Kinetics of aspartic acid isomerization and enantiomerization in model aspartyl tripeptides under forced conditions

Article abstract of DOI:10.1002/jps.22134

The aim of the present study was the determination of the isomerization and enantiomerization of aspartic acid (Asp) in tripeptides. Capillary electrophoresis (CE) assays were developed and validated allowing the simultaneous determination of the diastereomeric α-D/L-Asp and β-D/L-Asp peptides. Rapid isomerization and enantiomerization were noted for peptides with the Phe-Asp-GlyOH sequence at pH 10 and 80°C while Gly-Asp-PheOH proved to be more stable due to the steric influence of the phenyl side chain. A kinetic model assuming a central role of the succinimide intermediate was used to fit the concentration versus time data. In incubations of L-Phe-α-L-Asp-GlyOH the ratio of α-Asp/β-Asp peptides was about 1:4 in agreement with literature data. With regard to L-Asp and D-Asp peptides an α-Asp/β-Asp ratio of about 1:3 and 1:5, respectively, was observed. The stereochemistry of Phe at the X - 1 position affected the ratio of L-Asp/D-Asp implying an effect of the stereochemistry of neighboring amino acids on Asp enantiomerization. Modeling only overall Asp enantiomerization rate constants in accordance to literature data were observed for Asp peptides. In case of the asparagine (Asn) peptide the data could only be fitted to the models considering a direct conversion of L-Asn to a D-configured succinimide via an alternative pathway.

Full text of DOI:10.1002/jps.22134

Kinetics of Aspartic Acid Isomerization and Enantiomerization
in Model Aspartyl Tripeptides under Forced Conditions
UWE CONRAD,1 ALFRED FAHR,2 GERHARD K.E. SCRIBA1
1Department of Pharmaceutical Chemistry, School of Pharmacy, Friedrich Schiller University Jena, Philosophenweg 14, 07743 Jena,
Germany
2Department of Pharmaceutical Technology, School of Pharmacy, Friedrich Schiller University Jena, Lessingstraße 8, 07743 Jena,
Germany
Received 4 April 2009; revised 1 February 2010; accepted 7 February 2010
Published online 5 April 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22134
ABSTRACT: The aim of the present study was the determination of the isomerization and
enantiomerization of aspartic acid (Asp) in tripeptides. Capillary electrophoresis (CE) assays
were developed and validated allowing the simultaneous determination of the diastereomeric a-
D/L-Asp and b-D/L-Asp peptides. Rapid isomerization and enantiomerization were noted for
peptides with the Phe-Asp-GlyOH sequence at pH 10 and 808C while Gly-Asp-PheOH proved to
be more stable due to the steric influence of the phenyl side chain. A kinetic model assuming a
central role of the succinimide intermediate was used to fit the concentration versus time data.
In incubations of L-Phe-a-L-Asp-GlyOH the ratio of a-Asp/b-Asp peptides was about 1:4 in
agreement with literature data. With regard to L-Asp and D-Asp peptides an a-Asp/b-Asp ratio of
about 1:3 and 1:5, respectively, was observed. The stereochemistry of Phe at the X ꢀ 1 position
affected the ratio of L-Asp/D-Asp implying an effect of the stereochemistry of neighboring amino
acids on Asp enantiomerization. Modeling only overall Asp enantiomerization rate constants in
accordance to literature data were observed for Asp peptides. In case of the asparagine (Asn)
peptide the data could only be fitted to the models considering a direct conversion of L-Asn to a
D-configured succinimide via an alternative pathway. ß 2010 Wiley-Liss, Inc. and the American
Pharmacists Association J Pharm SciJ. Pharm. Sci. 99:4162–4173, 2010
Keywords: enantiomerization; isomerization; deamidation; peptides; aspartyl peptide;
aspartic acid; isoaspartic acid; kinetics; stability; capillary electrophoresis
INTRODUCTION
space.1,2,6–9 For example, the reaction occurs fast in
the case of glycine compared to sterically hindered
amino acids. Furthermore, protein structure and
conformation can affect the reaction rates of aspartyl
and asparaginyl residues10–13 as can buffer type, pH,
temperature, etc.5,8,14,15 Generally, the relative rate
of succinimide formation of an Asp residue is slower
compared to Asn as the carboxyl group must be
protonated for the nucleophilic attack of the backbone
amide nitrogen.4
The succinimide is prone to enantiomerization due
to the increased acidity of the proton of the a-carbon
compared to Asp or Asn resulting in an approxi-
mately 105-fold increased enantiomerization rate in
comparison to unmodified Asp or Asn residues.5,16
Hydrolysis of the D-succinimide yields the D-a-Asp
and D-b-Asp peptides. Recently, Li et al.17 provided
evidence that enantiomerization may also occur in
the tetrahedral intermediate preceding the succini-
mide. Isomerization and Asp enantiomerization are
processes of aging of natural proteins2,18,19 as well as
pharmaceutical peptides and proteins20 but may also
Asparagine (Asn) and aspartic acid (Asp) are among
the most unstable amino acids in peptides especially
at weakly acidic to alkaline pH.1,2 This has been
attributed to the formation of aminosuccinimidyl
(Asu) peptides (Scheme 1) arising from the intramo-
lecular nucleophilic attack of the side chain CO group
by the a-nitrogen of the peptide backbone amide.3,4
The succinimide can subsequently hydrolyze to yield
the respective aspartyl (Asp or a-Asp) peptides and
iso-aspartyl (iso-Asp or b-Asp) peptides favoring the
b-Asp peptide over the ‘‘normal’’ a-Asp linkage at
a ratio between 5:1 and 3:1.5,6 Formation of the
succinimide and subsequent isomerization largely
depend on steric hindrance and conformational
Correspondence to: Gerhard K.E. Scriba (Telephone: þ49-3641-
949830; Fax: þ49-3641-949802;
E-mail: gerhard.scriba@uni-jena.de)
Journal of Pharmaceutical Sciences, Vol. 99, 4162–4173 (2010)
ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association
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ASPARTIC ACID ENANTIOMERIZATION IN MODEL ASPARTYL TRIPEPTIDES
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Scheme 1. Isomerization and enantiomerization of Asn and Asp peptides.
possess implications in diseases. For example, b-Asp
linkage and/or D-Asp have been detected in neuro-
fibrillary tangles and plaques in Alzheimer’s
disease.21–23
containing pseudo-tetrapeptide klerval in neutral
and basic aqueous solutions at 808C. The degradation
of Gly-Asp-PheNH2 and Phe-Asp-GlyNH2 in solution
at pH 10 and 808C yielded the respective iso-aspartyl
peptides containing b-L-Asp and b-D-Asp linkages as
well as the a-D-Asp peptide.30,31 Moreover, deamida-
tion of the C-terminal amide of the peptides occurred
under the experimental conditions applied.
The present study was conducted in order to
determine the isomerization and enantiomerization
kinetics of the model tripeptide Phe-Asp-GlyOH
at pH 10 and 808C. The effect of the stereochemistry
of Asp and Phe was investigated by substitution of
L-amino acids by the D-enantiomers in the tripeptide.
Furthermore, the respective Asn peptide was
included as well as a Gly-Asp-PheOH as a peptide
with a sterically hindered amino acid attached to the
C-terminus of Asp. DeHart and Anderson32 recently
reported the degradation of Phe-Asn-GlyOH and Phe-
Asp-GlyOH at alkaline pH and 258C. While isomer-
ization to Phe-b-Asp-GlyOH was observed, the main
degradation product proved to be the diketopiper-
azine cyclo(Phe-Asp)-GlyOH which was found to
epimerize under the experimental conditions. The
enantiomerization of Asp in the course of the
isomerization via the succinimide was not addressed
in detail in this study.
While factors affecting the isomerization have been
investigated in detail, most studies on the concomitant
enantiomerization reported only total D-Asp.19,23–27
Only a few studies distinguished between a-D-Asp
and b-D-Asp peptides. Geiger and Clarke5 studied the
isomerization and enantiomerization of the model
hexapeptides Val-Tyr-Pro-Asn-Gly-AlaOH and Val-
Tyr-Pro-Asp-Gly-AlaOH at pH 7.4. HPLC analysis
was able to separate all isomers. L-Asu and D-Asu
peptides were found to epimerize with the same rate
constant while the rates of hydrolysis differed for both
succinimide stereoisomers.
Schowen and coworkers determined the epimeriza-
tion of Asn and Asp at pH 10 and 708C using the model
peptides Gly-Gln-L-Asn-L-Glu-GlyOH and Gly-Gln-D-
Asn-L-Glu-GlyOH.17 Although HPLC separation of
all four L/D-Asp and L/D-b-Asp peptide isomers was
achieved the data were reported as total L-Asp or
D-Asp peptides. The authors concluded that enantio-
merization did not occur exclusively at the succini-
mide level but also in the tetrahedral intermediate
preceding the succinimide formation. Cloos and
Fledelius28 studied the isomerization and enantio-
merization of the collagen fragment Ala-His-Asp-Gly-
Gly-ArgOH at pH 7.4 and 378C. All four Asp peptide
isomers containing either L-Asp, D-Asp, b-L-Asp, or
b-D-Asp were observed. Of the D-configured peptides
the isomers with the b-linkage predominated under
equilibrium conditions. Won et al.29 observed the
formation of D-Asp and b-D-Asp isomers from the Asp-
MATERIALS AND METHODS
Chemicals
Fmoc-L-Phe-OH, Fmoc-D-Phe-OH, Fmoc-L-Asp(OtBu)-
OH, Fmoc-D-Asp(OtBu)-OH, Fmoc-L-AspOtBu, Fmoc-
DOI 10.1002/jps
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CONRAD, FAHR, AND SCRIBA
D-AspOtBu, Fmoc-L-Asn-OH, Fmoc-GlyOH, Fmoc-
Gly-Wang resin, and Wang resin were purchased
from Bachem AG (Heidelberg, Germany). The pep-
tides L-Phe-a-L-Asp-GlyOH, D-Phe-a-L-Asp-GlyOH,
L-Phe-a-D-Asp-GlyOH, D-Phe-a-D-Asp-GlyOH, L-Phe-
b-L-Asp-GlyOH, D-Phe-b-L-Asp-GlyOH, L-Phe-b-D-
Asp-GlyOH, D-Phe-b-D-Asp-GlyOH, L-Phe-a-L-Asn-
GlyOH, Gly-a-L-Asp-L-PheOH, Gly-a-D-Asp-L-PheOH,
Gly-b-L-Asp-L-PheOH, and Gly-b-D-Asp-L-PheOH
were synthesized by standard solid-phase synthesis
using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethy-
luronium hexafluorophosphate (HBTU) as a coupling
reagent and 1-hydroxybenzotriazole (HOBt) as an
additive. L-Phe-L-Asu-GlyOH and Gly-L-Asu-L-PheOH
were prepared from L-Phe-a-L-Asp-GlyOH and Gly-a-
L-Asp-L-PheOH, respectively, by treatment with
1.8 M HCl in glacial acetic acid.33 The crude peptides
were purified by preparative RP-HPLC using an
acetonitrile–0.1% aqueous trifluoroacetic acid (TFA)
gradient. Collected fractions were combined and
lyophilized. The peptides were characterized by
capillary electrophoresis (CE), HPLC, and mass
spectrometry. The purity was at least 99% by HPLC
and CE analysis.
and 5% acetonitrile. To avoid the evaporation of
acetonitrile the buffer was covered with a thin layer of
liquid paraffin. Normalized peak areas were used for
quantitative determinations.
Kinetic Studies
The degradation of the Asp peptides was performed at
808C in 50 mM borate buffer, pH 10, adjusted to an
ionic strength of 0.2 M by the addition of sodium
chloride. The buffer pH was adjusted at 258C, the
actual pH at elevated temperatures can be estimated
using a value of dpKa/dT of ꢀ0.008 Kꢀ1. The peptides
had an initial concentration of 2 mmol/mL containing
0.09 mmol/mL PAMBA as the internal standard to
compensate for solvent evaporation during the incu-
bation process. At selected time intervals, 70 mL
aliquots were withdrawn and added to 140 mL of ice-
cold 100 mM phosphoric acid in order to stop the
degradation process. The samples were thoroughly
mixed, frozen, and stored at ꢀ208C until analyzed.
The Asu peptides were studied at 37, 40, 60, and
808C under otherwise identical conditions. A peptide
stock solution was added to preheated buffers
containing 0.09 mmol/mL PAMBA to achieve a final
concentration of 2 mmol/mL. The solutions were
incubated at the respective temperature for 1 min.
Seventy microliters of the solutions was withdrawn,
added to 140 mL ice-cold 100 mM phosphoric acid,
and immediately shock frozen in liquid nitrogen.
The samples were stored at ꢀ208C until analyzed. All
incubations were performed in triplicate.
HPLC quality isopropanol, acetonitrile, and TFA
were purchased from Merck (Darmstadt, Germany).
Carboxymethyl-b-cyclodextrin sodium salt was from
Wacker Chemie AG (Burghausen, Germany) and
4-(aminomethyl)benzoic acid (PAMBA) from Fisher
Scientific (Schwerte, Germany). All other chemicals
were of analytical grade. Buffers and solutions were
prepared in double distilled, deionized water, filtered
(0.2 mm), and degassed by sonication.
Data Analysis
The variables in the kinetic model were estimated by
the fitting algorithms of the SAAM II v1.2.1 software
(SAAM Institute, Seattle, WA). This program was
intended to be mainly used in metabolism modeling
and pharmacokinetics, but because of its general
algorithms for solving not only systems of linear
differential equations and its graphical interface it is
also applied in other fields with kinetics problems,
especially in first-order kinetic problems. The pro-
gram can simulate and fit models to data. It can also
handle simultaneous and multiple data sets.
The optimizer function of SAAM II is based on the
Gauss–Newton method.34 Convergence is achieved
when the difference between the value of the linear
approximation of the objective function at the current
parameter estimate and the minimum value of
the linear approximation is less than a specified
value (default value 10ꢀ4 multiplied by the value of
the linear approximation at the current parameter
estimate). The algorithm also can be used to minimize
the variance by introducing weighting schemes for
experimental data with different reliabilities. More-
over, the program allows the use of a Bayesian term
enabling the use of rate constants obtained from other
Capillary Electrophoresis
CE was performed on a Beckman P/ACE MDQ
Capillary Electrophoresis System (Beckman Coulter,
Unterschleißheim, Germany) equipped with a diode
array detector. Fifty micron ID fused-silica capillaries
(BGB Analytik Vertrieb, Schloßbo¨ckelheim, Germany)
with an effective length of 40.0 cm and a total length
of 50.2 cm were used. The capillary was thermostated
at 208C, UV detection was carried out at 215 nm at
the cathodic end of the capillary. Separations were
performed in 50 mM sodium phosphate buffer
adjusted to pH 3.0 by the addition of solid sodium
hydroxide to 50 mM phosphoric acid. The applied
voltage was 25 kV. Sample solutions were introduced
at the anodic end by hydrodynamic injections at
0.5 psi for 5 s. Between the analyses the capillary was
washed for 2 min with 100 mM sodium hydroxide,
2 min with water, and 3 min with the background
electrolyte. Separation of the diastereomeric pairs
L-Phe-a-L-Asp-GlyOH/L-Phe-a-D-Asp-GlyOH and D-
Phe-a-L-Asp-GlyOH/D-Phe-a-D-Asp-GlyOH were per-
formed in 50 mM phosphate buffer, pH 3.0, containing
16 mg/mL carboxymethyl-b-cyclodextrin sodium salt
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ASPARTIC ACID ENANTIOMERIZATION IN MODEL ASPARTYL TRIPEPTIDES
4165
data sets. The program also alerts the user if the
condition number of the variance–covariance matrix
is larger than a certain value indicating an unreliable
fit.
small injection errors and changes of the electro-
osmotic flow in CE analysis as well as for solvent
evaporation during the incubations. The compound
proved to be stable under the incubation conditions at
elevated temperatures.31 As PAMBA was complexed
strongly by the cyclodextrin and did not reach the
detector within 45 min, quantitation of the Phe-a-
Asp-GlyOH isomers was based on the sum of analytes
using the background electrolyte without cyclodex-
trin followed by the determination of the ratio of the
compounds obtained in the assay using the cyclodex-
trin in the run buffer.
The assay was subsequently validated in the range
of 0.015–3.0 mmol/mL with respect to linearity, limit
of quantification (LOQ), limit of detection (LOD), and
precision. The data are summarized in Table 1. The
lower limit of the calibrated concentration range
of 0.015 mmol/L was also the LOQ, the LOD was
0.005 mmol/L (17 mg/mL) for all analytes. Precision
was estimated by injecting concentrations of 0.06 and
0.75 mmol/L six times on the same day (intraday
precision) and three consecutive days (interday
precision). The relative standard deviation (RSD)
was better than 6% in all cases.
In the present setup, the compounds were treated
as compartments linked via the respective reactions
as outlined in Scheme 1. This results in a single
comprehensive model allowing the simultaneous fit of
all reactions. The means of the compound concen-
trations Æ SD were entered into the program for data
fitting. Data for the succinimides were not directly
available from the incubations because the respective
concentrations were below the detection limit of the
analytical method. Information on these compounds
were obtained from an incubation of the Asu peptides
and included in the optimization process as a starting
point. The program subsequently optimized all
functions using information from the concentration
versus time profiles of the incubated peptides as well
as the information from the separate incubation of the
succinimides. None of the parameters introduced was
fixed, all parameters were allowed to be varied by the
optimization procedure of SAAM II.
RESULTS AND DISCUSSION
Peptide Degradation
The peptides L-Phe-a-L-Asp-GlyOH, L-Phe-a-D-Asp-
GlyOH, D-Phe-a-L-Asp-GlyOH, L-Phe-a-L-Asn-GlyOH,
and Gly-a-L-Asp-L-PheOH were incubated in borate
buffer, pH 10, at 808C. The buffer pH was adjusted
at 258C, the actual pH at 808C can be estimated
using dpKa/dT of ꢀ0.008 Kꢀ1. Figure 1 shows the
electropherograms of an incubation of L-Phe-a-L-Asp-
GlyOH after 24 h analyzed in the absence (Fig. 1A)
and in the presence (Fig. 1B) of carboxymethyl-b-
cyclodextrin (for the resolution of the diastereomers
L-Phe-a-L-Asp-GlyOH and L-Phe-a-D-Asp-GlyOH) as
well as after 120 h (Fig. 1C). Peak identification in the
samples was performed by coinjection of synthetic
Method Development and Validation
The CE analysis was based on the experimental
procedures reported in earlier studies.30,31 Except
for the pairs of diastereomers L-Phe-a-L-Asp-GlyOH/
L-Phe-a-D-Asp-GlyOH and D-Phe-a-L-Asp-GlyOH/
D-Phe-a-D-Asp-GlyOH, all compounds could be sepa-
rated using a phosphate buffer, pH 3.0, as back-
ground electrolyte. Separation of the respective
diastereomers was achieved by the addition of
16 mg/mL carboxymethyl-b-cyclodextrin and 5% acet-
onitrile to the phosphate buffer, pH 3.0. PAMBA was
selected as the internal standard to compensate for
Table 1. Calibration Data of the Capillary Electrophoresis Assay
Intraday Precision (RSD) (%)
Interday Precision (RSD) (%)
Peptide
Range (mmol/L)
Slope
R2
0.06 mmol/L
0.75 mmol/L
0.06 mmol/L
0.75 mmol/L
L-Phe-a-L-Asp-GlyOH
L-Phe-b-L-Asp-GlyOH
L-Phe-a-D-Asp-GlyOH
L-Phe-b-D-Asp-GlyOH
L-Phe-a-L-Asn-GlyOH
D-Phe-a-L-Asp-GlyOH
D-Phe-b-L-Asp-GlyOH
D-Phe-a-D-Asp-GlyOH
D-Phe-b-D-Asp-GlyOH
Gly-a-L-Asp-L-PheOH
Gly-b-L-Asp-L-PheOH
Gly-a-D-Asp-L-PheOH
Gly-b-D-Asp-L-PheOH
L-Phe-a-L-Asu-GlyOH
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
0.015–3.0
3.1966
3.0090
3.1348
2.9958
2.4001
2.5226
2.7880
2.9596
2.8676
3.0227
2.9602
3.0050
2.9859
3.1699
0.9998
0.9995
0.9991
0.9997
0.9979
0.9998
0.9995
0.9997
1.0000
0.9989
0.9991
0.9994
0.9970
0.9979
3.74
1.16
1.80
2.96
5.54
5.11
3.89
2.15
0.58
4.85
2.44
3.90
5.47
2.42
3.90
1.12
2.40
2.29
2.16
0.92
1.29
2.46
1.08
1.66
2.40
1.15
0.85
1.79
1.99
3.04
2.18
2.63
2.95
3.23
2.40
5.15
0.69
4.09
2.39
3.67
3.44
1.62
2.49
1.32
1.72
2.13
1.46
0.83
1.08
2.08
0.92
1.68
1.28
1.66
1.52
3.17
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CONRAD, FAHR, AND SCRIBA
Figure 1. Electropherograms of incubations of L-Phe-a-L-Asp-GlyOH (1) in borate
buffer, pH 10, at 808C after 24 h (A and B) or 120 h (C) and Gly-a-L-Asp-L-PheOH (7) in
borate buffer, pH 10, at 808C after 240 h (D). Background electrolyte: phosphate
buffer, pH 3, (A, C, and D) and phosphate buffer, pH 3, containing 16 mg/mL carbox-
ymethyl-b-cyclodextrin and 5% acetonitrile (B); the other compounds are L-Phe-a-D-Asp-
GlyOH (2), L-Phe-b-L-Asp-GlyOH (3), L-Phe-b-D-Asp-GlyOH (4), trans-DKP (5), cis-DKP
(6), Gly-a-D-Asp-L-PheOH (8), Gly-b-L-Asp-L-PheOH (9), Gly-b-D-Asp-L-PheOH (10), and
internal standard PAMBA (IS). For other CE conditions, see the Materials and Methods
Section.
standards as well as by CE–electrospray mass
spectrometry analysis of selected samples (data not
shown).
but are negatively charged at pH 3. Moreover, CE–
electrospray mass spectrometry yielded m/z ¼ 320 for
these peaks which is consistent with the [M þ H]þ-ion
of the diketopiperazine cyclo(Phe-Asp)-GlyOH. Thus,
the peaks were assigned the diastereomeric cis- and
trans-diketopiperazines (cis-DKP and trans-DKP)
identified by DeHart and Anderson. However, in
contrast to their study the peak areas of the diketo-
piperazines never exceeded 2% of the sum of all peak
areas under the present experimental conditions
during the entire incubation. Thus, the compounds
did not appear to be the major degradation products of
L-Phe-a-L-Asp-GlyOH at 808C in borate buffer, pH 10.
The diketopiperazines are formed from the succini-
mides via intramolecular attack of the amino
terminus.32 It can be speculated that higher tem-
peratures favor succinimide hydrolysis over diketo-
piperazine formation. This assumption is supported
by the fact that higher concentrations of the
diketopiperazines were observed in incubations of
the succinimide peptide L-Phe-L-Asu-GlyOH at lower
temperatures (see the discussion below).
Isomerization with concomitant enantiomerization
of Asp was observed for all peptides. For example,
incubation of L-Phe-a-L-Asp-GlyOH yielded the iso-
meric b-Asp peptide L-Phe-b-L-Asp-GlyOH as well
as the epimerization products L-Phe-a-D-Asp-GlyOH
and L-Phe-b-D-Asp-GlyOH. The succinimide inter-
mediates were not observed. In a similar way the
respective isomerization and epimerization products
were observed in experiments varying the stereo-
chemistry of Asp and Phe by incubation of L-Phe-a-D-
Asp-GlyOH and D-Phe-a-L-Asp-GlyOH. In the case
of L-Phe-a-L-Asn-GlyOH the parent peptide quickly
disappeared from the incubation solution yielding
both a-L/D-Asp and b-L/D-Asp diastereomeric pairs
of peptides. DeHart and Anderson32 described the
diketopiperazine derivative cyclo(Phe-Asp)-GlyOH as
the main degradation product of the incubations of
L-Phe-a-L-Asn-GlyOH and L-Phe-a-L-Asp-GlyOH at
258C in alkaline buffers (pH 7.5–9.4). The b-Asp-
containing isomerization product was detected at
lower concentrations. In contrast, at pH 10 and
higher temperatures the isomerization products
predominated. In the present study two small peaks
migrating after the EOF were observed (peaks 5 and 6
in Fig. 1A and C). According to the migration behavior
these compounds do not possess a basic functionality
As expected, Gly-a-L-Asp-L-PheOH degraded
slower compared to L-Phe-a-L-Asp-GlyOH due to
the steric hindrance of the phenyl side chain in
accordance with the literature.2,6 Nevertheless, the
diastereomeric pairs of a-L/D-Asp and b-L/D-Asp
peptides could be observed (Fig. 1D). The succinimide
intermediate was not detected presumably due to the
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low stability under the experimental conditions (pH
10, 808C). Small peaks migrating after the EOF were
also observed (data not shown) so that formation of
diketopiperazines from Gly-a-L-Asp-L-PheOH can be
assumed. However, as for the other peptides inves-
tigated the compounds appear to be only minor
degradation products at 808C with relative peak areas
which did not exceed 2% of the sum of all peptides at
any time point investigated.
treated as kinetic compartments. The model assumes
that Asp enantiomerization occurs at the succinimide
level. Collins et al.35 developed an analogous model
considering only the succinimide. The model was used
for published data to predict Asp enantiomerization
at elevated temperatures. In contrast, Geiger and
Clarke5 concluded from a study of the degradation of a
hexapeptide at 378C that most of the enantiomeriza-
tion could in fact be accounted for from epimerization
of the succinimide but that other mechanisms could
be involved as well. Li et al.17 provided evidence that
enantiomerization in the case of Asn peptides can also
occur in the tetrahedral intermediate preceding
the succinimide. A tetrahedral intermediate is also
formed in the case of Asp peptides so that enantio-
merization at this stage could be postulated as well.
However, the current model does not distinguish
between the individual contributions of these two
possible mechanisms. Therefore, only an ‘‘overall’’
enantiomerization can be derived independent of the
stage (tetrahedral intermediate or succinimide)
where enantiomerization occurs. The rate constants
of the enantiomerization of the Asu peptides were
assumed to be equal, that is, k5 ¼ kꢀ5. Identical
epimerization rates of L- and D-succinimides have
been reported by Geiger and Clarke.5
Kinetics of Isomerization and Enantiomerization
The time courses of the degradation of the peptides
and the appearance of the isomerization and epimer-
ization products at pH 10 and 808C are summarized in
Figure 2. The focus of the present study was the
isomerization and epimerization, and the respective
compounds were the major apparent degradation
products observed. Therefore, small peaks that could
be detected but never exceeded a concentration of
about 2% of the total peptide content at any time point
of the incubations such as the presumed diketopiper-
azines were not considered.
The experimental data were fitted to a model
according to Scheme 1 in which all compounds are
The succinimides could not be detected at any time
point studied. Therefore, the rates of the reactions
describing the degradation of the succinimides (kꢀ1 to
kꢀ4) are difficult to assess. In order to estimate these
rate constants, L-Phe-L-Asu-GlyOH was incubated at
37, 40, 60, and 808C for 1 min. During this short
period of time the amount of the succinimide formed
by back reaction from the Asp peptides is negligible.
Therefore, the concentrations of the a-L/D-Asp and
b-L/D-Asp peptides result solely from the degradation
of the Asu peptide. cis-DKP could be detected at
considerable concentrations at the lower tempera-
tures and was, therefore, also included. trans-DKP
could not be detected in any of these incubations. As
no reference material of cis-DKP was available, the
concentration was estimated using the calibration
data of the Asu peptide.
The formation rate constants of L-Phe-a-L-Asp-
GlyOH, L-Phe-b-L-Asp-GlyOH, and cis-DKP from the
L-Asu peptide can be calculated according to Eq. (1)
yielding the constants kꢀ1, kꢀ2, and kDKP (formation
of DKP, which is not included in Scheme 1):
½LꢀAsuŠ ¼ ½LꢀAsuŠ0  eꢀk t
(1)
n
t
Figure 2. Time course of compounds in solutions of
L-Phe-a-L-Asp-GlyOH (A), L-Phe-a-D-Asp-GlyOH (B), L-
Phe-a-L-Asn-GlyOH (C), D-Phe-a-L-Asp-GlyOH (D), and
Gly-a-L-Asp-L-PheOH (E) in borate buffer, pH 10, 808C.
&, a-L-Asp epimer; ~, a-D-Asp epimer; ^, b-L-Asp epimer;
!, b-D-Asp epimer; *, L-Asn peptide. The data are the
mean of three experiments; the SD values are smaller than
the symbols.
Enantiomerization at the succinimide level is calcu-
lated according to
ðk5 þ kꢀ5 ꢀ k5ð1 ꢀ eꢀtðk þk ÞÞ
5
ꢀ5
½LꢀAsuŠ ¼ ½LꢀAsuŠ
t
0
k5 þ k
ꢀ5
(2)
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CONRAD, FAHR, AND SCRIBA
Table 2. Rate Constants for the Hydrolysis of L-Phe-L-Asu-GlyOH at pH 10 and 37, 40, 60, and 808C
Rate Constants (hꢀ1
)
Activation Energy
(kJ/mol)
378C
408C
608C
808C
k
k
k
k
1.33 Æ 0.02
4.25 Æ 0.03
1.06 Æ 0.06
3.89 Æ 0.12
2.30 Æ 0.08
0.96 Æ 0.02
1.58 Æ 0.06
5.05 Æ 0.06
1.22 Æ 0.30
4.42 Æ 0.30
2.46 Æ 0.09
0.97 Æ 0.06
5.15 Æ 0.07
16.6 Æ 0.18
3.46 Æ 0.72
12.1 Æ 0.7
13.2 Æ 0.1
1.27 Æ 0.07
14.7 Æ 0.1
46.3 Æ 0.1
7.66 Æ 0.53
28.1 Æ 0.6
27.3 Æ 0.3
1.54 Æ 0.10
50.9
50.6
42.0
42.0
—
ꢀ1
ꢀ2
ꢀ3
ꢀ4
k5 and k
kDKP
ꢀ5
10.2
The SD values were calculated by the SAAM II software.
those of the hydrolysis reactions. Thus, at 808C the
rates of hydrolysis of the L-Phe-L-Asu-GlyOH greatly
exceeded the rate of the cyclization to cis-DKP. This
explains the fact that the diketopiperazines were only
minor degradation products in this study at 808C
while they were the dominant products in the pH
range 7.5–9.5 at 258C.32 At pH 10 and 258C the linear
peptides also exceeded the diketopiperazines.32
Extrapolating the plots of kꢀ1 and kꢀ2 to 258C results
in values of 0.58 and 1.89 hꢀ1, respectively. These
values are in good agreement with the data of DeHart
and Anderson,32 who reported values of 0.42–0.66 hꢀ1
for k and 1.14–1.98 hꢀ1 for k depending on the
Including the assumption k5 ¼ k
according to
ꢀ5
Geiger and Clarke5 simplifies Eq. (2) to Eq. (3)
1
½LꢀAsuŠ ¼ ½LꢀAsuŠ0ð1 þ eꢀt2k
Þ
(3)
5
t
2
Subsequently, formation of the D-Asp containing
peptides, k and kꢀ4, can be calculated by
ꢀ3
½LꢀAsuŠ ¼ ½LꢀAsuŠ
t
0
ꢀ
ꢁ
kn
k5
eꢀtk
þ
eꢀtk
(4)
n
5
Â
1 þ
k5 ꢀ kn
kn ꢀ k5
ꢀ1
ꢀ2
composition of the buffer. In contrast, the rate
constant estimated for the formation of cis-DKP
was about 2–3 times lower than the reported value
(0.89 vs. 1.92–3.0). This may be attributed to the
different buffers used in the studies and/or the fact
that the concentration of cis-DKP could only be
estimated due to the lack of reference compound for
assay calibration.
Subsequently, the rate constants determined for
the hydrolysis reactions at 808C were used as a
starting point in the modeling of the experimental
data of the concentration versus time plots shown in
Figure 2 according to Scheme 1. The initial hydrolysis
constants were also allowed to be varied by the SAAM
II software during the optimization process. The
resulting constants are summarized in Table 3. The
stability of the Asp peptides was estimated from
the constant Kn defined as the ratio of kꢀn/kn (Tab. 3).
Table 4 lists the calculated percentage of the
individual peptide isomers at equilibrium using the
respective rate constants given in Table 3.
All these equations influence each other. Therefore,
the modeling software was used to calculate the
respective rate constants at 37, 40, 60, and 808C
according to Scheme 1 as summarized in Table 2.
Using the slope of the line of best fit of the Arrhenius
plot (Fig. 3) the activation energies of the reactions
were determined (Tab. 2). Interestingly, the slope of
the Arrhenius plot of the reaction from the Asu
peptide to cis-DKP was much flatter compared to the
slopes of the reaction rates representing hydrolysis
to linear a-Asp and b-Asp peptides. Moreover, the
activation energy was 4–5 times lower compared to
Incubation of L-Phe-a-L-Asp-GlyOH at 808C yielded
the b-Asp as well as D-Asp peptides (Fig. 2A).
Complete equilibrium was not reached after the total
incubation period of 240 h. The ratio a-Asp/b-Asp
peptides calculated from the percent peptide content
at equilibrium conditions yielded a value of 1:4.0
which is comparable to the value of 1:4 reported for an
Asp hexapeptide containing the Asp-Gly sequence
after 70 h at pH 10 and 708C.6 A similar a/b ratio was
also found within the L-Asp and D-Asp isomers,
Figure 3. Arrhenius plot of the degradation of L-Phe-L-
Asu-GlyOH at 37, 40, 60, and 808C. Rate constants labeled
according to Scheme 1: &, kꢀ1; ^, kꢀ2; ~, kꢀ3; !, kꢀ4; *,
rate constant of the formation of cis-DKP. The data are the
mean of three experiments Æ SD.
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ASPARTIC ACID ENANTIOMERIZATION IN MODEL ASPARTYL TRIPEPTIDES
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Table 3. Pseudo-First-Order Rate Constants and Rate Constant Ratio of Asp Isomerization and Enantiomerization of
Model Tripeptides at pH 10 and 808C
Rate Constant (hꢀ1
)
L-Phe-a-L-Asp-GlyOH
L-Phe-a-D-Asp-GlyOH
L-Phe-a-L-Asn-GlyOH
D-Phe-a-L-Asp-GlyOH
Gly-a-L-Asp-L-PheOH
k1
k2
k3
k4
k
0.0263 Æ 0.0002
0.0285 Æ 0.0021
0.0184 Æ 0.0010
0.0148 Æ 0.0014
14.8 Æ 0.1
46.3 Æ 0.1
7.67 Æ 0.52
28.2 Æ 0.5
27.3 Æ 0.2
—
0.0265 Æ 0.0016
0.0269 Æ 0.0022
0.0186 Æ 0.0008
0.0150 Æ 0.0013
14.7 Æ 0.1
46.3 Æ 0.1
7.67 Æ 0.52
28.2 Æ 0.5
27.3 Æ 0.3
—
0.0273 Æ 0.0015
0.0257 Æ 0.0018
0.0185 Æ 0.0010
0.0139 Æ 0.0013
14.7 Æ 0.1
0.0185 Æ 0.0010
0.0156 Æ 0.0017
0.0249 Æ 0.0010
0.0270 Æ 0.0024
7.67 Æ 0.53
28.2 Æ 0.6
14.8 Æ 0.1
46.3 Æ 0.1
27.3 Æ 0.3
—
0.0026 Æ 0.0003
0.0075 Æ 0.0016
0.0142 Æ 0.0056
0.0088 Æ 0.0030
337 Æ 85
ꢀ1
k
ꢀ2
k
ꢀ3
k
ꢀ4
46.3 Æ 0.1
382 Æ 80
7.58 Æ 0.50
28.2 Æ 0.5
673 Æ 206
428 Æ 106
k5 and k
k6
k7
K1
K2
K3
K4
27.3 Æ 0.3
440 Æ 133
ꢀ5
1.07 Æ 0.14
0.37 Æ 0.08
541 Æ 30
—
—
—
—
—
562 Æ 5
557 Æ 33
416 Æ 36
128,214 Æ 23,027
145,263 Æ 8904
47,425 Æ 9463
30,184 Æ 10,394
1628 Æ 123
416 Æ 35
1719 Æ 142
411 Æ 31
1802 Æ 127
409 Æ 34
1812 Æ 199
593 Æ 25
1911 Æ 187
1879 Æ 165
2035 Æ 191
1713 Æ 154
The SD values were calculated by the SAAM II software.
respectively (1:3.3 for the L-Asp peptides and 1:4.6 for
D-Asp peptides). The rate constants for the formation
observed for L-Phe-a-L-Asp-GlyOH. The same applies
to the ratio of L-Asp/D-Asp peptides. Interestingly, the
rate constants of the individual reactions listed in
Table 3 did not differ significantly between L-Phe-a-D-
Asp-GlyOH and L-Phe-a-L-Asp-GlyOH. As succini-
mide formation proceeded slower from L-Phe-a-D-Asp-
GlyOH equilibrium conditions were reached at a later
time.
Rapid deamidation with concomitant formation of
Asp isomerization and epimerization products was
observed for L-Phe-a-L-Asn-GlyOH at 808C in borate
buffer, pH 10 (Fig. 2C). The data could not be fitted
well assuming only conversion of the L-Asn peptide
to the L-configured succinimide (k6). Thus, a ‘‘direct’’
of a-Asp peptides from the succinimide, k and k
and of the formation of the b-Asp peptides from
,
ꢀ1
ꢀ3
the Asu intermediate, k and kꢀ4, do not differ
ꢀ2
significantly within each set. The ratios of the stab-
ility constants K2/K1 and K4/K3 were 2.9 and 4.6,
respectively, corresponding to the above-mentioned
ratio of a-Asp/b-Asp peptides at equilibrium. The
D-Asp peptides exceeded the L-Asp peptides at a ratio
of about 1.19:1.
Upon incubation of L-Phe-a-D-Asp-GlyOH (Fig. 2B)
the b-Asp peptides exceeded the a-Asp peptides by a
factor of about 4 (Tab. 4), consistent with the values
Table 4. Peptide Content and Asp Isomer Ratio at Equilibrium Conditions at pH 10 and 808C
Peptide
Degradation Product
Peptide Content (%)
a-Asp/b-Asp
L-Asp/D-Asp
1:1.19
L-Phe-a-L-Asp-GlyOH
L-Phe-a-L-Asp-GlyOH
L-Phe-b-L-Asp-GlyOH
L-Phe-a-D-Asp-GlyOH
L-Phe-b-D-Asp-GlyOH
L-Phe-a-L-Asp-GlyOH
L-Phe-b-L-Asp-GlyOH
L-Phe-a-D-Asp-GlyOH
L-Phe-b-D-Asp-GlyOH
L-Phe-a-L-Asp-GlyOH
L-Phe-b-L-Asp-GlyOH
L-Phe-a-D-Asp-GlyOH
L-Phe-b-D-Asp-GlyOH
D-Phe-a-L-Asp-GlyOH
D-Phe-b-L-Asp-GlyOH
D-Phe-a-D-Asp-GlyOH
D-Phe-b-D-Asp-GlyOH
Gly-a-L-Asp-L-PheOH
Gly-b-L-Asp-L-PheOH
Gly-a-D-Asp-L-PheOH
Gly-b-D-Asp-L-PheOH
10.5
35.1
9.7
44.7
10.8
34.6
9.7
44.9
10.5
35.0
9.1
45.4
9.4
45.2
10.7
34.7
40.7
22.3
15.6
21.4
1:4.0
L-Phe-a-D-Asp-GlyOH
L-Phe-a-L-Asn-GlyOH
D-Phe-a-L-Asp-GlyOH
Gly-a-L-Asp-L-PheOH
1:3.9
1:4.1
1:4.0
1:0.77
1:1.20
1:1.20
1:0.83
1:0.59
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CONRAD, FAHR, AND SCRIBA
formation of the D-succinimide from L-Phe-a-L-Asn-
GlyOH was introduced represented by k7. This
considers enantiomerization at a different stage than
the Asu peptide, for example, via the tetrahedral
intermediate preceding the succinimide as suggested
by Li et al.17 The apparent deamidation rate
constants were k6 ¼ 1.07 Æ 0.14 hꢀ1 and k7 ¼ 0.37 Æ
0.08 hꢀ1. All other rate constants as well as the ratios
a-Asp/b-Asp peptides and L-Asp/D-Asp peptides are
essentially identical within experimental error to
those found for L-Phe-a-L-Asp-GlyOH (Tabs. 3 and 4).
Thus, identical pathways of degradation can be
assumed for both peptides, except the deamidation
in case of L-Phe-a-L-Asn-GlyOH.
dent of configuration of the amino acids of the starting
peptide, Asp enantiomerization proceeded at compar-
able rates with constants of 27.3 hꢀ1 (k5 and k in
ꢀ5
Tab. 3).
The effect of an amino acid with a spacious
substituent on the isomerization and enantiomeriza-
tion of Asp was investigated using Gly-a-L-Asp-L-
PheOH which has the inverse amino acid sequence
compared to the parent peptide. The hydrolysis rate
constants of Gly-L-Asu-L-PheOH were estimated
from short time incubations at 40, 60, and 808C as
described for L-Phe-L-Asu-GlyOH. The constants were
subsequently used to model the experimental data
of the concentration versus time plots shown in
Figure 2E.
Furthermore, the effect of the stereochemistry of
the amino acid attached to the amino group of Asp
was studied by incubating D-Phe-a-L-Asp-GlyOH
(Fig. 2D). Interestingly, a higher amount of L-Asp
peptides was observed compared to incubations of the
parent peptide L-Phe-a-L-Asp-GlyOH. Thus, a ratio
L-Asp/D-Asp of 1:0.83 was calculated at equilibrium
conditions (Tab. 4). The ratio of a-Asp and b-Asp
peptides was comparable to the ratio found for
the other peptides with the Phe-Asx-Gly sequence
(Asx ¼ Asp or Asn). The rate constants describing the
enantiomerization of the succinimide intermediates
were also comparable to the peptides containing
L-Phe (Tab. 3). The rates of the formation of the
succinimides changed their relative order (Tab. 3).
Rate constants in the range of 0.0263–0.0273 hꢀ1 for
k1 or between 0.0184 and 0.0186 hꢀ1 for k3 were found
in the case of L-Phe peptides. In contrast, values of
0.0185 and 0.0249 hꢀ1 were determined for k1 and k3,
respectively, in the case of D-Phe-a-L-Asp-GlyOH. The
same applied to the constants k2 and k4 as well as for
The degradation of Gly-a-L-Asp-L-PheOH proceed-
ed slower, equilibrium conditions were not reached
during the incubation period of 1000 h (Fig. 2E).
Equilibrium conditions would have been reached
after at least 2000 h. a-Asp peptides dominated the
degradation products resulting in an a-Asp/b-Asp
ratio of 1:0.77. Oliyai and Borchardt6 also found lower
concentrations of b-Asp peptides than a-Asp peptides
in incubations of Val-Tyr-Pro-Asp-Val-AlaOH at pH 10
and 708C. Therefore, the reversed a-Asp/b-Asp ratio
can also be attributed to steric hindrance by the
phenyl side chain of the amino acid adjacent to Asp
inhibiting the formation of the succinimide and
subsequently the isomerization reaction. The con-
stants K1 and K3 describing the stability of the
tripeptide increased by a factor of 115–240 compared
to L-Phe-a-L-Asp-GlyOH, K2 and K4 increased only
15- to 80-fold. The rate constants for the formation of
the succinimides from D-Asp and L-Asp peptides
decreased by a factor of about 1.3 and 10, respectively,
which can also be attributed to the steric hindrance
by the amino acid side chain.2,6,8,37 Interestingly,
the reaction constants for the D-Asp peptides (8.8–
14.2 Â 10ꢀ3 hꢀ1) exceeded the rate found for L-Asp
peptides (2.6–7.5 Â 10ꢀ3 hꢀ1) as observed for D-Phe-a-
L-Asp-GlyOH albeit with approximately two- to
sevenfold lower values. Asp enantiomerization pro-
ceeded at a rate constant of 440 Æ 133 hꢀ1 (Tab. 3).
Taking the overall degradation pathway of the
L- and D-Asu intermediates into account the respec-
tive half-lives can be calculated according to Eqs. (5)
and (6):
the constants of the hydrolysis reactions k to k
.
ꢀ1
ꢀ4
The data suggest an effect of the configuration of the
amino acid N-terminal of Asp on the degradation
kinetics. As can be concluded from the rate constants
in Table 3 peptides with opposite stereochemistry of
Asp and Phe appear to have a decreased tendency for
cyclization to form the Asu intermediates compared to
peptides with amino acids of identical configuration.
Only a small effect of the amino acids N-terminal
to Asn on peptide deamidation was reported.36
However, one has to keep in mind that L-Phe-a-D-
Asp-GlyOH and D-Phe-a-L-Asp-GlyOH are in fact
enantiomers which will have equal reaction rates in
an achiral environment. Thus, further experiments
will be required to establish a stereochemical effect
of neighboring amino acids on Asp isomerization and
enantiomerization.
ln 2
t1=2ðLꢀAsuÞ ¼
t1=2ðDꢀAsuÞ ¼
(5)
(6)
k
ꢀ1 þ kꢀ2 þ k5
ln 2
Cyclization to the succinimide intermediate was
generally the rate-limiting step in Asp enantiomer-
ization as the respective rate constants are by far
smaller compared to the constants determined for the
hydrolysis and enantiomerization (Tab. 3). Indepen-
k
ꢀ3 þ kꢀ4 þ k
ꢀ5
The half-lives obtained from the data of the
respective peptides are summarized in Table 5. As
expected, essentially identical values were found for
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ASPARTIC ACID ENANTIOMERIZATION IN MODEL ASPARTYL TRIPEPTIDES
4171
Table 5. Half-Lives of Asu Intermediates in Tripeptide
Incubations at pH 10 and 808C
Table 6. Rate Constants of Overall Asp
Enantiomerization according to Scheme 2
t1/2 of L-Asu
Peptide (s)
t1/2 of D-Asu
Peptide (s)
Incubated Peptide
kr  103 (hꢀ1
)
k
ꢀr  103 (hꢀ1
)
kr/k
ꢀr
Incubated Peptide
L-Phe-a-L-Asp-GlyOH
L-Phe-a-D-Asp-GlyOH
L-Phe-a-L-Asn-GlyOH
D-Phe-a-L-Asp-GlyOH
Gly-a-L-Asp-L-PheOH
7.93
8.84
8.29
7.15
1.75
6.60
7.40
6.75
9.00
2.06
1.20
1.19
1.23
0.79
0.85
L-Phe-a-L-Asp-GlyOH
L-Phe-a-D-Asp-GlyOH
L-Phe-a-L-Asn-GlyOH
D-Phe-a-L-Asp-GlyOH
Gly-a-L-Asp-L-PheOH
28.24 Æ 0.12
28.25 Æ 0.12
28.25 Æ 0.12
39.53 Æ 0.55
2.15 Æ 0.54
39.51 Æ 0.55
39.53 Æ 0.55
39.55 Æ 0.55
28.24 Æ 0.12
1.62 Æ 0.46
The SD values were calculated by the SAAM II software.
fit onto the Arrhenius plot constructed by Collins
et al.35 from published data of Asx enantiomerization.
The experimental data of the incubation of L-Phe-a-
L-Asn-GlyOH were fitted according to Scheme 2
considering a conversion of L-Asn to L-Asp and
D-Asp via the reactions with the rate constants kL
(L-Asn ! L-Asu) and kD (L-Asn ! D-Asu). This is in
analogy to Scheme 1. The deamidation of Asn to
L-Asu proceeded with a rate constant kL ¼ 1.08 Æ
0.01 hꢀ1 and to D-Asu with kD ¼ 0.56 Æ 0.01 hꢀ1. The
data are in good agreement with constants deter-
mined for the reactions according to Scheme 1 (k6 and
k7 in Tab. 3). Thus, about 33% of the D-Asp peptides
appear to result from direct conversion of L-Asn
through an alternative pathway. Collins et al.35 also
observed that fitting of published data of Asp
racemization starting from Asn peptides was only
possible considering an additional direct conversion
of L-Asn to D-Asn. Geiger and Clarke5 concluded
from incubations of an Asn hexapeptide that not all
enantiomerization of Asp could be accounted for by
sole enantiomerization at the Asu level. Evidence
that racemization may also occur at the stage of a
tetrahedral intermediate was provided by Li et al.17
the succinimide intermediates derived from peptides
containing the L-Phe-Asx-GlyOH sequence. The half-
life of the D-Asu peptides is approximately 1.4-fold
longer than the half-life of the L-Asu peptides due to
their slower hydrolysis compared to the L-configured
succinimides. In contrast, Gly-D/L-Asu-L-PheOH dis-
played extremely short half-lives due to the faster
hydrolysis compared to Phe-Asu-GlyOH peptides.
Overall Enantiomerization of Asp
Most studies described only the overall enantiomer-
ization of Asp and did not consider the individual
contributions from a-Asp or b-Asp peptides. In order
to compare the published data with the present
results of the tripeptides, a second model accounting
only for the overall enantiomerization according to
Scheme 2 was employed. The concentrations of the
individual peptides shown in Figure 2 were combin-
ed solely according to the stereochemistry of Asp
and used for the calculation of the rate constants, kr
and kꢀr. The data are summarized in Table 6. The
constants of the enantiomerization reactions of Phe-
Asx-GlyOH peptides all lie in the same range with
values between 6.6 Â 10ꢀ3 and 9.0 Â 10ꢀ3 hꢀ1. In
contrast to the enantiomerization constants obtained
according to Scheme 1 (k5 and kꢀ5), the constants
calculated from Scheme 2 are not equal. This is due to
the fact that kr and kꢀr represent combined constants
which include formation of the succinimide, enantio-
merization, and hydrolysis. Nevertheless, the ratio of
the respective rate constants reflects the ratio L-Asp/
D-Asp peptides determined according to Scheme 1
(Tab. 4). Epimerization of Gly-a-L-Asp-L-PheOH
occurred at a fivefold lower rate reflecting the steric
hindrance by Phe. The present data are estimated to
CONCLUSION
The isomerization and enantiomerization kinetics of
Asp and Asn tripeptides were studied at pH 10 and
808C. CE assays allowed the determination of the
individual L-Asp and D-Asp peptides containing the
a-Asp or b-Asp linkage. Rapid isomerization and
enantiomerization of Asp were observed in peptides
with the Asx-Gly sequence. In agreement with the
literature a reduced rate was noted for Gly-Asp-
PheOH due to steric hindrance of the succinimide
formation by the spacious phenyl group. Comparable
a-Asp/b-Asp ratios of about 1:4 were observed for all
Phe-Asx-GlyOH peptides. While the D-Asp epimers
exceeded L-Asp isomers in case of L-Phe containing
peptides a larger amount of L-Asp isomers than D-Asp
epimers was found in incubations of D-Phe-a-L-Asp-
GlyOH. Thus, although the number of investigated
peptides is rather limited, the configuration of the
amino acid at the X ꢀ 1 position of Asp may affect
the enantiomerization. It has been already noted that
Scheme 2. Kinetic model of overall enantiomerization of
Asp peptides.
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CONRAD, FAHR, AND SCRIBA
the microenvironment within a protein affects Asp
enantiomerization, that is, not only the amino acid
sequence but also higher order structures affect Asx
reactivity.10,12 The configuration of neighboring
amino acids may present an additional factor in
this reaction. However, as some of the investigated
peptides are in fact enantiomers, additional studies
will be necessary in order to assess the stereochemical
effect of amino acids at the X ꢀ 1 position. Considering
only overall Asp enantiomerization the observed data
are in agreement with published values.35
Data of the Asn peptide could only be fitted taking
also a direct conversion of L-Asn to D-configured
peptides in both models. A similar observation has
been made by Collins et al.35 when compiling
published data in order to predict Asp racemization
during protein decomposition. Li et al.17 provided
evidence of the formation of a D-Asn peptide from a
L-Asn peptide (and vice versa) under deamidation
conditions suggesting that enantiomerization may
also occur at the tetrahedral intermediate preceding
the succinimide. Our data are consistent with these
observations.
9. Radkiewicz JL, Zipse H, Clarke S, Houk KN. 2001. Neighboring
side chain effects on asparaginyl and aspartyl degradation: An
ab initio study of the relationship between peptide conforma-
tion and backbone NH acidity. J Am Chem Soc 123:3499–
3506.
10. Wakankar AA, Borchardt RT. 2006. Formulation considera-
tions for proteins susceptible to asparagine deamidation and
aspartate isomerization. J Pharm Sci 95:2321–2336.
11. Lura R, Schirch V. 1988. Role of peptide conformation in the
rate and mechanism of deamidation of asparaginyl residues.
Biochemistry 27:7671–7677.
12. Xie M, Schowen RL. 1999. Secondary structure and protein
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13. Athmer L, Kindrachuk J, Georges F, Napper S. 2002. The
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succinimide hydrolysis. J Biol Chem 277:30502–30507.
14. Capasso S, Mazzarella L, Zagari A. 1991. Deamidation via
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ACKNOWLEDGMENTS
The authors thank Dr. Tereza Souza, Department of
Pharmaceutical Technology, University of Jena for
helpful discussions during mathematical modeling.
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