Studies on the synthesis and stability of α-ketoacyl peptides

Article abstract of DOI:10.1007/s00726-020-02902-8

Oxidative stress, an excess of reactive oxygen species (ROS), may lead to oxidative post-translational modifications of proteins resulting in the cleavage of the peptide backbone, known as α-amidation, and formation of fragments such as peptide amides and α-ketoacyl peptides (α-KaP). In this study, we first compared different approaches for the synthesis of different model α-KaP and then investigated their stability compared to the corresponding unmodified peptides. The stability of peptides was studied at room temperature or at temperatures relevant for food processing (100?°C for cooking and 150?°C as a simulation of roasting) in water, in 1% (m/v) acetic acid or as the dry substance (to simulate the thermal treatment of dehydration processes) by HPLC analysis. Oxidation of peptides by 2,5-di-tert-butyl-1,4-benzoquinone (DTBBQ) proved to be the most suited method for synthesis of α-KaPs. The acyl side chain of the carbonyl-terminal α-keto acid has a crucial impact on the stability of α-KaPs. This carbonyl group has a catalytic effect on the hydrolysis of the neighboring peptide bond, leading to the release of α-keto acids. Unmodified peptides were significantly more stable than the corresponding α-KaPs. The possibility of further degradation reactions was shown by the formation of Schiff bases from glyoxylic or pyruvic acids with glycine and proven through detection of transamination products and Strecker aldehydes of α-keto acids by HPLC–MS/MS. We propose here a mechanism for the decomposition of α-ketoacyl peptides.

Full text of DOI:10.1007/s00726-020-02902-8

Amino Acids
https://doi.org/10.1007/s00726-020-02902-8
ORIGINAL ARTICLE
Studies on the synthesis and stability of α‑ketoacyl peptides
Johann Sajapin1 · Michael Hellwig1,2
Received: 29 April 2020 / Accepted: 6 October 2020
© The Author(s) 2020
Abstract
Oxidative stress, an excess of reactive oxygen species (ROS), may lead to oxidative post-translational modifcations of pro-
teins resulting in the cleavage of the peptide backbone, known as α-amidation, and formation of fragments such as peptide
amides and α-ketoacyl peptides (α-KaP). In this study, we frst compared diferent approaches for the synthesis of diferent
model α-KaP and then investigated their stability compared to the corresponding unmodifed peptides. The stability of
peptides was studied at room temperature or at temperatures relevant for food processing (100 °C for cooking and 150 °C
as a simulation of roasting) in water, in 1% (m/v) acetic acid or as the dry substance (to simulate the thermal treatment of
dehydration processes) by HPLC analysis. Oxidation of peptides by 2,5-di-tert-butyl-1,4-benzoquinone (DTBBQ) proved
to be the most suited method for synthesis of α-KaPs. The acyl side chain of the carbonyl-terminal α-keto acid has a crucial
impact on the stability of α-KaPs. This carbonyl group has a catalytic efect on the hydrolysis of the neighboring peptide bond,
leading to the release of α-keto acids. Unmodifed peptides were signifcantly more stable than the corresponding α-KaPs.
The possibility of further degradation reactions was shown by the formation of Schif bases from glyoxylic or pyruvic acids
with glycine and proven through detection of transamination products and Strecker aldehydes of α-keto acids by HPLC–MS/
MS. We propose here a mechanism for the decomposition of α-ketoacyl peptides.
Keywords Protein oxidation · Backbone cleavage · Ketoacyl peptide · Schif base · Transamination
Abbreviations
α-KaP
THF
TLC
Tetrahydrofuran
α-Ketoacyl peptide
Thin layer chromatography
DNPH
2,4-Dinitrophenylhydrazine
High-pressure liquid chromatography
Mass spectrometry
HPLC
MS
Introduction
NFPA
Nonafuoropentanoic acid
Pyruvoylglycine
(O)AG-OH
(O)GG-OH
Aerobic cellular respiration is common for all known ani-
mals. Though highly energetically benefcial, it still bears
some risks, since it makes the exposure of the organism to
reactive oxygen species (ROS) inevitable. For example, ROS
have been shown to be formed in vivo in mitochondria as a
byproduct of glucose metabolism due to leakage of electrons
from the transport chain (Turrens and Boveris 1980) or in
activated phagocytic cells as a response to microorganisms
(Curnutte and Babior 1987). These reactive compounds have
the potential to infict severe damage on cells, so aerobic
organisms use an abundance of protective endogenous anti-
oxidant systems to maintain a precise balance between the
formation and neutralization of ROS. In the case of insuf-
cient protective mechanisms (for example, under-expression
of superoxide dismutase (Schwartz et al. 1998)) or an excess
of ROS (e.g., through infux with cigarette smoke; Valava-
nidis et al. 2009) the balance can be disrupted, so diverse
Glyoxylglycine
(O)GGG-OH Glyoxylglycylglycine
(O)IG-OH
ROS
(3-Methyl-2-oxo)valerylglycine
Reactive oxygen species
Reversed phase
RP
TFA
Trifuoroacetic acid
Handling editor: P. Mefre.
* Michael Hellwig
M.Hellwig@tu-braunschweig.de
1
Chair of Food Chemistry, Technische Universität Dresden,
01062 Dresden, Germany
2
Institute of Food Chemistry, Technische Universität
Braunschweig, Schleinitzstraße 20, 38106 Braunschweig,
Germany
Vol.:(0123456789)
1 3

J. Sajapin, M. Hellwig
biomolecules may be oxidized. This process is generally
described as oxidative stress, and since proteins make up
to half of dry cell mass, they are likely to become a major
subject of an oxidative attack resulting in a post-translational
modifcation (Esterbauer et al. 1992; Davies 2005).
backbone cleavage mechanism can be described, as shown
in Fig. 1. The process is initiated by an attack of ROS on
the polypeptide and the subsequent abstraction of an α-C
hydrogen, leading to the formation of a carbon-centered
peptide radical (1), stabilized by resonance structures of
both neighboring peptide groups. In the presence of oxy-
gen, this compound becomes subject of conversion includ-
ing several highly reactive species such as peptide peroxyl
and peptide alkoxyl radicals, which can decompose via the
diamide pathway or α-amidation, the former leading to the
formation of an imide at the N-terminal truncated peptide
(2) and an isocyanate at the C-terminal peptide (3), which
can further react with water to restore the amino acid struc-
ture (4). The α-amidation leads to an unsaturated peptide
(5), further decomposing into a peptide amide (6) and an
α-ketoacyl peptide (7).
These reactions were predominantly studied regarding
their impact on human metabolism and health (Schöneich
et al. 2003; Dean et al. 1997). More recent studies examine
the susceptibility of food proteins to oxidation in the course
of processing and storage, as well as the consequences on
food quality and digestibility (Feng et al. 2015; Utrera and
Estévez 2013; Sante-Lhoutellier et al. 2007). Though many
studies predominantly focus on specifc oxidation mark-
ers and products, elucidation of reaction pathways was
sought after as well. Very often, protein oxidation in food is
assessed by quantifcation of protein carbonyls, which com-
prise a large set of individual structures among which glu-
tamic and aminoadipic semialdehydes predominate (Have-
lund et al. 2017; Hellwig 2019).
Some recent studies indicate that certain amino acid side
chains (especially that of methionine) are not only prone
to oxidation by reactive species but can further transfer
the oxidative damage to the peptide backbone. The subse-
quently accelerated formation of a carbon-centered radical
and the following α-amidation lead to higher yields of pep-
tide amides (Headlam et al. 2000; Hellwig et al. 2015) and
α-ketoacyl peptides. Some individual peptide amides are
known to be bioactive and participate in the nervous and
endocrine systems (Grimmelikhuijzen and Graf 1986; Chu-
fan et al. 2009). The α-ketoacyl peptides are predominantly
In general, the post-translational oxidative modifcation
of proteins is represented by modifcation on side chains
of amino acids (Stadtman 1993), cross-linking (Mirzaei
and Regnier 2006) and fragmentation (Davies 1996). Espe-
cially oxidative fragmentation reactions of proteins under
the conditions of food processing need to be explored in
more detail. Based on previous studies (Garrison 1987; Gar-
rison et al. 1962, 1970; Stadtman 2006), a simplifed peptide
Fig. 1 Mechanism of peptide backbone cleavage, initiated by attack
of ROS on a peptide, abstraction of the α-C hydrogen atom, crea-
tion of a carbon-centered radical (1) and rapid decomposition via the
diamide pathway or α-amidation. The former leads to the formation
of an N-terminal carbonyl compound (2) and a C-terminal isocyanate
(3), which can further react with water to an amino compound (4).
The α-amidation leads to an unsaturated peptide species (5), further
decomposing into a peptide amide (6) and an α-ketoacyl peptide (7).
R1, R2, R3, R4 are amino acid side chains
1 3

Studies on the synthesis and stability of α-ketoacyl peptides
considered as a part of the protein carbonylation reaction
(Utrera and Estévez 2012), so the reactivity of these com-
pounds remains to be explored.
Model incubations
Each substance was incubated as 1 mM solution in water or
in 1% (m/v) acetic acid. Unmodifed peptides and α-ketoacyl
peptides were also incubated as dry substances. For that,
1 mM solutions were divided in 1 mL fractions and sepa-
rately lyophilized in glass vials. Aqueous solutions were
incubated in screw-cap tubes at room temperature for up to
48 h to simulate the treatment during the marination pro-
cess, and at 100 °C and 150 °C for up to 120 min to mimic
the respective thermal treatment in the course of cooking
and roasting in oven, then rapidly cooled on ice and refrig-
erated at – 32 °C. The incubations of dry substances were
conducted directly in sealed glass vials at 100 °C and 150 °C
to imitate the overall process of intensive dehydration under
exposure to heat. The residue was rapidly cooled to room
temperature, dissolved in 1 mL water and refrigerated at
–32 °C.
In the present study, we, therefore, compared diferent
synthetic approaches to α-ketoacyl peptides regarding their
complexity and feasibility. Five α-ketoacyl peptides were
synthesized in sufcient amounts to allow for a study on
their reactivity. For the frst time, the stability of model
α-ketoacyl peptides with each other and corresponding
unmodifed peptides was compared, discovering a crucial
efect of the α-carbonyl group. A new catalytic mechanism
is proposed. We further identifed and quantifed the decom-
position products of α-ketoacyl peptides and propose a com-
bined pathway of advanced decomposition reactions.
Materials and methods
Chemicals
Thin layer chromatography
Pyruvic acid and silica gel 60 were obtained from Acros
Organics (Geel, Belgium), and 5-(dimethylamino)-naphtha-
lene-1-sulfonyl chloride (dansyl chloride), pyridine, glycine,
and 2,5-di-tert-butyl-1,4-benzoquinone (DTBBQ) from Alfa
Aesar (Kandel, Germany). Glycylalanine (H-GA-OH), and
Boc-Isoleucylglycine (Boc-IG-OH), were purchased from
Bachem (Bubendorf, Switzerland). Ethyl acetate from BCD
Chemie (Hamburg, Germany), ethanol from Berkel Alkohol-
handel (Berlin, Germany) and acetic acid for synthesis, and
formic acid for HPLC from Carl Roth (Karlsruhe, Germany)
were used. Acetonitrile LC–MS grade and n-hexane were
from Th. Geyer (Renningen, Germany). Trifuoroacetic acid
(for HPLC) was obtained from Fisher Scientifc (Loughbor-
ough, UK), glyoxylic acid monohydrate and acetaldehyde
from Fluka Chemicals (Buchs, Switzerland). 1-Butanol,
potassium iodide, ammonium formate, cupric sulfate pen-
tahydrate, sodium carbonate, sodium hydrogen carbon-
ate and sodium hydroxide were purchased from Grüss-
ing (Filsum, Germany), and o-dianisidine from Lancaster
Synthesis (Morecambe, UK). Acetone, 2,4-dinitrophenyl
hydrazine (DNPH), deuterium oxide, alanylglycine (H-AG-
OH), nonafuoropentanoic acid (NFPA), hydrochloric acid,
glycylglycine, and glycylglycylglycine (H-GGG-OH) were
from Sigma-Aldrich (Steinheim, Germany). Sodium sul-
fate (anhydrous), 4-methylbenzenesulfonylchloride (tosyl
chloride), acetonitrile (HPLC grade), methanol and tetrahy-
drofuran were obtained from VWR (Darmstadt, Germany).
2,2-Dihydroxy-1H-indene-1,3(2H)-dione (ninhydrin) was
from Serva Feinbiochemica (Heidelberg, Germany). Water
for solutions, buffers, and HPLC eluents was obtained
from a Bi 18 E double distillation system (QCS, Maintal,
Germany).
TLC was used for quick reaction controls. Reaction mixtures
(2 µL) were applied on a silica gel 60 plate (Merck, Darm-
stadt, Germany) and developed in a double-trough cham-
ber. A solution of 1-butanole, glacial acetic acid and water
(3/1/1, v/v/v) was used as the mobile phase. The developed
plates were dried at 50 °C for 10 min and treated with a
coloring agent. All substances containing carbonyl groups
were sprayed with a 0.1% (m/v) solution of DNPH in 2 M
hydrochloric acid. Yellow–orange colored spots revealed
the presence of carbonyl compounds. Secondary amines
and peptides were treated with chlorine gas for 30 min fol-
lowed by evaporation of excessive chlorine and spraying
with a saturated solution of o-dianisidine and 0.05 M potas-
sium iodide in 2% (v/v) acetic acid. Primary and secondary
amines appeared as black spots. Specifc detection of pri-
mary amines was carried out by spraying of developed plates
with a 0.1% (m/v) solution of ninhydrin in 0.05 M ethanolic
acetic acid and subsequent incubation at 50 °C in a drying
chamber for 10 min. Primary amines showed violet spots.
High‑performance liquid chromatography
with ultraviolet detection
All high-performance liquid chromatography analyses were
performed on a high-pressure gradient system, consisting
of a pump P1000 with an online degasser (Knauer, Berlin,
Germany), a column oven and a diode array detector Azura
DAD 2.1L. Analyses were carried out using a stainless steel
column (250 mm×4.6 mm; Knauer, Berlin, Germany) flled
with Eurospher-100 RP C-18 material (5 µM particle size)
with a guard column (5 mm× 4 mm) flled with the same
material.
1 3

J. Sajapin, M. Hellwig
Peptides and amino acids
Agilent) as the nebulizing gas with a fow rate of 11 L/min
and a pressure of 35 psi. Product ion spectra were recorded
at a fragmentor voltage of 100 V and a collision energy of
10 eV. Data acquisition and evaluation were conducted with
the software Mass Hunter B.02.00 (Agilent).
The analyses of peptides were carried out after derivati-
zation with dansyl chloride. For this, 50 µL of the sample
solution was added to 75 µL of 80.8 mM lithium carbonate
bufer, pH 9.5 and mixed with 100 µL of 0.5% (m/v) solu-
tion of dansyl chloride in acetonitrile for 1 h at 40 °C. The
reaction was stopped afterwards by adding 10 µL of 3 M
hydrochloric acid. The whole mixture was centrifuged for
5 min at 10,600g and the supernatant was directly used for
the analysis. The injection volume was 20 µL, the separa-
tion was performed at 40 °C and at a fow rate of 1 mL/
min. As mobile phases, 0.1 M formic acid in water (solvent
A), and 0.1 M formic acid in a mixture of acetonitrile and
water (90/10, v/v; solvent B) were used in a gradient pro-
gram (0 min, 20% B; 3 min, 20% B; 11 min, 75% B; 14 min,
80% B; 16 min, 80% B; 19 min, 20% B; 24 min, 20% B).
The absorbance was read at 340 nm. External calibrations
were performed with peptide and amino acid solutions in a
concentration range between 100 and 1300 µM under inclu-
sion of the dansylation step.
α‑Ketoacyl peptides
Before analysis, 50 µL of sample taken during the incuba-
tion experiments was mixed with 450 µL of 10 mM NFPA,
centrifuged at 10,600g for 5 min, and 250 µL of the super-
natants were subjected to analysis. The injection volume
was 10 µL, and the separation was performed at 35 °C and
at a fow rate of 0.25 mL/min on a stainless steel column
Zorbax 300 SB (50 mm×2.1 mm; Agilent) flled with RP
C-18 material (particle size 3.5 µm) using 10 mM NFPA in
water (solvent A) and 10 mM NFPA in acetonitrile (solvent
B) as mobile phases in a gradient program (0 min, 10% B;
15 min, 66% B; 19 min, 66% B; 20 min, 10% B; 28 min,
10% B). The absorbance was read at 230 and 280 nm, and
spectra were recorded between 200 and 350 nm (increment,
1 nm). Product ion spectra were recorded with the conditions
listed in Table 1.
Carbonyl compounds
The analyses of α-ketoacyl peptides and other carbonyl com-
pounds were carried out after derivatization with DNPH.
For this, 100 µL of fltered samples was mixed with 100 µL
of 0.1% (m/m) DNPH solution in 2 M hydrochloric acid
and incubated at room temperature for 5 min followed by
addition of 300 µL acetonitrile and diluted with 500 µL of
solvent A for analysis. The injection volume was 20 µL, the
separation was performed at room temperature and at a fow
rate of 1 mL/min. As mobile phases, 0.1% (v/v) trifuoro-
acetic acid in a mixture of water and acetonitrile (90/10, v/v;
solvent A), and 0.1% (v/v) trifuoroacetic acid in a mixture
of acetonitrile and water (90/10, v/v; solvent B) were used
in a gradient program (0 min, 0% B; 10 min, 0% B; 20 min,
10% B; 60 min, 100% B; 65 min, 0% B; 75 min, 0% B). The
absorbance was read at 214 and 365 nm. External calibra-
tions were performed with peptide and amino acid solutions
in a concentration range between 10 and 1200 µM.
Schif bases
Prior to mass spectral analysis, 100 µL of a sample taken
during the incubation experiments was mixed with 400 µL
of HILIC solvent B, membrane fltrated (0.45 µm) and sub-
jected to spectral analysis. The injection volume was 10 µL.
The separation was performed at 35 °C and at a fow rate
of 0.2 mL/min on a stainless steel column SeQuant ZIC-
HILIC (100 mm×2.1 mm, 3.5 µM, 100 Å; Merck KGaA,
Darmstadt, Germany) using 5 mM ammonium formate and
0.1% (v/v) formic acid in water (HILIC solvent A) and 2 mM
ammonium formate and 0.1% (v/v) formic acid in a mixture
of acetonitrile and water (80/20, v/v; HILIC solvent B) in a
gradient program (0 min, 95% B; 2 min, 95% B; 6 min, 85%
Table 1 Operating conditions for recording of product ion spectra by
HPLC–MS/MS
High‑performance liquid chromatography
with mass spectrometric detection
Substance
Column
Precursor
ion (m/z)
(3-Methyl-2-oxo)-valerylglycine
Glyoxylglycine
Glyoxylglycylglycine
Pyruvoylglycine
Pyruvoylglycylalanine
N-(1-Carboxyethylidene)-glycine SeQuant ZIC-HILIC 146
N-(Carboxymethylidene)-glycine SeQuant ZIC-HILIC 132
Zorbax 300 SB
Zorbax 300 SB
Zorbax 300 SB
Zorbax 300 SB
Zorbax 300 SB
188
132
189
146
217
The high-pressure gradient system 1200 series (Agilent
Technologies, Böblingen, Germany) consisting of a binary
pump, a column oven, an autosampler, and a diode array
detector, coupled with a mass spectrometer 6410 Triple
Quad (Agilent) with Electrospray Ionization (ESI) was
used. The mass spectrometer was run in the positive mode,
with a source temperature of 350 °C and a capillary voltage
of 4000 V using nitrogen (nitrogen generator 5183–2003,
All spectra were recorded in the positive mode
1 3

Studies on the synthesis and stability of α-ketoacyl peptides
B; 7 min, 65% B; 9 min, 65% B; 10 min, 95% B; 18 min,
95% B) as published previously (Kölpin and Hellwig 2019).
Product ion spectra were recorded with the conditions listed
in Table 1.
(2.4377 g, 12.8 mmol) was dissolved in 6.25 mL acetone
(dried over anhydrous sodium sulfate) and cooled down to
0 °C in an ice bath. Pyruvic acid (0.875 mL, 12.6 mmol)
was slowly added to the solution with stirring, and then
pyridine (1.025 mL, 12.7 mmol) was mixed in at 0 °C. The
mixture was stirred for 20 min and afterwards, a solution
of glycine (0.9375 g, 12.5 mmol) and pyridine (2.125 mL,
26 mmol) in water (8.75 mL) was added dropwise at 0 °C.
The mixture was allowed to warm to room temperature and
rapidly stirred until the previously formed precipitate had
dissolved. The solution was reduced to dryness in vacuo.
The residue was dissolved in 20 mL water, acidifed with
1 mL 12 M hydrochloric acid and extracted with ethyl ace-
tate (4×100 mL). The organic phases were combined, dried
over sodium sulfate and evaporated to dryness in vacuo. The
crude product was dissolved in 10 mL water, neutralized
with 50 µL 5 M sodium hydroxide and chromatographed
on a 1.5×50 cm glass column flled with 80 mL of strongly
basic anion-exchange resin (AG 1-X8, BioRad Lab, Munich,
Germany), previously regenerated and preconditioned by
successive application of 300 mL 1 M sodium hydroxide,
600 mL water, 300 mL 1 M hydrochloric acid and washed
neutral with 600 mL water. The elution was performed with
4 M acetic acid (500 mL). The fractions were collected in
separate 5 mL tubes using a BioRad 2110 Fraction collector.
Samples (50 µL) of each fraction were mixed with 50 µL of
0.1% (m/v) solution of DNPH in 2 M hydrochloric acid and
incubated at room temperature for 5 min. The mixtures were
then extracted with 250 µL ethyl acetate. The organic phases
were separated and extracted with 350 µL 0.4 M sodium
carbonate bufer, pH 9.6. Of each aqueous solution, 100 µL
was transferred to a microtiter plate, and the absorbance at
365 nm was measured using the plate reader Tecan Infnite
M200 Pro (Tecan Group, Männedorf, Switzerland) The frac-
tions 51–67 (out of total 100) were tested positively (photo-
metrically) and further characterized using HPLC–MS/MS.
Fractions that contained the product were combined, dried in
vacuo and lyophilized. The yield was 27.9 mg (0.192 mmol,
molar yield=3%).
Amino acid analysis
All samples were subjected to acid hydrolysis prior to amino
acid analysis. For that, 5 mg of dry substance was mixed
with 2 mL of 6 M hydrochloric acid in screw-cap tubes and
incubated in a drying chamber at 110 °C for 23 h. An ali-
quot of 300 µL of the hydrolysates was dried afterwards in a
vacuum centrifuge (SPD Speed Vac; Thermo Fischer Scien-
tifc, Karlsruhe, Germany). The dry residues were dissolved
in 600 µL of loading bufer (0.12 N lithium citrate, pH 2.20),
membrane fltered (0.45 µm) and subjected to amino acid
analysis. Quantifcation of amino acids was performed with
the amino acid analyzer S 433 (Sykam, Fürstenfeldbruck,
Germany). The injection volume was between 10 and 100
µL, and a PEEK column flled with the cation exchange resin
LCA K07/Li (150 mm×4.6 mm, 7 µM) was used for separa-
tion according to a custom gradient program. Loading and
running bufers were purchased from Sykam. Post-column
derivatization with ninhydrin was applied, followed by VIS
detection with an integrated two-channel photometer at 440
and 570 nm. External calibration was performed with com-
mercial amino acid standards (Sigma-Aldrich, Steinheim,
Germany).
Characterization of products
The 1H spectra of synthesized products were recorded with
an Avance III HDX 500 MHz Ascend instrument (Bruker,
Rheinstetten, Germany) at 500.13 MHz. For that, 5.0 mg of
dry substance was dissolved in 650 µL of deuterium oxide.
All chemical shifts are given in parts per million (ppm) rela-
tive to an internal HOD signal (1H; 4.70 ppm).
Statistical treatment
All samples were analyzed at least in triplicate. Grubbs test
was performed for outlier elimination. Normal distribution
was tested with the Shapiro–Wilk test. Comparison of mean
values was performed by one-way ANOVA. All statistical
calculations were performed with the software Origin Pro
2019 (OriginLab).
Synthesis of pyruvoylglycylalanine (8b)
Tosylation was performed using the same approach as
described above. For the reaction, 0.325 g (1.7 mmol)
of tosyl chloride (in 0.833 mL dry acetone) was mixed
with pyruvic acid (0.177 mL, 1.66 mmol) and pyridine
(0.135 mL, 1.67 mmol) at 0 °C. After 20 min of stirring, a
solution of glycylalanine (0.246 g, 1.68 mmol) and pyri-
dine (0.283 mL, 3.6 mmol) in 1.167 mL water was added.
After stirring, warming up and dissolving of the precipi-
tate, the mixture was dried to syrup in vacuo, dissolved
in 2 mL water and acidifed with 100 µL of 12 M hydro-
chloric acid. The solution was then extracted with ethyl
Synthesis of α‑ketoacyl peptides via tosylation
Synthesis of pyruvoylglycine (8a)
Tosylation was performed according to a literature approach
(Dixon 1964) with some modifications. Tosyl chloride
1 3

J. Sajapin, M. Hellwig
acetate (4 × 2.5 mL), and the combined organic phases
were dried in vacuo. The residue was dissolved in 2 mL
of water and neutralized with 2 µL of 5 M sodium hydrox-
ide and chromatographically purifed as described above.
DNPH-positive fractions were identified as described
above. The fractions 13–31 out of total 100 were tested
positively (photometrically) and further characterized
using HPLC–MS/MS. Fractions that contained the product
were combined, dried in vacuo and lyophilized.
Analytical data: Content by amino acid analysis was
56% based on glycine. Yield of pure substance was 53.8 mg
(0.371 mmol, molar yield 54%).
Synthesis of glyoxylglycine (8c)
The synthesis was performed as described for pyruvoylg-
lycine, but starting from glycylglycine (0.132 g, 1 mmol).
The reagent solution consisted of glyoxylic acid monohy-
drate (0.75 g, 8 mmol), 2.4 mL of 0.5 M cupric sulfate solu-
tion and pyridine (1.5 mL, 7.4 mmol) flled up with water
to 10 mL. After ion-exchange chromatography, the frac-
tions 101–159 were shown to contain the target product by
spotting test and HPLC–MS/MS. They were worked up as
described above.
Analytical data: HPLC–ESI–MS/MS (α-KaP system): tR,
1.1 min; fragmentation, positive mode (100 V, 10 eV) of
[M + H]+ (m/z 217): 44 (100), 90 (59), 100 (14), 30 (11);
1H-NMR (500 MHz, D2O), δ [ppm]: 1.37 (dd, 3H, J=2.1,
9.1, Ala-CH3); 1.51 (s, 1.3 H, COCH3, hydrated form), 2.40
(s, 1.7 H, COCH3), 3.93 (d, 0.8H, J = 1.6 Hz, Gly-CH2,
hydrated form); 3.98 (s, 1.2H, Gly-CH2), 4.33 (q, 1H, Ala-
CH, J = 9.1). The yield was 4.2 mg (0.019 mmol, molar
yield=3%).
Analytical data: Content by amino acid analysis was
17% based on glycine. Yield of pure substance was 15.6 mg
(0.119 mmol, molar yield 12%).
Synthesis of (3‑methyl‑2‑oxo)valerylglycine (8e)
Synthesis of α‑ketoacyl peptides
via metal‑ion‑catalyzed transamination
Prior to synthesis, the protecting group of Boc-IG-OH was
removed. For that, Boc-IG-OH (0.306 g, 1.06 mmol) was
dissolved in 3 mL THF. Then, 3 mL of 6 M hydrochloric
acid was added and the mixture was stirred at room tem-
perature for 3 h. The progress of removal of the Boc group
was verifed using TLC and detection with ninhydrin. After
the complete removal of the Boc group, the solution was
dried in vacuo, dissolved in 1 mL water and evaporated
again. This process was repeated threefold until the product
was free of hydrochloric acid. The crude dipeptide solution
was lyophilized. Then, isoleucylglycine (0.189 g, 1 mmol)
was dissolved in 10 mL of water, and the transamination
procedure was conducted as described for pyruvoylglycine.
The reagent solution was the same as for glyoxylglycine.
After ion-exchange chromatography and characterization of
fractions by spotting test and HPLC–MS/MS, the fractions
193–256 were combined and worked up as described above.
Analytical data: Content by amino acid analysis was
57% based on glycine. Yield of pure substance was 97.4 mg
(0.512 mmol, molar yield 49%).
Synthesis of pyruvoylglycine (8a)
Transamination reactions were performed using modifed
literature approaches (Dixon 1964; Nishimura et al. 1998).
Alanylglycine (0.100 g, 0.68 mmol) was dissolved in 50 mL
of water. Then, a reagent solution was mixed containing
glyoxylic acid monohydrate (3.75 g, 41 mmol), 1.2 mL
of a 0.5 M cupric sulfate solution, and pyridine (1.5 mL,
7.4 mmol) flled up with water to 10 mL in total. The reagent
solution was added to the peptide solution and the mixture
stirred for 60 min at 25 °C. Reaction progress was verifed
by TLC and DNPH spraying reagent. To each synthesis mix-
ture, 5 mL of 12 M hydrochloric acid was added. Acidifed
solutions were extracted with ethyl acetate (3× 100 mL).
The combined organic extracts were dried over sodium sul-
fate, fltrated and evaporated in vacuo. The residues were
dissolved in 10 mL water and the pH was adjusted to 7 with
0.1 M acetic acid. The crude product solutions were chroma-
tographed on a 1.5×50 cm glass column flled with 80 mL
of strongly basic resin AG 1-X8 previously equilibrated as
described above. The elution was conducted with 500 mL of
each 1 M, 2 M, and 4 M acetic acid. Fractions of 5 mL were
collected and target fractions were identifed by TLC with
DNPH spraying reagent. The positive fractions 110–150
were further characterized in scan and product ion scan
measurements with HPLC–MS/MS. Combined fractions
were dried in vacuo, the residue was dissolved in 10 mL of
water and dried again until the product was free of acetic
acid. Then the product was dissolved in 5 mL water and
lyophilized.
Synthesis of α‑ketoacyl peptides via reaction
of peptides with 3,5‑di‑tert‑butyl‑o‑benzoquinone
(DTBBQ)
Synthesis of pyruvoylglycine (8a)
This set of transamination reactions was performed using
a modifed literature approach (Vinšova et al. 2005). Ala-
nylglycine (0.290 g, 2 mmol) was dissolved in 20 mL of
water, and a solution of DTBBQ (0.440 g, 2 mmol) in
1 3

Studies on the synthesis and stability of α-ketoacyl peptides
40 mL ethanol was added at room temperature. Then, the
reaction mixture was stirred at 50 °C for 30 min under nitro-
gen atmosphere. The reaction progress was monitored by
TLC and further by HPLC–MS/MS. Then, the solution was
evaporated to dryness in vacuo and purifed by silica gel
chromatography. The elution was conducted with 500 mL
of each hexane, 1/1 (v/v) ethyl acetate in hexane, ethyl ace-
tate, and fnally 1/1 (v/v) ethyl acetate in methanol. Frac-
tions of 10 mL were collected, and DNPH-positive fractions
were combined and evaporated to dryness in vacuo. The
residue was dissolved in 10 mL of water, and this solution
was subjected to anion-exchange chromatography exactly
as described above for metal-ion catalyzed transamination.
The DNPH-positive fractions 120–140 were combined and
lyophilized. The residue was dissolved in 20 mL of 50% (v/v)
acetonitrile in water and further purifed by semipreparative
HPLC–UV on a Wellchrom high-pressure gradient system
consisting of two pumps (K-1001), an online degasser, a UV-
detector (K-2501), and a 16-port fractionation valve (K-16;
all from Knauer). Fractionations were carried out using a
stainless steel column (300 mm×8 mm; Knauer) flled with
Eurospher-100 RP-18 material (5 µM particle size) with a
guard column (300 mm×8 mm) flled with the same mate-
rial. The injection volume was 2 mL, and the separation was
performed at room temperature and at a fow rate of 1.4 mL/
min. As mobile phases, the same eluents as for HPLC–UV
of carbonyl compounds (see above) were used in a gradi-
ent program (0 min, 0% B; 10 min, 0% B; 35 min, 10% B;
70 min, 100% B; 75 min, 0% B; 80 min, 0% B). The absorb-
ance was read at 214 nm. The eluates were collected at the
retention time between 50 and 70 min, combined, lyophi-
lized and dissolved in 10 mL of water. Afterwards, another
purifcation by semipreparative HPLC was conducted under
the same conditions using only water as the mobile phase.
Product eluates were collected at the retention times between
6 and 10 min, combined and lyophilized.
Analytical data: HPLC–ESI–MS/MS (α-KaP system): tR,
0.9 min; fragmentation, positive mode (100 V, 10 eV) of
[M+H]+ (m/z 132): 30 (100), 86 (95), 58 (31), 114 (5), 76
(2); 1H-NMR (500 MHz, D2O), δ [ppm]: 3.87 (s, 2H, CH2),
5.28 (s, 1H, CHO, hydrated form). Content by amino acid
analysis was 69% based on glycine, yield of pure substance
was 154.6 mg (1.181 mmol, molar yield=59%).
Synthesis of glyoxylglycylglycine (8d)
This compound was synthesized as described above, but
starting from glycylglycylglycine (0.378 g, 2 mmol). After
ion-exchange chromatography, DNPH-positive fractions
57–120 were combined and subjected to semipreparative
HPLC.
Analytical data: HPLC–ESI–MS/MS (α-KaP system): tR,
0.8 min; fragmentation, positive mode (100 V, 10 eV) of
[M + H]+ (m/z 189): 79 (100), 30 (58), 86 (49), 143 (18),
1
114 (5), 58 (5); H-NMR (500 MHz, D2O), δ [ppm]: 3.97
(s, 2H, CH2); 3.99 (s, 2H, CH2), 5.31 (s, 1H, CHO, hydrated
form). Content by amino acid analysis was 63% based on
glycine, yield of pure substance was 57.3 mg (0.304 mmol,
molar yield=15%).
Synthesis of (3‑methyl‑2‑oxo)‑valerylglycine (8e)
This compound was synthesized as described above, but
starting from isoleucylglycine (0.376 g, 2 mmol), which had
previously been obtained from Boc-Ile-Gly by the proce-
dure described above. After ion-exchange chromatography,
DNPH-positive fractions 179–186 were combined and sub-
jected to semipreparative HPLC.
Analytical data: HPLC–ESI–MS/MS (α-KaP system):
tR, 5.9 min; fragmentation, positive mode (100 V, 10 eV)
of [M + H]+ (m/z 188): 57 (100), 76 (11), 41 (10), 85 (2);
1H-NMR (500 MHz, D2O), δ [ppm]: 4.03 (s, 2H, Gly-CH2);
1.64 (m, 1H, Ile-CH), 1.38 (m, 2H, Ile-CH2), 1.04 (d, 3H,
Ile-CH3, J=7.1), 0.82 (t, 3H, Ile-CH3, J=7.9). Content by
amino acid analysis was 86% based on glycine, yield of pure
substance was 291.5 mg (1.559 mmol, molar yield=78%).
Analytical data: HPLC–ESI–MS/MS (α-KaP system):
tR, 1.0 min; fragmentation, positive mode (100 V, 10 eV)
of [M+H]+ (m/z 146): 43 (100), 76 (54), 30 (31), 100 (9);
1H-NMR (500 MHz, D2O), δ [ppm]: 1.51 (s, 1.3H, Gly-
CH2, hydrated form); 2.40 (s, 1.7H, Gly-CH2); 3.97 (s, 0.9
H, CH3, hydrated form); 4.02 (s, 1.1H, CH3). Content by
amino acid analysis was 63% based on glycine, yield of pure
substance was 130.8 mg (1.181 mmol, molar yield=59%).
Results and discussion
Comparison of synthesis approaches
Synthesis of glyoxylglycine (8c)
The synthesis approaches were compared based on
the overall complexity of the process and the yield of
α-ketoacyl peptides (Table 2). Tosylation couplings were
successfully carried out, but the molar yields of products
were lower than using other pathways. Furthermore, this
reaction is only occurring in the absence of water, which
is disadvantageous for synthesis of α-ketoacyl peptides
This compound was synthesized as described above,
but starting from glycylglycine (0.264 g, 2 mmol). After
ion-exchange chromatography, DNPH-positive fractions
105–156 were combined and subjected to semipreparative
HPLC.
1 3

J. Sajapin, M. Hellwig
Table 2 Comparison of approaches for the synthesis of α-ketoacyl peptides
Yield (pure substance) [mol%]
Tosylation (Dixon 1964)
Transamination
Transamination
(Vinšova et al.
2005)
(Nishimura et al. 1998)
Glyoxylglycine
Pyruvoylglycine
Pyruvoylglycylalanine
Glyoxylglycylglycine
(3-Methyl-2-oxo)-valerylglycine
(O)GG-OH
(O)AG-OH
(O)AGA-OH
(O)GGG-OH
(O)IG-OH
Not applicable
3
3
N/A
N/A
12
54
N/A
N/A
49
59
45
N/A
15
78
Data are given as molar yields (pure substance in mol%) of synthesized α-ketoacyl peptides. The most successful approach for each product is
highlighted in italic
N/A not available
group is replaced by an “O” for oxygen written in brackets.
For the α-ketoacyl peptide pyruvoylglycine (Fig. 2), which
can formally be derived from alanylglycine (H-AG-OH),
the notation would be (O)AG-OH.
Study on the stability of α‑ketoacyl peptides
The α-ketoacyl peptides formed during α-amidation of pro-
teins are expected to be more reactive than regular peptides,
since the α-carbonyl carbon atom is an electrophile and
can react with nucleophiles such as water (Bell 1966). To
examine the reactivity of α-ketoacyl peptides, incubations
were carried out either in water, in 1% (m/v) acetic acid or
as dry substance at room temperature, 100 °C and 150 °C
as described above. Furthermore, we carried out a direct
comparison of α-ketoacyl peptides and the correspond-
ing unmodifed peptides (with a terminal amino group) in
regards of their stability and the occurrence of decomposi-
tion products.
Fig. 2 Structural formulas of pyruvoylglycine (8a, (O)AG-OH),
pyruvoylglycylalanine (8b, (O)AGA-OH), glyoxylglycine (8c, (O)
GG-OH), glyoxylglycylglycine (8d, (O)GGG-OH), and (3-methyl-
2-oxo)-valerylglycine (8e, (O)IG-OH)
The examined α-KaPs (Fig. 2) showed a decreasing sta-
bility at higher temperatures. At room temperature, no signif-
icant decomposition of (O)GG-OH (8c) or (O)AG-OH (8a)
was detected. However, the stability of examined substances
decreased drastically at higher temperatures, as shown in
Fig. 3. To maintain the comparability of the results only
the residual contents of peptides after 2 h incubation were
depicted, although it was observed that at high temperatures
especially the dry substances decomposed very fast leading
to up to 98% of the overall degradation in the frst 30 min of
incubations. The highest stability was demonstrated by (O)
IG-OH (8e) only showing a signifcant decomposition as dry
substance. (O)AG-OH (8a) proved to be stable in solution at
100 °C, while a signifcant decrease of concentration could
be detected at 150 °C. The compound (O)GG-OH (8c) was
less stable in solution even at 100 °C. The only structural
diference between the examined α-ketoacyl dipeptides is
the length of the acyl side chain at the carbonyl terminus
with terminal glyoxylic acid, due to its availability only
in hydrated form. The synthesis approach via transamina-
tion with DTBBQ (Vinšova et al. 2005) resulted in high-
est yields and purity for most products. Complexity of
the synthesis and purifcation eforts were comparable to
transamination with metal ions, leading to the conclusion
that α-ketoacyl peptides are preferably synthesized using
DTBBQ.
Diferently from “normal” peptides, α-ketoacyl pep-
tides have a carbonyl terminus, where an α-keto acid is
bound to an amino acid or a peptide. To clearly denomi-
nate α-ketoacyl peptides and to distinguish them from the
intact parent peptides while conserving the information
about the side chain of the carbonylterminal α-keto acids,
we propose that the sequence of the corresponding peptide
is noted, but the hydrogen atom indicating the intact amino
1 3

Studies on the synthesis and stability of α-ketoacyl peptides
Fig. 3 Residual content of
(3-methyl-2-oxo)valerylglycine
(black columns, (O)IG-OH),
pyruvoylgycine (white columns,
(O)AG-OH), glyoxylglycine
(gray columns, (O)GG-OH) and
glyoxylglycylglycine (hatched
column, (O)GGG-OH) in
mol% of initial substance after
2 h incubation at 100 °C and
150 °C. The incubations were
carried out as 1 mM solutions
in either water, 1% (m/v) acetic
acid, or as dry substances. Data
are mean values SD, n=2–4
relative to the overall size of the substances, so it can be
safely assumed that the involvement of comparatively big
side chains increases the stability of α-KaP. It was also
observed that the compound (O)GGG-OH (8d) proved to
be even less stable than (O)GG-OH (8c), which could be
explained by generally higher reactivity of the triglycine
chain compared to diglycine as described for unmodifed
compounds (Lu et al. 2005) and the ability to form cyclic
glycylglycine as further degradation intermediate accelerat-
ing the overall decomposition of the α-KaP (Lee et al. 2015).
The examined α-ketoacyl dipeptides are not able to undergo
the cyclization reactions, which could explain their generally
higher stability in comparison to (O)GGG-OH.
to reactive amides, though this assumption must be further
studied in the future.
The decomposition of α-KaPs in the presence of water
was proven to be represented by hydrolysis of the peptide
bond resulting in the formation of an α-keto acid and an
amino compound, as shown exemplarily for (O)GG-OH at
150 °C in water (Fig. 4a) by a signifcant increase in the con-
tent of glyoxylic acid and glycine. In addition, the assump-
tion can be made, that the hydrolysis primarily takes place
on the peptide bond closest to the α-keto group, since the
decomposition of (O)GGG-OH predominantly leads to for-
mation of glyoxylic acid and glycylglycine (e.g., reaction in
water at 150 °C, Fig. 4b).
The decomposition process was further examined by ana-
lyzing the products formed in the process. The incubations
of dry substances did not result in any signifcant increase
in the content of carbonyl or amino components. One of
the probable reaction pathways could be a pyrolysis fol-
lowed by formation of azomethine ylides and their dimeri-
zation (Guerra and Yaylayan 2010) or further degradation
Since the hydrolysis of regular peptides takes place only
in strongly acidic (Roach and Gehrke 1970) or basic milieu
(East 2018), direct comparisons of α-ketoacyl peptides
and the corresponding regular peptides were carried out.
At room temperature, no deviations could be verifed after
48 h, since neither (O)GG-OH (8c) and (O)AG-OH (8a) nor
the corresponding dipeptides glycylglycine (H-GG-OH) and
Fig. 4 a Content of glycine (black triangles) and glyoxylic acid (gray
triangles) formed as the result of hydrolysis of glyoxylglycine ((O)
GG-OH) at 150 °C in water in mol% of initial α-KaP amount. b Con-
tent of glycylglycine (black circles, H-GG-OH) and glyoxylic acid
(gray circles) formed as the result of hydrolysis of glyoxylglycylgly-
cine ((O)GGG-OH) at 150 °C in water in mol% of initial substance.
Data are mean values SD, n=2–4
1 3

J. Sajapin, M. Hellwig
Fig. 5 Residual content of
glyoxylglycine (black columns,
(O)GG-OH) and glycylglycine
(white columns, H-GG-OH)
in mol% of initial substance
after 2 h incubation at room
temperature, 100 °C and 150 °C
in water, in 1% (m/v) acetic acid
(1 mM peptide solutions) and
as dry substance. Data are mean
values SD, n=2–4
alanylglycine (H-AG-OH) showed any signifcant decom-
position. However, at higher temperatures, a signifcant dif-
ference between regular peptides and α-KaPs was observed
(e.g., (O)GG-OH and H-GG-OH, Fig. 5). Glycylglycine
showed higher stability under all examined conditions. The
most signifcant diferences between these two peptides
were observed at 150 °C, when the regular peptide remained
comparatively stable losing 10–22 mol% of its initial con-
tent, whereas up to 93 mol% of the α-ketoacyl peptide were
decomposed. Other α-ketoacyl peptides and corresponding
regular peptides were incubated under the same conditions
showing similar trends. Since the α-keto-group is the only
structural deviation of the corresponding peptides, it can be
assumed that this group is able to provoke the decomposi-
tion of peptides.
signals of respective geminal diols instead of aldehydes,
proving a complete hydration similar to glyoxylic acid. On
the contrary, (O)IG-OH remained in the keto-form, and
no diol could be detected. The stability of (O)AG-OH was
proven to be between that of (O)IG-OH and (O)GG-OH,
and correspondingly, the signals of geminal diol and keto-
form revealed ca. 40% of hydrated form for this α-ketoacyl
peptide.
Based on these observations, we postulate the hydroly-
sis mechanism shown in Fig. 6. We assume a nucleophilic
addition of water to α-ketoacyl peptides (9) resulting in a
geminal diol (10). The hydroxyl group is expected to form a
pseudo-pentagonal complex with the peptide oxygen atom
in the process, interfering with the resonance stabilization
of the amide bond. With subsequent application of energy,
a further water molecule can attack the peptide carbon atom
hence initiating the hydrolysis and leading to the formation
of an α-keto acid (11) and an amino compound (12).
Proposed mechanism of peptide cleavage
and catalytic role of the α‑keto‑group
Carbonyl groups are prone to nucleophilic addition, which
can be slowed down by steric hindrance (Charton 1975)
or by a positive inductive effect of neighboring groups
(Taskinen and Kukkamäki 1977). The α-ketoacyl peptides
examined in the present study demonstrate higher stability
with increasing length of their alkyl side chains (Fig. 3),
and consequently, with increasing occupied volume (Rich-
ards 1977) and inductive effect (Dwyer 2005) of those
side chains. These fndings lead to the assumption that the
hydrolysis of the peptide bond nearest to the carbonyl ter-
minus in α-KaP is initiated by a nucleophilic attack on the
α-keto group, with the most probable reaction partner being
water, since the hydrolysis could only be detected in aqueous
solutions. Further evidence in favor of this hypothesis was
Advanced degradation reactions
The α-keto acids released in hydrolysis of α-KaPs are elec-
trophilic compounds, hence, they can react as well with
water forming geminal diols (Sørensen et al. 1974; Grif-
fths and Socrates 1967). A further reaction partner can be
the amino compound, so the formation of an imine (Schön-
berg and Moubacher 1952; Chu and Yaylayan 2008) can be
expected. The plausibility of this assumption was verifed
through equimolar incubation of pyruvic and glyoxylic acids
with glycine followed by a successful qualitative search for
respective imines (Schif bases) using HILIC-MS/MS, as
exemplarily shown in Fig. 7 for a mixture after 2 h of incu-
bation at 150 °C.
1
obtained by H-NMR spectroscopy, showing a clear cor-
We were further able to detect a rise in concentration
of Strecker aldehydes in the course of decomposition of
α-ketoacyl peptides, e.g., acetaldehyde formed from (O)
AG-OH (Fig. 8a). These substances can only originate from
relation between hydration rate and reactivity of the exam-
ined substances. The spectra of (O)GG-OH and (O)GGG-
OH, the most reactive α-keto acyl peptides, only contained
1 3

Studies on the synthesis and stability of α-ketoacyl peptides
Fig. 6 Proposed hydrolysis
mechanism of α-ketoacyl pep-
tides (9) with initial hydration to
a geminal diol (10), subsequent
cleavage of the peptide bond
closest to the carbonyl terminus
and formation of an α-keto acid
(11) and an amino compound
(12). R1 and R2 are amino acid
side chains, R3 is -OH or amino
acid
Fig. 7 a HILIC chromatogram
with UV and MS detections of
glycine (10 mM), pyruvic acid
(10 mM) and a mixture of both
(each 10 mM) after 2 h incuba-
tion at 150 °C. b Mass spectrum
of the peak at the retention time
3.4 min (Schif base)
Fig. 8 a Content of acetaldehyde (MeCHO) formed in advanced deg-
radation reaction of 1 mM solutions of pyruvoylglycine ((O)AG-OH)
at 150 °C in water (black triangles) and 1% (m/v) acetic acid (HOAc,
gray triangles) in mol% of initial amount of α-KaP. b Content of gly-
oxylglycine ((O)GG-OH) formed in advanced degradation reaction of
1 mM solution of glyoxylglycylglycine ((O)GGG-OH) at 150 °C in
water (black circles) and 1% (m/v) acetic acid (HOAc, gray circles).
Data are mean values SD, n=2–4
1 3

J. Sajapin, M. Hellwig
Fig. 9 Proposed decomposition
pathway of α-ketoacyl peptides
(9) in the absence of water lead-
ing to carbonyl-free compounds
and in the presence of water
leading to hydrolysis into α-keto
acid (11) and C-terminal amino
compound (12), which can
further react with each other
forming a Schif base (13). The
latter can cleave and decar-
boxylate forming the Strecker
aldehyde (14) of the α-keto acid
or hydrolyze causing transami-
nation and the formation of
amino acid (15) and C-terminal
α-keto compound (16). R1 and
R2 are amino acid side chains,
R3 is -OH or amino acid
α-keto acids, previously emerged during hydrolysis. Since
glyoxylic acid as well as pyruvic acid are not decomposed
under the examined conditions, advanced degradation reac-
tions are the only possible reason for the formation of alde-
hydes, the most likely being the Strecker-like decomposition
of previously formed Schif bases (Chu and Yaylayan, 2008)
with subsequent decarboxylation of α-keto acids.
We have determined a signifcant decline in stability of
α-KaPs in comparison to their corresponding regular pep-
tides resulting in higher hydrolysis rates. Furthermore, we
have characterized and quantifed the products of advanced
degradation of examined modifed peptides. Based on these
observations, we propose the degradation pathway shown
in Fig. 9. In the absence of water, the α-ketoacyl peptides
(9) are expected to decompose into products without free
carbonyl groups, but degradation into volatile compounds is
also possible. These reactions will be subject of our further
investigations. By application of energy, the prevalent reac-
tion in the presence of water is a hydrolysis of the peptide
bond next to the carbonyl terminus initiated by the hydration
of the latter leading to the formation of an α-keto acid (11)
and C-terminal amino compound (12). These are expected
to react with each other and form a Schif base (13), which
can further be cleaved by decarboxylation of the previous
α-keto acid, releasing a Strecker aldehyde (14). The hydro-
lytic cleavage of the Schif base leads to transamination of
intermediate compounds, forming the corresponding amino
acid (15) and a new C-terminal α-keto compound (16).
Further approaches should be made to confirm and
complete the proposed decomposition pathway, the most
interesting being the carbonyl-free degradation products
in absence of water. Degradation to various reactive inter-
mediates should be confrmed and attempts for identifca-
tion of products made. For more insights into formation of
volatile degradation compounds, analysis with GC–MS/MS
systems should be considered. Another important aspect is
A closer look at the degradation of (O)GGG-OH further
displayed a formation of (O)GG-OH, as shown in Fig. 8b.
Glycylglycine (H-GG-OH) was stable at examined condi-
tions, showing almost no oxidation, so no α-KaP could be
formed in that pathway. Another possible reason could be
the hydrolysis of the peptide bond closer to the C-terminus
in (O)GGG-OH catalyzed by the α-keto group; however, we
consider it unlikely owing to the long distance between both
reaction partners. Therefore, we assume another nucleo-
philic reaction between initial hydrolysis products (glyoxylic
acid and glycylglycine), leading to the formation of a Schif
base with subsequent hydrolysis resulting in a transamina-
tion (Hidalgo et al. 2013) of both reactants.
Conclusion
In summary, optimal synthesis conditions for the prepara-
tion of α-ketoacyl peptides were identifed. The studies on
diferent α-ketoacyl peptides have confrmed our postulated
hypothesis, that the α-keto group has a crucial infuence on
the reactivity and reaction pathways of α-ketoacyl peptides.
1 3

Studies on the synthesis and stability of α-ketoacyl peptides
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Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
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