
(PNA) whichwhich
monomers, act as binding
act as
sites for the Mb (Figure 29) [105]. Varieties of linkers were inserted between the
binding sites for the Mb (Figure 29) [105]. Varieties of linkers were inserted between the PNA PNA binding
bindingsite
and the catalytic
site and cyclen
the catalytic sitesite
cyclen forfor
thethe
formation
formationofofMb-cleaving
Mb-cleavingcatalysts. MALDI-TOFMS
catalysts. MALDI-TOF MSshowed
showed
that the cleavage of the peptide backbone chain of Mb takes place at Leu89-Ala90. Various
that the cleavage of the peptide backbone chain of Mb takes place at Leu89-Ala90. Various chelating chelating
ligands
ligands were
were tested
tested for
for determining
determining the the activity
activityofof the
theMb-cleaving
Mb-cleavingcatalyst
catalystbut
butonly
onlycyclen
cyclenandanditsits
triaza-monooxo
triaza-monooxo analog
analog showed catalytic activity.
showed efficient catalytic 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 NH N NH N
N
N N NH2 N S N N NH2 N O
H H H H
A* T* G C

Figure
Figure 29.
29. Catalyst
Catalystdesign
designfor
forprotein
proteincleavage.
cleavage.

Mechanism: step involves

Mechanism: The first step involves the
thebinding
bindingof ofthe
thecarbonyl
carbonylgroup
groupofofthe
theamide
amidetotothetheCo(III)
Co(III)
metal
metal center
center to
to form
form a CS complex (Figure
(Figure 30).
30). The
Theselectivity
selectivityofofone
oneamide
amidecarbonyl
carbonyloveroverthe
therest
restofof
the amidic carbonyls
the amidic carbonyls is based on the other half of the catalyst, which is responsible for the recognition
the other half of the catalyst, which is responsible for the recognition
of
of aa particular
particular protein
protein and
and a particular This Co(III)
particular site. This Co(III) carbonyl
carbonylcoordination
coordinationactivates
activatesthetheamidic
amidic
carbonyl for
carbonyl for aa nucleophilic attack by
by the
the hydroxide
hydroxideionionononthe
themetal
metalcenter,
center,resulting
resultingininthe
theformation
formation
of tetrahedral
of tetrahedral intermediate
intermediate T.T. This
This is
is followed
followed byby the
the collapse
collapseofofthe
thetetrahedral
tetrahedralintermediate
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

Molecules 2018,
resulting 23, xbreakage of the amide bond to generate a peptide amine and corresponding peptide
in the 21 of 43
Molecules 2018, 23, x 21 of 43
acid [106].
Suh and
Suh co-workers
co-workersshowed thatthat
the cleaving capability of the Co(III)/Cu(II) complexescomplexes
of cyclen
Suhand
and co-workers showed
showed the cleaving
that the cleaving capability
capability of the Co(III)/Cu(II)
of the Co(III)/Cu(II) complexes of cyclen
increased
ofincreased by the
cyclen increased replacement of one
by the replacement nitrogen atom
of one of cyclen
nitrogen atomwith an
of an oxygen
cyclen atom.
with The
an The
oxygenCo(III)-
atom.
by the replacement of one nitrogen atom of cyclen with oxygen atom. Co(III)-
oxacyclen
The complexes complexes
Co(III)-oxacyclen (1-oxa-4,7,10-triazacyclododecane Co(III)) cleaved
(1-oxa-4,7,10-triazacyclododecane thecleaved
Co(III)) proteins
thesuch as BSA,
proteins such
oxacyclen complexes (1-oxa-4,7,10-triazacyclododecane Co(III)) cleaved the proteins such as BSA,
HEWL, Mb, and bovine serum-globulin with a 4–14 times higher catalytic efficiency compared to the
asHEWL,
BSA, HEWL,
Mb, and Mb, and bovine
bovine serum-globulin
serum-globulin withtimes
with a 4–14 a 4–14 timescatalytic
higher higher catalytic efficiency
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' III O N R'
R N
H X X Co
III O
NH
NH H X X Co O R
O
H R
III OH2 NH H
X XXCoIII OH2 NH
X Co OH
OH
NH CS
NH CS
XX == NH
NH or
or O
O

NH
NH H
RCH - H
RCH22OO
OO- III O N R'
N R'
III
XCo O
XX
X Co O
O R
R
H
NH H
NH
NH
NH T
T
III
III O
O
XXXXCo R
R
Co
OO
NH
NH H22
H
NH
NH N R'
N R'
IIIO
III O
XCo
XX Co
O
O R
R
R'NH
R'NH2 NH
NH

Figure 30.
Figure 30. Mechanism
Mechanism of
Mechanism of Co(III)-metal
Co(III)-metal complexes.
Co(III)-metalcomplexes.
complexes.

PDF-Selective
PDF-SelectiveArtificial
PDF-Selective ArtificialProtease
Artificial Protease
Protease
The
Thecatalyst
The catalystfor
catalyst forthethe
for cleavage
the cleavage
cleavage of the protein,
of the
of peptide
the protein, deformylase
peptide deformylase
deformylase(PDF), was obtained
(PDF),
(PDF), was in a selective
was obtained
obtained ininaa
manner
selectiveby
selective screening
manner
manner by the library
byscreening
screening oflibrary
the
the catalysts
library (Figure 31).
of catalysts The31).
(Figure catalyst
31). The cleavedcleaved
The catalyst
catalyst the backbone
cleaved the peptide
thebackbone
backbone
chain of the
peptide
peptide PDF
chain
chain ofatthe
of position
the PDF at
PDF Gln152-Arg153.
at Docking simulations
position Gln152-Arg153.
position Gln152-Arg153. Docking showed that
Docking simulations
simulations multiple
showed
showed that
thatinteractions
multiple
multiple
were responsible
interactionswere
interactions for the
wereresponsible formation
responsible for of
for the a complex
the formation between the catalyst
formation of a complex between
between theand PDF.
the catalyst Fifteen
catalystand other
andPDF. proteins
PDF.Fifteen
Fifteen
other
were
other proteinswere
examined,
proteins were
but none examined, but
of thembut
examined, none of
underwent
none of them
them underwent
cleavage by this cleavage
underwent Co(III)
cleavage by
by this
thisCo(III)
complex, which complex,
Co(III) further
complex, which
confirmed
which
further
the highly confirmed
selective the highly
nature of selective
these metalnature of these
complexes metal
[107]. complexes
further confirmed the highly selective nature of these metal complexes [107]. [107].

NH
NH
X OH O O OH
OH
XX
X Co
C o OH22
OH
OH22
X == NH
NH N
N
X

O
O
N
N NH
NH
O
H N O
H22N

Figure 31. Metal-assisted catalyst for PDF.

Metal-assistedcatalyst
31. Metal-assisted
Figure 31. catalystfor
forPDF.
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
Molecules
such as 2018, 23, 2615
Alzheimer’s and type 2 diabetes. Based on the above concept, Suh et al. synthesized various 22 of 43

other metal complexes for the selective cleavage of amide bonds in these proteins. The main
Molecules 2018, 23, x 22 of 43
advantage of this
AmPs-Selective work is
Artificial that such amyloidogenic proteins cannot be targeted by conventional
Protease
approaches due to the lack of an active site (Figure 32) [108–110].
AmPs-Selective
AmPs Artificial Protease
Soaresare et amyloidogenic peptides or
al. utilized mononuclear proteins which
copper(II) lack active
complexes [Cu(HL sites
1)Cl2and
] andare[Cu(L
related to for
1)Cl] diseases
the
such as
AmPs
cleavage Alzheimer’s
ofare and type
amyloidogenic
unactivated 2 diabetes.
amidepeptides
bonds of Based
orthe on
proteins the
which
proteins above concept,
lack active
bovine Suh
serum sites et al.
and are
albumin synthesized
related
(BSA) and to various
diseases
Taq DNA
other
such metal
as
polymerase, complexes
Alzheimer’s
under mild fortype
and the and
pH selective cleavage
2 diabetes. of
onamide
Basedconditions
temperature bonds
the above in these
concept,
(Figure proteins.
33). Suh
The et Theoccurred
main advantage
al. synthesized
cleavage various
at the
of this metal
other
specificwork
site iscomplexes
that
on thesuch amyloidogenic
for the
solvent- proteins
selective
exposed cannot
cleavage
portions be targeted
of amide
of the proteinbonds byinconventional
theseparticular
to generate approaches
proteins. The main due
proteolytic
to the lack
advantage of an active site (Figure 32) [108–110].
of this work is that such amyloidogenic proteins cannot be targeted by conventional
fragments [111].
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
(R).(LCo 3+)
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 generateNHparticular H proteolytic
fragments [111]. N R'
O
X XCo
OH R
n NH
(R).(LCo3+)

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

NH H
N R'
O
X XCo
OH R
n NH

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

+ +
n-1

+ +
Metal complexes
Figure 32. Metal complexesfor
forthe
thecleavage
cleavageof
ofamyloidgenic
amyloidgenicpeptides.
peptides.
n-1
Soares et al. utilized mononuclear copper(II) complexes [Cu(HL1 )Cl2 ] and [Cu(L1 )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. Cu complexes

33. Cu complexesfor
foractivation
activationofofamide
amidebonds.
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, x 23 of 43
at the N-terminus of the serine and threonine residues. Such a cleavage was catalyzed by the N→O
rearrangement
rearrangement and and does
does not
not employ the Lewis
employ the Lewis acid
acid properties
properties of
of the
the metal
metal atom.
atom. Based
Basedononthe the
proposed
proposed mechanism,
mechanism, the the first
first step involves
involves the
the formation
formation of
of the
theNi(II)
Ni(II)complex
complexwithwith4 4NNofofthe the
backbone amide chain and the side chain of the His residue with the (Ser/Thr)-Xaa-His
backbone amide chain and the side chain of the His residue with the (Ser/Thr)-Xaa-His sequence sequence
(Figure
(Figure 34).
34). The
The second
second step involves
involves the N→Orearrangement
the NO rearrangementfromfromthe theside
sidechain
chainofofthe
theSerSerororThr
Thr
that
thattransfers
transfers thethe N-terminal
N-terminal R1 moiety from thethe peptide
peptide bond
bondto
toform
forman anester
esterbond.
bond.This
Thisisisfollowed
followed
by
by the
the hydrolysis
hydrolysis of of the
the resulting ester, leading
leading to
to the
the formation
formationofoftwotworeaction
reactionproducts,
products,the theR1R1
peptide
peptide acid
acid and
and the
the Ni(II)
Ni(II) complex
complex with the peptide
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 H 2N Ni N Zaa
N N O N N O
R1 H H
HN R2 HN R2

OH

R O Xaa
O
O
HO N
R1 O O
+ H2N Ni N Zaa
N N
H O
R = H or CH3
HN R2

Figure 34. Non

Non lewis
lewis acid
acidmediated
mediatedN,
N,OOAcyl
Acylrearrangement.
rearrangement.

Scandium(III)
Scandium(III) Triflate-Promoted
Triflate-Promoted Serine/Threonine SelectivePeptide
Serine/Threonine Selective PeptideBond
BondCleavage
Cleavage
Kanai
Kanai et
et al. reportedthe
al. reported thehydrolysis
hydrolysisofofthe
thepeptide
peptide bond
bond at at
thethe N-terminus
N-terminus of Ser/Thr
of Ser/Thr residue
residue by
by using
using scandium
scandium triflate.
triflate. ThisThis chemical
chemical cleavage
cleavage relies
relies on on Sthe
Sthe c triflate
c triflate mediated
mediated N NtotoOOacyl
acyl
rearrangement ◦ (Figure 35).
rearrangementfollowed
followedby bythe
thesubsequent
subsequenthydrolysis
hydrolysisofofthe
theester
esterbyby
heating
heating it it
at at
80–100
80–100C°C (Figure
Complete hydrolysis
35). Complete hydrolysistooktook
place in 18–20
place h. The
in 18–20 authors
h. The authorshave used
have usedthis approach
this approach forforthe
thecleavage
cleavageof
various peptides
of various peptides including
including posttranslationally
posttranslationallymodified
modified(PTM)
(PTM) peptides and the
peptides and thecleavage
cleavageofofnative
native
protein Aβ1-42, which
protein Aβ1-42, which is closely related to Alzheimer’s disease
Alzheimer’s disease [114].[114].

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

Figure 35.
Figure N, O
35. N, O Acyl
Acylrearrangement.
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 Table 3.
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]
(Table 4) of peptide
H2 O2 responsive C-terminal side
8 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/H2 O N-acyl group mediated oxazolinium specie [144]
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
S H 2N Peptide HN N Peptide
H
O O

heat, H+

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

Figure Edman’s degradation

Figure 36. Edman’s degradation approach
approachfor
forcleavage
cleavageof
ofpeptide
peptidebonds.
bonds.

4.1.2.
4.1.2. Cyanogen
Cyanogen Bromide
Bromide for
for Cleavage at Met
Cleavage at Met Residue
Residue
Cyanogen
Cyanogen bromide
bromide ledled to the cleavage
to the cleavage of of the
the peptide
peptidebond
bondat atthe
theC-terminus
C-terminusofofthe themethionine
methionine
residue
residueinina selective manner.
a selective manner.The The
first step
first involves the nucleophilic
step involves attack ofattack
the nucleophilic the sulfur of methionine
of the sulfur of
on cyanogen bromide (Figure 37) [116]. This displaces the bromide from
methionine on cyanogen bromide (Figure 37) [116]. This displaces the bromide from cyanogen cyanogen bromide, followed
by the attack of the amide carbonyl on the cyano group, resulting in the formation
bromide, followed by the attack of the amide carbonyl on the cyano group, resulting in the formationof the five-membered
ring,
of theiminolactone,
five-membered comprising a double bond
ring, iminolactone, in the aring
comprising between
double bond nitrogen and
in the ring carbon.
between This double
nitrogen and
bond results in a rigid ring conformation, thus activating the amide bond
carbon. This double bond results in a rigid ring conformation, thus activating the amide bond towards cleavage at the
C-terminus of Met, resulting in the generation of homoserine lactone. This approach
towards cleavage at the C-terminus of Met, resulting in the generation of homoserine lactone. This has widely been
utilized
approach forhas
thewidely
sequencing of proteins
been utilized [116].
for the sequencing of proteins [116].

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

SCH3 H3CS
N Br
N

O R' R'
RHN + H2O
O HN
H2N COOH RHN COOH
homoserine O
lactone
iminolactone

Figure 37. Cyanogen

Cyanogen bromide
bromidefor
forselective
selectivecleavage
cleavageatatMet.
Met.

4.1.3.
4.1.3. 2-Nitro-5-Thiocyano
2-Nitro-5-Thiocyano Benzoic Acid for
Benzoic Acid for Cleavage
Cleavage at
at Cys
Cys
2-Nitro-5-thiocyano
2-Nitro-5-thiocyano benzoic acid led
benzoic acid led to
to the
the hydrolysis
hydrolysisof ofthe
theamide
amidebond
bondatatthe
theN-terminal
N-terminalside side
of
ofthe
the cysteine
cysteine residue. The first
residue. The first step
step is
is the
the cyanylation
cyanylationof ofthe
theside
sidechain
chainofofcysteine
cysteineonona apeptide
peptidebyby
2-nitro-5-thiocyano benzoic acid, which is followed by the attack of the cysteine amidic
2-nitro-5-thiocyano benzoic acid, which is followed by the attack of the cysteine amidic nitrogen nitrogen to to
the
cyano group on the side-chain of cysteine, resulting in the formation of the 5-membered
the cyano group on the side-chain of cysteine, resulting in the formation of the 5-membered thiolactone
ring. This, inring.
thiolactone turn, activates
This, theactivates
in turn, amide bond towards
the amide bondhydrolysis under basicunder
towards hydrolysis conditions (Figure 38).
basic conditions
This is again
(Figure dueistoagain
38). This the inability
due to theof cysteine
inability ofamidic nitrogen
cysteine amidicinnitrogen
a thiolactone to form a resonating
in a thiolactone to form a
structure with the carbonyl of peptide bond [117].
resonating structure with the carbonyl of peptide bond [117].
Molecules 2018, 23, 2615 26 of 43
Molecules 2018, 23, x 26 of 43

O NCS O
H H
R N R' R N R'
N + N
H H
O O
SH COOH S N
NO22 cleavage

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

2-nitro-5-thiocyanobenzoic
Figure 38. 2-nitro-5-thiocyano benzoicacid
acidselective
selectivecleavage
cleavageatatCys.
Cys.

4.1.4.
4.1.4. 2-Iodosobenzoic
2-Iodosobenzoic Acid for Cleavage
Acid for Cleavage at
at Trp
Trp
2-Iodosobenzoic acid has
2-Iodosobenzoic acid has been
been used
used for
for the
thehydrolysis
hydrolysisof ofthe
theamide
amidebond
bondatatthe
theC-terminal
C-terminalside
side
of
of the Trp residue.
the Trp residue. The
Themechanism
mechanismofofthe the cleavage
cleavage is aistwo-step
a two-step process.
process. The The
first first
step step involves
involves the
the oxidation
oxidation of side-chain
of the the side-chain of tryptophan
of tryptophan by 2-iodosobenzoic
by 2-iodosobenzoic acid followed
acid followed by the nucleophilic
by the nucleophilic attack
attack fromneighboring
from the the neighboring carbonyl
carbonyl groupgroup
of theof the
amide amide bond,
bond, leading
leading to to
thethe formationofofanan
formation
iminospirolactone
iminospirolactone which hydrolyzes the peptide
which hydrolyzes peptide chain
chain ininthe
thepresence
presenceofofwater
water(Figure
(Figure39)39)[118,119].
[118,119].

HN OH HN
I O
O NH
NH HO
HN
O O HN
O O
N N OO N
H H H

O
O
HN HN
H22N +
O O N
HN O HN H
O O
iminospirolactone

Figure 39.
Figure Iodosobenzoicacid
39. Iodosobenzoic acidfor
forhydrolysis.
hydrolysis.

4.1.5.
4.1.5. TBC
TBC for
for Cleavage
Cleavage at Trp
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-
methylcyclohexadienone
methylcyclohexadienone (TBC) (TBC)atatthethe C-terminus
C-terminus (Figure
(Figure 40). 40).
TyrosylTyrosyl and histidyl
and histidyl peptide peptide
bonds
bonds which are usually cleaved by other brominating agents (such
which are usually cleaved by other brominating agents (such as α-bromosuccinimide, α- as α-bromosuccinimide,
α-bromoacetamide,
bromoacetamide, etc.) etc.)
areare stable
stable toto this
this reagent.
reagent. Additionally,other
Additionally, otheramino
aminoacids,
acids,which
whichare
aresensitive
sensitive
to
to oxidation,
oxidation, react
react with
with TBCTBC but
but do
do not
not cleave the peptide
cleave the peptide bonds.
bonds. This
Thismethod
methodwaswassuccessfully
successfully
applied to a variety of peptides and proteins [118,119].
applied to a variety of peptides and proteins [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
NHR2
NHR2 NHR1Br NHR1
H2O Br
O O
NH2R2 +N
H HN O O N O NHR2
HN H
HBr
OH Br

NHR1 H O NHR1
2
Figure 40. TBC for selective cleavage at Trp residue.
NH2R2 +
O O O NHR2
HN HN
According to the reaction mechanism suggested HBrby 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 (Figure Figure 40).
Figure40. These
40.TBC
TBC for reactions
for led to the
selective cleavage
selective cleavage atatformation
Trp residue.
Trp of an oxindole derivative,
residue.
which cleaves the peptide bond.
4.2. N-Amidination
According for Cleavage
to the reaction of mechanism
the N-Terminal Amideby
suggested Bond
Patchornik et al. (1960), oxidative bromine
4.2. N-Amidination for Cleavage of the N-Terminal Amide Bond
participates in the modification-cleavage reaction [118,119]. 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.
The N-amidination of reported
peptides the cleavage
leads theof
toThese the amide bonds
formation by moiety
the N-amidination of peptides. The
and hydrolysis reactions (Figure 40). reactions of ledatocyclic
the formation which resulted
of an oxindole in the cleavage
derivative,
N-amidination
of thewhich
amide of
bondthe peptides
at room leads
temperatureto the formation of a cyclic moiety which resulted in the
(Figure 41) [120]. The rate of cleavage was slow under ambient cleavage of
cleaves peptide bond.
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. N-Amidination
conditions for Cleavage
(2% NaOH of thet N-Terminal
aq) with = 1.5 min.Amide
To Bondevaluate the broad applicability of this cleavage
basic conditions (2% NaOH aq) with 1/2 t1/2 = 1.5 min. To evaluate the broad applicability of this cleavage
reaction, a series
Hamada
reaction,
of peptides
et of
a series al. peptides with
reported with different
the cleavage amino
differentofamino
the amideacids
acidsbonds
at the
at theby
N-terminus
the N-amidination
N-terminus
such of
aspeptides.
such as the
the Lys, Glu,
Lys, Glu,The
Ser,
Ser,
Cys,Cys,
Tyr,Tyr,
Val,Val,
andand
N-amidination Pro residues
of peptides
Pro residues were
leads cleaved
to the
were with
formation
cleaved withoftta1/2 values
1/2cyclic
values from
moiety
from 1 min
which
1 min to 10
resulted
to 10 min.
in
min. the A slightly
cleavage
A slightly of slow
slow
cleavage
cleavage was observed with bulky amino acids at the terminus such as Val or Pro, which might be be
the was
amide observed
bond at roomwith bulky
temperature amino
(Figureacids
41) at the
[120]. terminus
The rate of such
cleavageas Val
was or
slowPro, which
under might
ambient
conditions
hindering
hinderingthethe (PBS
path
pathbuffer,
of pH 7.4) at 37 °C with t1/2 = 35.7 h, but a rapid cleavage was observed under
cyclization.
of cyclization.
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-terminusR1such 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 or O Pro, which might be
hindering theOpathRof 2 cyclization. O O R2 HN N R2
H
H2N H2N
N N H2N
H amidination H R1
novel N-terminal
R1 O NH R1 O O
degradation
HN reaction
N-amidinopeptide O
O R2 O O R2 HN NR2
H
H2N H2N
N Figure N strategy.
N-amidination
Figure 41. N-amidination strategy. H2N
H amidination H novel N-terminal
R1 O NH R1 O degradation reaction O
N-amidinated peptide
N-amidinated with
peptide a Cys
with a CysresidueN -amidinopeptide
residueatat
the N-terminus
the N-terminusalsoalsogenerated
generatedaafive-membered
five-membered ring,
ring, thiazolidine
thiazolidine by pathby b path b (intermediate
(intermediate B) which
B) which did lead
did not not lead to any
to any cleavage,
cleavage, therefore,the
therefore, thett1/2
1/2 of
of the
the peptide with Cys at the N-terminus
peptide with Cys at the N-terminusFigure in
in 2% 2% NaOH aq at 37 ◦
°C was 3.4 min (slower than with
NaOH aq at 37 C was 3.4 min (slower than with other other
41. N-amidination strategy.
amino
amino acids
acids at the
at 23,
the N-terminus) (Figure 42) [120].
Molecules 2018, 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].

Figure42.
Figure 42.N-amidination
N-amidination strategy
strategywith
withN-terminal
N-terminalcysteine.
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
 Molecules 2018, 23, 2615 28 of 43
Figure 42. N-amidination
Figure 42. N-amidination strategy withstrategy withcysteine.
N-terminal N-terminal cysteine.

4.3. Lactonization
4.3. Lactonization
4.3. Lactonization Mediated
Mediated Cleavage Cleavage
of Amide
Mediated of
of Amide
Bonds
Cleavage Amide Bonds
Bonds
Otaka et al.Otaka et al. developed
developed an auxiliaryanwith
auxiliary
specialwith special groups
protecting protecting groups
(PGs), which (PGs), which of
is capable is capable of
Otaka et al. developed an auxiliary with special protecting groups (PGs), which is capable of
formingwith
forming a lactone a lactone with the
the carbonyl ofcarbonyl
an amideofbond,
an amide bond,inresulting
resulting in the
the cleavage ofcleavage
the amide of bond
the amide bond
forming a lactone with the carbonyl of an amide bond, resulting in the cleavage of the amide bond
(Table 4)Depending
(Table 4) [121–128]. [121–128]. Depending
on the natureon of
thethe
nature of the groups,
protecting protecting
an groups, an amide
amide bond bond
cleavage cleavage can
can
(Table 4) [121–128]. Depending on the nature of the protecting groups, an amide bond cleavage can be
be initiatedbe
in initiated
peptidesin bypeptides by using
using different different responsive
responsive reagents forreagents for the deprotection
the deprotection of PGs (Tableof PGs
4). (Table 4).
initiated in peptides by using different responsive reagents for the deprotection of PGs (Table 4). Table 4
Tablevarious
Table 4 showed 4 showedPGsvarious
and thePGs and the corresponding
corresponding responsive responsive
reagents forreagents for their deprotection.
their deprotection. The The
showed various PGs and the corresponding responsive reagents for their deprotection. The thiol
thiol responsive
thiol responsive reagent was reagent
appliedwasforapplied for theofcleavage
the cleavage of the PNA/DNA
the PNA/DNA complex using complex
thiol-using thiol-
responsive reagent was applied for the cleavage of the PNA/DNA complex using thiol-responsive
responsive responsive protecting
protecting groups groups [121–128].
[121–128].
protecting groups [121–128].
Table 4. Lactonization
Table 4. Lactonization mediated
mediated cleavage cleavage
of amide of amide bonds.
bonds.
Table 4. Lactonization mediated cleavage of amide bonds.

Reagent/Condition
Reagent/Condition PG PGR1 R2 R1 R3 R2 R3
Reagent/Condition PG R1 R2 R3
NO2 H NO2 H H H
NO2 NOOMe
2 H
NO2 OMeOMe OMeH
Ultraviolet Near-infrared H NOOTBDPS
2 H OMe
OTBDPS
H HOMe
Ultraviolet Near-infrared
Ultraviolet Near-infrared Fluoride hypoxia
Fluoride hypoxia
Fluoride hypoxia H OTBDPS H
H H NO2 H H
NONO
2 2 H H

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

4.4.Peroxide-Induced
4.4. Hydrogen
4.4. Hydrogen Peroxide-Induced
Hydrogen Peroxide-Induced Amide
Amide BondAmide Bond Cleavage
Cleavage
Bond Cleavage
Later,the
Later, the hydrogen
Later, theperoxide
hydrogen
hydrogen (Hperoxide (H2O
2O2)-responsive
peroxide (H 2)-responsive protecting
protecting group was group was introduced
introduced to the amino to the amino
2 O2 )-responsive protecting group was introduced to the amino
acid.
acid. Thisacid. This
protecting protecting
group group
contains a contains
boronate a boronate
or boronic or boronic
acid acid
moiety moiety
which which
underwent
This protecting group contains a boronate or boronic acid moiety which underwent deprotection underwent
in oxidative stress because of the release of hydrogen peroxide followed by the formation ofby
deprotectiondeprotection
in oxidativein oxidative
stress stress
because of because
the of
release the
of release
hydrogen of hydrogen
peroxide peroxide
followed byfollowed
the the
lactone
formation offormation
andlactone of lactone
and
the cleavagetheof and
cleavage
the the
of cleavage
peptidethebond
peptideof the peptide
bond
(Figure bond
(Figure
43) [128]. 43) (Figure
[128]. 43) [128].
Molecules 2018, 23, x 29 of 43

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

Figure
Figure 43. Hydrogen peroxide
43. Hydrogen peroxideresponsive
responsiveprobes.
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)
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, which
Molecules arex unsuitable substrates for enzymes.
2018, 23, 30 of 43

Glutamic acid
Figure 44. Glutamic acidselective
selectiveactivation
activationofofpeptide
peptidebonds.
bonds.

4.6.
4.6. Asparagine
Asparagine Selective
Selective Cleavage of Amide
Cleavage of Amide Bonds
Bonds
Kanai
Kanai et
et al.
al. described the method
described the method for for the
the site-selective
site-selectivechemical
chemicalactivation
activationofofpeptide
peptidebonds
bondsforfor
hydrolysis
hydrolysis atat the
the asparagine
asparagine residue using diacetoxyiodobenzene
residue using diacetoxyiodobenzene(DIB) (DIB)[131].
[131].The
Thereaction
reactionofofthe
the
side-chain
side-chain of Asn with DIB leads to the formation of isocyanate by Hofmann rearrangement. This isis
of Asn with DIB leads to the formation of isocyanate by Hofmann rearrangement. This
followed
followed byby the attack of
the attack of the
theN-terminal
N-terminalamidic
amidicnitrogen
nitrogenofof the
the peptide
peptide backbone
backbone chain,
chain, affording
affording a
afive-membered
five-membered N-acylurea
N-acylurea intermediate,
intermediate, thus thus activating
activating the bond
the amide amidetowards
bond towards
hydrolysishydrolysis
(Figure
(Figure 45). Asn-selective
45). Asn-selective peptide
peptide bond bondwas
cleavage cleavage
proceededwas in
proceeded
an aqueousin neutral
an aqueous neutral
solution at 37 solution
°C and
at 37 ◦ C and exhibited a broad substrate scope. The Gln-site was not cleaved under the reaction
exhibited a broad substrate scope. The Gln-site was not cleaved under the reaction conditions.
conditions.
Specifically,Specifically,
this methodthis method is applicable
is applicable to peptides
to peptides containing
containing unnatural
unnatural amino
amino acidsand/or
acids and/or
posttranslational modifications
posttranslational modifications where enzymatic cleavage is not very efficient.
cleavage is not very efficient.

O R O O R O
H H
N Peptide B N Peptide B
Peptide A N N Peptide A N N
H H H H
O O O
N
NH2 C
Hofmann rearrangement O
O R cyclization
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.
Specifically,
Molecules this
2018, 23, 2615method is applicable to peptides containing unnatural amino acids and/or
30 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 Peptide A N N
H H 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 O
HN O

Figure 45. Asparagine

Asparagineselective
selectivecleavage
cleavageofofpeptide
peptidebonds.
bonds.

4.7.
4.7. Cyclic
Cyclic Urethane
Urethane Mediated Activation of
Mediated Activation of Amide
Amide Bonds
Bonds
We
Wehave
havedeveloped
developedaamethod
methodfor forthe
thecleavage
cleavage ofof
thetheamide
amidebackbone
backbonechain at the
chain N-terminal
at the N-terminal side
ofMolecules
Ser, Thr, and
2018, 23,Cys
x
side of Ser, Thr, and byCys
the formation of a five-membered
by the formation cyclic urethane
of a five-membered moiety [132].
cyclic urethane moietyThe[132].
formation
31 of 43
The
of the cyclic urethane moiety with an amide backbone makes the amidic carbonyl group susceptible
toformation of the
nucleophilic cyclicThis
attack. urethane moiety with
is presumably anto
due amide backbone
the twist in themakes the amidic
backbone amidecarbonyl groupby
chain caused
susceptible to nucleophilic attack. This is presumably due to the twist in the backbone amide chain
the cyclic urethane moiety. Thus, it was no longer able to form a resonating structure. To achieve
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
cyclic urethane
formation of theintermediate B makes
cyclic urethane the amide
intermediate bond susceptible
B makes the amide bondto nucleophilic
susceptible attack and led to
to nucleophilic
the
attack and led to the cleavage of the amide bond in neutral aqueous conditions (room temp, There
cleavage of the amide bond in neutral aqueous conditions (room temp, pH 7.5, 12 h). pH 7.5,is a
possibility of is
12 h). There nucleophilic
a possibilitydisplacement
of nucleophilic ofdisplacement
the intermediateof theAintermediate
by the amidicA nitrogen through
by the amidic path b.
nitrogen
This could lead to the formation of six-membered ring B 0 , but we did not observe the formation of any
through path b. This could lead to the formation of six-membered ring B′, but we did not observe the
six-membered ringsix-membered
formation of any as analyzed by NMR
ring studies. by NMR studies.
as analyzed

O backbone O
H H
N activation N
N N
H H
O O
OH O
path a path b

path b O O
A N
O O O
H
N
N
O
B’ O O path a

O O

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

Figure 46. Cyclic

Figure46. Cyclic urethane mediated
mediatedactivation
activationofofpeptide
peptidebond.
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
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
epimerization
Molecules
Molecules 2018, 23,free
2018, 23, x C-functionalized peptides.
x 32
32 of
of 43
43

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

O
O
O
O O
O

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

Figure 47.
Figure 47. Pyroglutamyl
Pyroglutamyl imide
Pyroglutamyl imidemediated
imide mediatedactivation
mediated activationof
activation ofofpeptide
peptidebond.
peptide bond.
bond.

O
O
CH
CH33OH
OH
OCH
OCH33
O
O
H O
O NH
H NH22CH
CH22Ph
Ph NHCH
N
N NHCH22Ph
Ph
N O
O
N
O NaBH
NaBH44
O OH
OH
O
O

RSH
RSH O
O
=
= peptide
peptide SR
SR

Figure 48.
Figure 48. Cyclic
Cyclic urethane
Cyclic urethane mediated
urethane mediatedsynthesis
mediated synthesisof
synthesis ofC-terminal
of C-terminalpeptides.
C-terminal peptides.
peptides.

Later,
Later,this
Later, thiscyclic
this cyclicurethane
cyclic urethaneamide-activation
urethane amide-activationapproach
amide-activation approachwas
approach wasapplied
was appliedfor
applied forthe
for thecleavage
the cleavageofof
cleavage ofaaavariety
varietyof
variety
cyclic
of and lasso peptides obtained from nature to determine their sequence, which is
of cyclic and lasso peptides obtained from nature to determine their sequence, which is difficult to
cyclic and lasso peptides obtained from nature to determine sequence, which is difficult
difficult totobe
bebe
determined
determined
determined by by conventional approaches
by conventional approaches (Figure
approaches (Figure 49)
(Figure 49) [135,136].
49) [135,136]. We
[135,136]. We have
We have also
have also applied
also applied this
applied this method
this method
method for for
for
the
the synthesis
the synthesis of
synthesis of aa peptide-based molecular machine
peptide-based molecular
molecular machine(rotaxanes)
machine (rotaxanes)for
(rotaxanes) forthe
for thefirst
the firsttime
first time(Figure
time (Figure50)
(Figure 50)
50) [135,136].
[135,136].
[135,136].

X
X X X
X X
X
X X X
X X
O
O O
X X DIEA,
X DIEA, DMAP,
DMAP, O X
X
X DMF, DSC
X
X DMF, DSC O
O X
N HN
HN X
N N O
O X
X
N
H
H O
OH
OH O O
O
O
O
 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
determined
Molecules by2615
2018, 23, conventional 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 O X
X DIEA, DMAP, O X
X
X DMF, DSC O HN X
N O X
N
H O
OH O
O

O
O X X X
O phosphate X X
NH
buffer X
X X OH N
X X O O H O
2
X X O
Molecules 2018, 23, x 33 of 43

Figure
Figure 49.
49. Cyclic
Cyclicurethane
urethanefor
forcleavage
cleavageofofcyclic
cyclicpeptides.
peptides.

H
N
O
S9 I10
O
L11
L11
I10 Q12 Q12
OH
S9 P7Q13 R6 P7Q13 R6
O 1
D8
D8 DSC,H2O
G5 G5
G1 E14 F4 G1 E14 F4
V2 G3 V2 G3

Q15 Q15

A17 A17
K17 K17

P18 P18

M19 M19

Figure 50.
Figure Synthesisof
50. Synthesis ofrotaxane
rotaxanefrom
fromlasso
lassopeptide.
peptide.

4.8.
4.8. Intein-Inspired
Intein-Inspired Amide Cleavage Chemical
Amide Cleavage Chemical Device
Device
A
A photoresponsive
photoresponsive device
device was
wasdeveloped
developedfor forthe
thecleavage
cleavageofofthe
theamide
amidebond
bondatatthe
theC-terminus
C-terminusof
the AsnAsn
of the residue [137].
residue This This
[137]. approach was inspired
approach by intein-mediated
was inspired proteinprotein
by intein-mediated splicingsplicing
and its chemical
and its
environment was mimicked by the incorporation of geminal dimethyl groups
chemical environment was mimicked by the incorporation of geminal dimethyl groups and and a secondary amine
a
on the asparagine scaffold.
secondary amine on the asparagine scaffold.
The secondary amine
The secondary amineactsactsasasanan intramolecular
intramolecular base,
base, which
which enhances
enhances the nucleophilicity
the nucleophilicity of theof
the amide nitrogen (Figure 51). The geminal dimethyl groups led to a
amide nitrogen (Figure 51). The geminal dimethyl groups led to a Thorpe-Ingold effect, which Thorpe-Ingold effect,
which enhances the intramolecular attack, thus assisting in the formation of the
enhances the intramolecular attack, thus assisting in the formation of the succinimide ring [138–140]. succinimide
ring [138–140]. The o-nitrobenzyloxycarbonyl
The o-nitrobenzyloxycarbonyl (o-NBnoc) masks(o-NBnoc) masks the
the basic character ofbasic characteramine
the secondary of the[141,142],
secondary
amine
thus leading to the photo triggered cleavage of an amide bond by the deprotection of the secondaryof
[141,142], thus leading to the photo triggered cleavage of an amide bond by the deprotection
the secondary
amine amine unit
unit containing the containing the o-nitrobenzyloxycarbonyl
o-nitrobenzyloxycarbonyl group. group.

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

O O H2N

O2N
 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],
thus leading
Molecules to2615
2018, 23, the photo triggered cleavage of an amide bond by the deprotection of the secondary
33 of 43
amine unit containing the o-nitrobenzyloxycarbonyl group.

O UV H O
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.
Figure Intein-inspiredamide
51. Intein-inspired amidebond
bondcleavage.
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 HCO2 H) 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).
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.
This strategy
Molecules 2018, 23,is2615
widely distinct from Lewis acid, promoted by the Ser-selective peptide hydrolysis
34 of 43
through N-to-O rearrangement.

H2O NO CuI + keto-ABNO-H

H2 NO2
N N
HO O CuII
O
H O
N N O
Peptide A N Peptide B
H H
O
(1e + 1e) oxidation

O O O
O O
H O H
N O2 (Cu)
N N
Peptide A Peptide B Peptide A N Peptide B
H H B
A O O

HCO2H
O O O O
H H
+ N hydrolysis N
X = NH2 or OH X X N
D’ D H C
O O

Molecules 2018, 23, x Serine selective

selectiveaerobic
aerobiccleavage
cleavageofofpeptide
peptidebonds.
bonds. 35 of 43
Figure 52. Serine

O O
H by the Formation
4.10. Hydrolysis of Amide Bonds of Oxazolinium H
Specie: Function of Acyl Protecting
R2 N R3 TFA/H2O R2 N
Group N OH + R3NH2
H
O R1 1 O R
Peptides containing a simple N-acyl group activates the amide bond four bonds away from an
R3 = H, Ala-NH2
acyl group for cleavage under acidic conditions [144]. First, TFA leads to the protonation of amide
R2 = alkyl,
carbonyl followed by the nucleophilic attack from the oxygen G acyl carbonyl to generate a five-
of the
Mechanism
membered oxazolinium specie A in the peptide chain. Second, the collapse of the oxazolinium
R1
intermediate A leads toOthe cleavage O
of an unactivatedRamide
1 bond (FigureR53).
3NH
NHR NHR OH
The nature of R
the aromatic group,
3 G, was responsible for the
3 O hydrolysis of the peptide
rate of
2 N R2 N R1
bond. Electron-donating Hgroups H
O increase the rate of hydrolysis
O whereasN electron withdrawing
H R2
substituents decrease it (Figure 53). AH

O R1 O
OH O
R2 N R3NH2 + R1
H N
O R2 H

Figure 53.
Figure Oxazoliniumspecies
53. Oxazolinium speciesformation.
formation.

4.11. Hydrazinolysis for the Cleavage of Amide Bonds

4.11. Hydrazinolysis for the Cleavage of Amide Bonds
The
The hydrazinolysis
hydrazinolysis of of unactivated
unactivated amide
amide bonds
bondswas
wasaccelerated
acceleratedby
bythe
theaddition
additionofofammonium
ammonium
salts. The reaction proceeds at 50–70 ◦ C to give peptide cleavage products and exhibits a broad
salts. The reaction proceeds at 50–70 °C to give peptide cleavage products and exhibits a broad
substrate
substrate scope
scope that
that out-performs existing amide
out-performs existing amide bond
bond cleavage
cleavagereactions
reactions[145].
[145].This
Thisapproach
approachwas was
applied for the cleavage of the peptide bonds without racemization at the α-position
applied for the cleavage of the peptide bonds without racemization at the α-position of the amino of the amino
acids
acids (Figure
(Figure 54).
54). It
It was
was also applied to
also applied to the
the cleavage
cleavage of
of the N-acetylgroup
theN-acetyl groupfrom
fromthe
theamino
aminosugar.
sugar.

O O NH4I
H H H
N N hydrazine hydrate N
N N cleavage H2N
H H
O O O

OH OH
O NH4I O
HO hydrazine hydrate HO
HO HO
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
applied2018,
Molecules for 23,
the2615
cleavage of the peptide bonds without racemization at the α-position of the amino
35 of 43
acids (Figure 54). It was also applied to the cleavage of the N-acetyl group from the amino sugar.

O O NH4I
H H H
N N hydrazine hydrate N
N N cleavage H2N
H H
O O O

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

Hydrazinolysis for
Figure 54. Hydrazinolysis forthe
thecleavage
cleavageof
ofpeptide
peptidebonds.
bonds.

4.12.
4.12. Amide
Amide Bond
Bond Cleavage
Cleavage of the N-Methylcysteinyl
of the N-Methylcysteinyl Peptide
Peptide
Tam
Tam et et al. developeda aselective
al. developed selective bi-directional
bi-directional peptide
peptide bond bond cleavage
cleavage approach
approach utilizing
utilizing N-
N-methylcysteine
methylcysteine (MeCys) (MeCys)in in
thethe Xaa-MeCys-Yaa
Xaa-MeCys-Yaa peptides
peptides (Xaa
(Xaa andand Yaa,
Yaa, non-cysteine
non-cysteine residues)
residues) [146].
[146].
Under
Under strong
strong acidic
acidic conditions, peptide Xaa-MeCys-Yaa
conditions, peptide Xaa-MeCys-Yaa led led to
to the
the formation
formationofofan anoxazolinium
oxazolinium
intermediate, resulting in the cleavage of the Xaa-MeCys bond. The oxazolinium
intermediate, resulting in the cleavage of the Xaa-MeCys bond. The oxazolinium intermediate was intermediate was
later
later trapped
trapped by by thiocresol
thiocresol (TC)
(TC) toto form
form aa Xaa-MeCys-TC thioester(Figure
Xaa-MeCys-TC thioester (Figure55).
55).The
Thereplacement
replacementofof
MeCys by Cys residue did not result in the peptide bond cleavage, suggesting
MeCys by Cys residue did not result in the peptide bond cleavage, suggesting the important role the important roleofof
N-methylation
N-methylation
Molecules in MeCys residue for the formation of oxazolone.
2018, 23, xin MeCys residue for the formation of oxazolone. 36 of 43

O R1 O O R1 O
H H
N protonated N
Peptide N NH2 Peptide N NH2
O R2 O R2
O R1 O R1 H
S OH
Peptide N ptol Peptide N
O O
4-Mercaptotoluene H 2O
O O HO O
O
H2N NH2 + O Peptide N NH2
R1 H
R2 N N R2
Peptide R1

N-methylcysteinylpeptide
Figure 55. N-methylcysteinyl peptidecleavage.
cleavage.

4.13.
4.13. N-MeAib
N-MeAib Induced
Induced Unusual Cleavage of
Unusual Cleavage of Amide
Amide Bonds
Bonds
Peptides
Peptides containing acylated N-methyl-aminoisobutyryl
containing acylated N-methyl-aminoisobutyryl (NMeAib)(NMeAib)residues
residuesshowed
showedunusual
unusual
cleavage
cleavage of amide bonds
of amide bonds under
underacidic
acidicconditions.
conditions.The The cleavage
cleavage takes
takes place
place at the
at the C-terminal
C-terminal sideside
of
of the NMeAib residue (Figure 56) [147]. X-ray diffraction studies of the NMeAib
the NMeAib residue (Figure 56) [147]. X-ray diffraction studies of the NMeAib containing molecules containing
molecules
showed that showed that atom
the oxygen the oxygen atom of group
of the carbonyl the carbonyl group ofresidue
of the preceding the preceding residue
is close to is close
the carbonyl
to
carbon of the NMeAib residue, thus acting as an internal nucleophile forming a tetrahedral a
the carbonyl carbon of the NMeAib residue, thus acting as an internal nucleophile forming
tetrahedral
intermediate.intermediate. Once this intermediate
Once this tetrahedral tetrahedral intermediate
was formed, wasloneformed, lone pair
pair electrons electrons
on the nitrogenon of
the
nitrogen of the phenylalanine
the phenylalanine werebe
were no longer noable
longer be able
to form to form resonating
resonating structures
structures with with thegroup
the carbonyl carbonyl
of
group of NMeAib. In fact, the phenylalanine nitrogen becomes a proton acceptor
NMeAib. In fact, the phenylalanine nitrogen becomes a proton acceptor like the amines, which like the amines,
which resulted
resulted in theincleavage
the cleavage of amide
of the the amide
bondbond followed
followed by by
thethe removal
removal of of phenylalanine
phenylalanine andthe
and the
formation
formation ofof an
an oxazolinium intermediate,which
oxazolinium ion intermediate, whichfurther
furtherreacts
reactswith
withwater
watertotoform
formaacarboxylic
carboxylic
acid product.
acid product.

O O O O
H H
N protonated N
N OCH3 N OCH3
O O
H
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
formation
Molecules of23,
2018, an2615
oxazolinium ion intermediate, which further reacts with water to form a carboxylic
36 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 work up O
N
NH
O N OH
PheOMe O
oxazolinium ion
N
tetrahedral intermediate

Figure 56.
56. N-Me
N-MeAib
Aibmediated
mediatedamide
amidebond
bondcleavage.
cleavage.

5.
5. Conclusions
Conclusions
This
This mini-review
mini-review highlights the methods
highlights the methods for for the
the activation
activation ofof unactivated
unactivatedamide
amidebonds
bondsinin
biomolecules.
biomolecules. This Thisreview
reviewfurther
furtherhighlights
highlights thethe application
application of of these
these methods
methods in the
in the sequencing
sequencing of
of proteins
proteins andandthethe synthesis
synthesis ofof peptideacids,
peptide acids,thioesters,
thioesters,alcohols,
alcohols,and
andamides.
amides.These
Thesestudies
studiesshowed
showed
that
that there
there is
is still
still aa lot
lot to
to learn
learn from enzymes catalyzed pathways
enzymes catalyzed pathwaysand andhow howwewecan
candevelop
developenzyme
enzyme
mimetics
mimetics for catalyzing the cleavagecleavage ofof unactivated
unactivated and and highly
highlystable
stableamide
amidebonds
bondsatatparticular
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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Where Have All the New Reactions Gone? J. Med. Chem. 2016, 59, 4443–4458. [CrossRef] [PubMed]
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expression and protein purification. Methods Enzymol. 2000, 326, 601–617.
8. Kemnitz, C.R.; Loewen, M.J. Amide Resonance correlates with a breadth of C-N rotation barriers. J. Am.
Chem. Soc. 2007, 129, 2521–2528. [CrossRef] [PubMed]
9. Mujika, J.I.; Mercero, J.M.; Lopez, X. Water-promoted hydrolysis of a highly twisted amide: Rate acceleration
caused by the twist of the amide bond. J. Am. Chem. Soc. 2005, 127, 4445–4453. [CrossRef] [PubMed]
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