43
Peptide Side Reactions Yi Yang, Chemical Development 21 st Apr. 2016, IPC
Peptide Side ReactionsYi Yang, Chemical Development21st Apr. 2016, IPC
2
Amino Acid in Peptide SPPS
H2N
O
OH
R-amino group/N
backbone amino group
backbone carboxylate
-Carbon/C
Side chain
NH O
OH
R1
PG1
PG2
NH O
OH
HN
Fmoc
Boc
Base labile
Acid labile
H2N
O
OH
R1
+H2N
O
R2H2N
O
R1HN
O
R2
H2O
3
Solid Phase Peptide Synthesis (SPPS)X X = Cl, OH, NH2
NH O
OH
R1 PG1
Fmoc
NH O
O(NH)
R1 PG1
Fmoc
piperidine
HN
H2N
O
O(NH)
R1 PG1
Fmoc
HN
O
OH
R2
PG2
couplingreagent
Fmoc
HN
O
O
R2
PG2
Y
Y: activating group
NH O
O(NH)
R1 PG1
Fmoc
HN
O
R2
PG2
Fmoc deprotection
Fmoc-Amino acid coupling
repetitive Fmoc deprotection and AA coupling
NH O
O(NH)
R1 PG1
HN
O
R2
PG2
Peptide segment
PG3
PG4
O PGn-1
H2N
Rn
PGn
TFA/Scavenger
NH O
OH(NH2)
R1
HN
O
R2Peptide segment
O
H2N
Rn
Loading
Cleavage and Global deprotection
Peptide Assembly
4
A. Peptide Fragmentation/Deletion Side Reactions
A-1: N-Ac-N-alkyl peptide acidolysis
‣ Peptides with a motif of N-Ac-N-alkyl-Xaa sequence at the N-terminus have the distinctively high propensity to acidolysis
O
N
R1
O
NH
H+
N
OOH
R1
+Peptide OH H2N Peptide OH
Dyn A(1-11) H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-NH2
Arodyn 1: Ac-Phe-Phe-Phe-Arg-Leu-Arg-Arg-D-Ala-Arg-Pro-Lys-NH2
Arodyn 2: Ac-N-Me-Phe-Phe-Trp-Arg-Leu-Arg-Arg-D-Ala-Arg-Pro-Lys-NH2
Arodyn 3: CH3OCO-N-Me-Phe-Phe-Trp-Arg-Leu-Arg-Arg-D-Ala-Arg-Pro-Lys-NH2
Arodyn 4: N-Me-Phe-Phe-Trp-Arg-Leu-Arg-Arg-D-Ala-Arg-Pro-Lys-NH2
Ac-N-Me-Phe-Phe-Trp-Arg-Leu-Arg-Arg-D-Ala-Arg-Pro-Lys-NH2
H+
H-Phe-Trp-Arg-Leu-Arg-Arg-D-Ala-Arg-Pro-Lys-NH2
O
N
R
O
5
A. Peptide Fragmentation/Deletion Side Reactions
A-2: -N-Acyl-N-alkyl-Aib-Xaa acidolysis
‣ endo-peptide bond scission at ‑N-acyl-N-alkyl-Aib-Xaa- sequence upon acid treatment.
H2N
HN
O
O
NH
HN
O
O
NOH
O
HN
HNN
NH
NH
O
O
OO
O
O
OTFA
O
OLabile Bond
O
N
O
HN
O
H+O
N
OH+
HN
O
N+
O NH
OH
O
Tetrahedral Intermediate
N+
OO
O
NOH
O
H2O
H2NO
oxo-oxazolinium derivative
6
A. Peptide Fragmentation/Deletion Side Reactions
A-3: Acidolysis of -Asp-Pro- bond
‣ -Asp-Pro- peptide bond is labile under acidic conditions such as in TFA, HF, formic acid and acetic acid, even at weak acidic milieu (pH 4)
Segment 1 Asp-Pro Segment 2H+
Segment 1 Asp-OH
+
Segment 2H-Pro
HN
O
O
OH
N O
NH
H+
H2O
N+
HNO
O
ONH
2 H2O
NH
HN O
HN
O
OH
O
OH
Segment 1 Segment 1
Segment 2
Segment 2
Segment 1Segment 2
+
protein E298D eNOS has been identified to suffer from acidolytic fission at -Asp298-Pro299- sequence, giving rise to 100 kDa and 35 kDa fragments, while its native protein counterpart eNOS (Glu298) is exempted from acidolysis under the same conditions.
Herpes simplex virion-originated peptide might suffer from -Asp-Pro- cleavage during FAB-MS analysis; while –Asn-Pro- is exempted from such degradation.
7
A. Peptide Fragmentation/Deletion Side Reactions
A-4: Auto-degradation of peptide N-terminal H-His-Pro-Xaa- moiety
‣ The amide bond between Pro and the amino acid on its C-terminal side in peptide sequence could undergo fragmentation process catalyzed by the imidazole group on N-terminal His.
H-His-Pro- PeptideH+
His-Pro fragment
Peptide
[M] [M-234]
50% AcOH, H2N
O
RO
N
NH
N
ONH
N
NH
H2N
O
NHO
R
NH
HO
8
A. Peptide Fragmentation/Deletion Side Reactions
A-5: Acidolysis of the peptide C-terminal N-Me-Xaa
Peptides containing C-terminal N-Me-Xaa residues are less stable in acidic condition, giving rise to deletion sequence.
Peptide
O
NHO
OH
R
H+
Peptide
O
HN
H
O
OH
R
OH
+
O
N
R
O
HO
H+HO
NR
O
O
O
O
O
R
HN O
OHR
HNH3O+
O+
O
H
9
A. Peptide Fragmentation/Deletion Side Reactions
A-6: Deguanidination side reaction on Arg
If the guanidino moiety from Arg side chain is acylated by amino acid derivatives, it could be decomposed into Orn side product
NH O
NH
NH2HN
NH O
NH2
[M] [M-42]
H2N
O
N
HNHN
PG
PG
'
PG = Protecting GroupX = Leaving Group
NH
R
OX
O
Fmoc
NH O
N
HNN
PG
PG
'
OHN
R
Fmoc
O
NH
R
FmocNH O
HN
NH
N
PG
PG
O
H2N
R
OHN
RBase
NH O
N
HNN
PG
PG
'
O
H2N
R
O
H2N
R
Orn
10
A. Peptide Fragmentation/Deletion Side Reactions
A-7: DKP (2,5-diketopiperazine) formation The nucleophilic attack of the Nα group from the peptide N-terminal amino acid on the carbonyl functionality, either in the form of amide or ester moiety from the second amino acid, gives rise to the fission of the affected amide or ester bond. The N-terminal dipeptide is split off the peptide backbone in the form of a six-member ring derivative diketopiperazine.
NH
O
NH
O
HNO
NH
O N
O
HN
NH
OO
NH
O
HN
ONH
SO
HN
OS
H2N
N
OHO
O
HN
N O
O
NH2
Exact Mass: 1424.60
NH
O
NH
O
HN
O
NH
HN
O
O
HN
O
HN
O
NH
SO
HN
O
S
H2N
N
OH
OO
HN
N
O
O
NH2
Exact Mass: 1270.53
FE 205030
[M-154]
FE 205030 Des Gly-Pro
H2N
R1
O
HN
R2
O
X
R3
O
HN R1
ONH
R2
O
HX
R3
O
X = O, NH
+
11
B. β-Elimination Side Reactions
• β-elimination is a group of common side reactions that predominantly affect peptides bearing electron-withdrawing substituent located on the side chain Cβ position, such as Cys and phosphorylated Ser/Thr.
• These peptides could suffer from β-elimination mostly under base treatment.
• The consequence of this side reaction is the elimination of substituent on Cβ and the formation of dehydroalanine and/or corresponding relevant adducts.
NH O
EWGBase
NH O
H
12
B. β-Elimination Side Reactions
B-1: β-Elimination of Cys sulfhydryl side chainHα on Cys residue in the parental peptide is vulnerable to the base treatment and the protected sulfhydryl derivative suffers from the degradation by means of splitting off the β-position on Cys side chain, giving rise to the formation of a dehydroalanine intermediate.
NH O
SPiperidine
NH O
H
PG
PG: Protecting Group
PG-SH
O NH
O R1
O
HN
O
O
S
PG
H
HN
H2N
R1
O
HN
O
O
HN
H2N
R1
O
HN
O
O
N
dehydroalanine intermediate 3-(1-piperidinyl)alanine adduct
H
PG = Protecting GroupDBF = Dibenzofulvene
DBF, CO2
[M+51]
13
B. β-Elimination Side Reactions
B-2: β-Elimination of phosphorylated Ser, Thr
Utilization of Fmoc-Ser/Thr(PO3R2)-OH (R=methyl, ethyl, tert-butyl, benzyl) building blocks for the preparation of phosphopeptides could potentially cause β-elimination side reaction resembling Cys β-elimination.
O
O
NH
O
OR1
P OO
OR R
Fmoc-Ser/Thr(PO3R2)-OH(Thr: R1=CH3; Ser: R1=H)
R= methyl, ethyl, tert-butyl or benzyl
Base
H2N
O
R1
O-
P OO
OR R
O
O
NH
O
OR1
P OO
OR R
Fmoc-Ser/Thr(PO3R2)-OH(Thr: R1=CH3; Ser: R1=H)
R= methyl, ethyl, tert-butyl or benzyl
O NH
O R1
O
HN
O
NH
O
H
HN
H
R2
O
X
P
O
OOR R
H2N
R1
O
HN
O
NH
HN
dehydroalanine intermediate
O
X
R2
3-(1-piperidinyl)alanine adduct
H2N
R1
O
HN
O
NH
NO
X
R2
Thr: R3=CH3; Ser: R3=HR=methyl, ethyl, tert-butyl or benzylX=Peptide Fragment, O, NH
R3
R3
R3
P
O
O O
O-
R R
[M-98] [M-13]
14
C. Peptide Rearrangement Side Reactions
• Undesirable peptide rearrangement represents a category of common side reactions occurred in the process of peptide manufacture as well as storage.
• pH is one of the most important factors that drive peptide rearrangement process.
• One of challenges inherent to these types of side reactions is that the derived side products are frequently isomer to the target peptides and the development of analytical methods with respect to the re-arranged peptide impurities poses a critical task.
• Only acyl O N migrations are discussed herein.
15
C. Peptide Rearrangement Side Reactions
C-1: Acid catalyzed acyl NO migration and the subsequent peptide acidolysis
• Acyl NO migration process was initially detected under the circumstances of substrate treatment by strong acid such as H2SO4, HF or HCl.
• TFA-catalyzed acyl NO migration is becoming one of the most frequently detected side reactions in peptide synthesis.
• Its severity is evidently correlated to the sequences from the parental peptide. • The acceptors of the acid-catalyzed acyl shift in peptide synthesis are normally those residues that
bear nucleophilic substituents like Ser, Thr or Cys.
NH
R
O
HN
OH+
HO X
NH
R
HO
HN
O
O X
H+
NH
R NH3
O
XO O
X = H or CH3
NH
R
O
HN
O
HO X
H+
NH
R NH3
O
XO O
X = H or CH3
Segment 1
H2O
NH
R
OH
O
H2N
O
HO X
+
Segment 1
Segment 1
Segment 2 Segment 2
Segment 2
16
C. Peptide Rearrangement Side Reactions
C-1: Acid catalyzed acyl NO migration and the subsequent peptide acidolysis
NH O
HN
O
R
O
Fmoc piperidineH2N
O
HN
O
R
OAc2O
NH O
HN
O
R
OO
Pyridine
TFA
NH O
HN
OH
R
OOH+
OH-H3N
O
HN
O
R
O
O
H3O+
H3N
O
HN
OH
R
O
+ +
Targtet product [M]
O-Ac Isomer Imputiy [M]
Deacetyl impurity [M-42]
17
C. Peptide Rearrangement Side Reactions
C-2: Base catalyzed acyl ON migration
Base catalyzed acyl ON shift could be regarded as the reverse reaction of acid catalyzed acyl NO migration.
H2N
O
O
HN
O
X
R
HN
O
O
X
HO
R
HN
base baseHN
HO
O
XO
R
NH
X = H or CH3
H2N
O
O X
X = H or CH3
(TFA)
H2N
O
O X
O
FF
F
neutralization HN
O
HO XO
FF
F
O
OHF
FF
H3N
O
HO X
(TFA)
O
OHF
FF
[M] [M+96]
N-trifluoroacetylated impurity
18
C. Peptide Rearrangement Side Reactions
C-2: Base catalyzed acyl ON migration
HN
O
O
NH
O
O
O
OFmoc
O
HN
O
O1) Pd(PPh3)42) PyBOP/HOBt/DIEA
HN
O
O
Fmoc
O
HN
O
HN O
O
H2N
O
O
O
HN
O
HN O
O
PiperidineO
HN
O
HN O
O
O N acyl shift
HN
OH
O
Peptide
PeptidePeptide
Peptide
Piperidine
Target Intermediate (Ester)Amide Isomer impurity
19
D. Intramolecular Cyclization Side Reactions
D-1: Aspartimide formation
• Asp converted to imide by repelling a H2O molecule [M-18].• One of the most severe side reactions on peptides.• Both acid and base-catalyzed.• Occurred both in peptide synthesis, formulation, and storage.• Sequence dependent -Asp-Xaa-• Could also affect Glu, but to a much lesser extent (glutarimide).
NH
HN
O
O
O
OH
NH
O
N
OO
R R
[M] [M-18]
NH
HN
O
O
O
OH Acid or Base
NH
O
N
OO
R R
NH O
HN
R
O
HN
O OH
HN
O
O
R
(L/D)--Asp-peptide
(L/D)-iso-Asp-peptide 5
NH O
HN
R
O
HN
O
HN
O R
(L/D)--Asp-peptide piperidide
(L/D)-iso-Asp-peptide piperidide 6
N
O
N
O
Racemization
Nu
Nu
Path A
Nu = H2O Nu = piperidine
O
OHPath A
Path B
+
+
Path B
Nu = H2O Nu = piperidine
[M-18]
[M] [M+67]
[M] [M+67]
[M]
20
D. Intramolecular Cyclization Side Reactions
D-1: Aspartimide formation
OO
ONH
NH
OO
ROO
NH
O
HN
O
NHO
OH
Activation
R
O
HN
O
NHO
X
R
O
HN
O
N
O
R
O
HN
O
NHO
R
O X = Leaving GroupPG = Protecting Group
PG
PG
PG PG
PG
PG
HN
O
NHO
OH
R
O
peptide
HN
NH2
OPG PGHN
O
NHO
X
R
O
HN
NH2
OPG PGActivation H
N
O
NH
O
R
O
HN
HN
OPG PG
HN
O
N
O
R
O
HN
NH2
OPG PG
X = Leaving GroupPG = Protecting Group
peptide
peptide
peptide
21
D. Intramolecular Cyclization Side Reactions
D-2: Asn/Gln deamidation
• Asn/Gln-containing peptides are frequently involved in deamidation side reactions • Amide side chains are converted into the corresponding carboxylates [M+1].• Diverse mechanism.• Both acid- and base-catalyzed.• Sequence dependent -Asn-Xaa- N
H
HN
O
O
O
NH2
R
[M] [M+1]
NH
HN
O
O
O
OH
R
NH O
HN
O
NH2
R
O
NH O
N
O
NH2
R
Obase
NH O
N
-ONH2 O
R
NH3
NH O
N
O O
R
Racemization
NH O
N
O O
R
H2OPath A
H2OPath B
NH O
HN
O O
R
OH
NH
O
NH
OO
R
HO
Path A
Path B
32
22
E. Side Reactions on Amino Groups
Nα-acetylation H2N
R
O
NH
R
O
O
[M] [M+42]
Nα-trifluoroacetylation
F3C
O
OH HX+ F3C
O
X
N = O or NH
R R
[M] [M+96]
H2N
HN
+
HN
O
NH
O
OH
F3C
O
OH
Coupling Reagent
NH
O
F3C
O
O
O
O
O
O
Segmen ASegmen A
Segmen B
Segmen B
Segmen B
TFAO
O
HO
peptide
O
NH
R
Boc O
O
O
O
H3N
R
O
F3C
DIEA
O
O
O
O
H2N
R
O
F3C
O N acyl shift O
O
HO
O
NH
R
F3C
O
peptide
peptidepeptide
23
E. Side Reactions on Amino Groups
Nα-formylation
Nα-alkylation
H2N
R
O
N
O
H
NH
R
O
O
H
[M] [M+28]
H2N RH H
ONH
HORH+
NRH+
HN
O
N+
HN
O
N
+
[M] [M+30] [M+12]
R1
NH2
O
HN
O
R2
HCHO H2O
R1
N
O
HN
O
R2
R1
HN
O
N
O
R2
[M] [M+12]
24
E. Side Reactions on Amino Groups
Nα-alkylationHN
O
NH2
HN
O
NH
H2N NH
+HCHO
NH
ON
NH
ONH
NH
N
HN
O
NNH
O
N
NH
NH
and/or
25
E. Side Reactions on Amino Groups
N-alkylation on N-terminal His via acetone-mediated enamination
H2N
O
NH
N
OH+
H2N
O
N
N
OH
H+
H2O
H2N
O
N
N
[M] [M+40]
26
F. Peptide Oxidation Side Reactions
Cys Oxidation
NH O
SH
NH O
S
OH
NH O
S
O
OH
NH O
SO
OH
O
Cys (-2) Cys sulfenic acid (0) Cys sulfinic acid (+2) Cys sulfonic acid (+4)
NH O
SH
[O]NH O
SNH
O
HSOH
NH O
S
NH
O
S
NH O
S
O
NH O
NH O
S
NH O
S
OH
S
OH
H2O
thiosulfinite
NH
S
O
OH
H2N R
NH
S
O
HNR
H2O
HN
O
NH
SO
R'
OH
HN
O
NS
O
R'
H2O
sulfenamide sulphenyl amide
27
F. Peptide Oxidation Side Reactions
Met OxidationNH O
S
[O]
NH O
SO
[O]
NH O
SO
O
Met-sulfoxide Met-sulfone
Trp OxidationNH
HN
O
NH
HN
O
O
Oia
NH
HN
O
CHO
ONH2
HN
O
O
NH
HN
O
CHO
OHO NH2
HN
O
O
HO
KynNFK
3-hydroxykynureninehydroxy-N-formylkynurenine
Trp
NH
HN
O
HO
5-hydroxy-Trp
[M] [M+16] [M+32] [M+4]
28
F. Peptide Oxidation Side Reactions
His Oxidation
HN
O
N
NH
HN
O
N
NH
OH
HN
O
NH
NH
O
[O]
29
G. Cys Disulfide-related Side Reactions
Disulfide Scrambling
S SR1
R2
-S R3S S
R2
R3R1 S- +
S S
-S
SH
S
SH
S
-S
Thiol-Cystine disulfide exchange
S S
S S
-S R
-S
S S
S
SR
SS
SRS
-S
/ R-S-S-R
Redox buffer induced disulfide bond scrambling
30
G. Cys Disulfide-related Side Reactions
Disulfide Degradation
Homolytic degradation
NH O
S
S
HN
O
OH-
NH O
S-
S
HN
O
HO
OH-
-S
HN
O
H2O
+
S
HN
O
O
-O
NH O
S
S
HN
O
OH-H
NH O S-
S
HN
O
H2O
+
HS
HN
O
S
OH-
O-
S
HN
O
HS- -S
HN
O
S
HN
O
O
-O
+Cys persulfide
β-elimination
NH O
S
S
HN
O
OH-
H NH O
S
-S
HN
O H
+
OH-
NH O
OH
thioaldehyde
α-elimination
31
G. Cys Disulfide-related Side Reactions
Trisulfide Formation
HN
O
S
S
NH
O
HN
O
SH
peptide
peptideNH
O
HS
-eliminationHN
O
S
S-
NH
O
HN
O
SH
peptide
peptideNH
O
HS
HN
O
S S
HN
O
S
peptide
HN
O
S
HN
O
S
peptide
disulfide scrambling
Trisulfide
[M]
[M+32]
LanthionineFormation -S
HN
OHN
O
S
HN
O
HN
ONH O
S
S
HN
O
-elimination
Lanthionine[M][M-32]
32
G. Excipient-induced Side Reaction
Excipient Impurity Potential Side ReactionsPeroxide Oxidation on Cys, His, Trp, Met, etc.
Formic acid Fomylation on amino, hydroxy group
FormaldehydeImine formation on amino group, cross linking between
amino acid, N-alkylation, etc.Aldehyde (Furfural, 5-
hydroxymethyl furfural)Imine formation on amino group
glucose Maillard Reaction with amino groupFormic acidacetic acid acetylation on amino and hydroxy group
Benzyl alcohol BenzaldehydeAldehyde Peroxide
Starch Formaldehyde
MannitolReducing sugar (mannose,
glucose)Imine formation on amino group
arylmethylamine degardaion
Povidone, Polysorbate (Tween)
Lactose
PEG
HN
O
NH
O
NH2
O
R H
HN
O
NH
O
N
R
HN
O
NH
O
N
R
H2O
HN
O
NH
O
O
H2NR
H
[M][M-1]
33
34
Average Δ Mass
Modification Proposed Side Reaction Scheme
-98 β-elimination of phosphopeptide
-80 Peptide dephosphorylation
-42 Conversion of Arg to Orn via deguanidination
-34 Cysteine β-elimination
-32 Disulfide desulfurization
NH
O
O
P
O
HO OH
NH
O
NH
O
O
P
O
HO OH
NH
O
OH
NH
O
NH
HN NH2
NH
O
NH2
S
HN
O
HN
ONH O
S
S
HN
O
NH O
SH
NH O
35
Average Δ Mass
Modification Proposed Side Reaction Scheme
-32 Disulfide desulfurization
-26 Reduction of Nva(N3) to Orn
-18 Pyroglutamate formation from Glu
-18Aspartimide/Glutarimide formation
from Asp/Glu
-18 β-elimination of Ser
S
HN
O
HN
ONH O
S
S
HN
O
H2N
OHO
O
HN
O
RNH
HN
O R
O
O
NH
HN
O
O
O
OH
NH
O
N
OO
R R
NH
O
OH
NH
O
NH O
N3
NH O
NH2
36
Average Δ Mass
Modification Proposed Side Reaction Scheme
-18 dehydration of Asn/Gln
-17 Pyroglutamate formation from Gln
-17Aspartimide/Glutarimide formation
from Asn/Gln
-16 H -phosphonate formation
-14 Thioanisole-induced Tyr demethylation
-2 Cysteine oxidation to Cystine
H2N
OH2N
O
HN
O
RNH
HN
O R
O
O
NH
HN
O
O
O
NH2
NH
O
N
OO
R R
S
NH O
O
NH O
O+
H+
S+NH O
OH
+
H
NH
NH2
O
O
OHNH
N
OH
O
1-2 1-2
NH O
O
PHO
O
H
NH O
O
PHO
O
OH
NH O
SH
NH O
HS
NH O
S
NH O
S
peptide peptide
37
Average Δ Mass
Modification Proposed Side Reaction Scheme
+1 Asn/Gln hydrolysis
+1 Peptide amide hydrolysis
+2 Cystine reduction
+2 Trp reduction
+4 Oxidation of Trp to Kynurenine
+12 Imine formation on amino-containing peptide
+12 Imidazolin-4-one formation on peptide N-terminus
NH
HN
O
O
O
NH2
RNH
HN
O
O
O
OH
R
NH O
NH2
R
NH O
OH
R
HN
O
NH
HN
O
NH
NH
HN
O
NH2
HN
O
O
H2N RH H
ON
R+
R1
NH2
O
HN
O
R2
HCHO H2O
R1
N
O
HN
O
R2
R1
HN
O
N
O
R2
NH O
SH
NH O
HS
NH O
S
NH O
S
peptidepeptide
38
Average Δ Mass
Modification Proposed Side Reaction Scheme
+12Formaldehyde-induced crosslinking of peptide N-terminal Cys, Trp, Lys(Nma)
+14 Methylation of amino group
+14 methylesterfication on carboxyl group
+16 Oxidation of Cys to Cysteine sulfenic acid
+16 Oxidation of Trp to Oia (Oxindolylalanine)
+16 Oxidation of Met to Met sulfoxide
+16 Oxidation of His to 2-oxo-His
OHCHO
O
NH
NH2
HS
O
NHS
NH2
HCHO
O
NH
NH
NHO
NH2
NH
O
NO
NH2
N
O
HCHO
R NH2 NH
R
R
O
OH R
O
O
NH O
SH
NH O
S
OH
NH
HN
O
NH
HN
O
O
NH O
S
NH O
SO
HN
O
N
NH
HN
O
N
NH
OH
HN
O
NH
NH
O
39
Average Δ Mass
Modification Proposed Side Reaction Scheme
+25 Cys cyanilation
+26Schiff base formation from amino group and
acetaldehyde
+27 Cyanohydrin formation
+28 Peptide formylation at N α, Lys-N ε, Trp-N in or His-N im
+28 Carboxylate ethylation
+28 N -dimethylation
+32 Trisulfide formation
+32 Oxidation of Met to Met sulfone
+32 Oxidation of Cys to Cysteine sulfinic acid
HN
O
SH
HN
O
S
CN
H2N R NR
R2R1
OR2R1
OH
CN
H2N R NH
R
O
H
R
O
OH R
O
O
R NH2 NR
HN
O
S S
HN
O
S
HN
O
S
HN
O
S
NH O
S
NH O
SO
O
NH O
SH
NH O
S
O
OH
40
Average Δ Mass
Modification Proposed Side Reaction Scheme
+32 Oxidation of Trp to N -formylkynureine
+34 Chlorination of Tyr
+40 Enamination of His imidazolyl side chain by acetone
+40Acetone induced peptide N -terminal imidazolidinone
formation
+42 Acetylation on N α, Lys-N e , O -Ser/Thr
+44 Trp carbamate
NH
HN
O
NH
HN
O
CHO
O
NH O
OH
Cl
NH O
OH
H2N
O
NH
N
H2N
O
N
NO
+
H2N
O
HN
R1 O
R2
HN
O
NR1O
R2
X
O
X = leaving group
OHR NH2R'or
O
O
R orNH
O
R'
HN
O
N
OO
TFA
HN
O
N
OHO
HN
O
NH
+
41
Average Δ Mass
Modification Proposed Side Reaction Scheme
+48 Oxidation of Cys to Cys sulfonic acid
+51 Cys beta-elimination and piperidide adduct formation
+56tert-butylation on nucleophilic amino acid or insuffi cient removal of tBu protecting group
+67 Asp-piperidide
+71 endo-β-alanine
R-XH
X = NH, O, S or indolyl
+ RX
NH
HN
O
O
O
XAcid or Base
NH
O
N
OO
R R NH O
HN
R
O
HN
O
HN
O R
(L/D)--Asp-peptide piperidide (L/D)-iso-Asp-peptide piperidide
N
ON
O
Racemization
piperidine
piperidine
Path A
Path A
Path BX = OH, OAlkyl, OAryl, NH2
1 2
+
Path B
H2N
O
R1
Fmoc
HN
O
OH
R2
NH O
R1
Fmoc
HN
O
R2
NH O
R1
Fmoc
HN
O
R2
coupling reagent
(contaminated by Fmoc--Ala-Xaa2-OH) +
O
HN
NH O
SH
NH O
SO
OH
O
HN
O
HS
HN
O
HN H
N
N
HN O
42
Average Δ Mass
Modification Proposed Side Reaction Scheme
+74 esterification of Asp/Glu by glycerol
+ 80 Sulfonation
+ 90 Benzylation
+96 trifluoroacetylation of amino or hydroxyl group
NH O
O
OH OH
OH
HO1-2
NH O
O
1-2OH
OH
O
HN
O
NH OHS
O
O
HN
O
NH
NH
O
O
S OO
OH
NH
O
O
S OO
OH
NH
O
OH
NH
O
OH
HN
HNHN
NHO
S
O
O
OH
HN
HNNH2
NHO
NH O
HO
NH O
OS
O
OHO
R XH
X = O or S
R X
F3C
O
OH HX+ F3C
O
X
N = O or NH
RR
43
Further Reading
