Reversible hydrogen transfer between cysteine thiyl radical and glycine and alanine in model peptides: Covalent H/D exchange, radical-radical reactions, and l - To D -Ala conversion

Article abstract of DOI:10.1021/jp101508b

The reversible intramolecular hydrogen transfer reaction of peptide Cys thiyl radicals with Gly and Ala residues was studied in model peptides, where thiyl radicals were either generated through photochemical cleavage of disulfide bonds or through the rea

Full text of DOI:10.1021/jp101508b

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J. Phys. Chem. B 2010, 114, 6751–6762
6751
Reversible Hydrogen Transfer between Cysteine Thiyl Radical and Glycine and Alanine in
Model Peptides: Covalent H/D Exchange, Radical-Radical Reactions, and L- to D-Ala
Conversion
Olivier Mozziconacci,† Bruce A. Kerwin,‡ and Christian Scho¨neich*,†
Department of Pharmaceutical Chemistry, 2095 Constant AVenue, UniVersity of Kansas,
Lawrence, Kansas 66047, and Department of Process and Product DeVelopment, Amgen Inc.,
1201 Amgen Court West, Seattle, Washington 98119
ReceiVed: February 18, 2010
The reversible intramolecular hydrogen transfer reaction of peptide Cys thiyl radicals with Gly and Ala residues
was studied in model peptides, where thiyl radicals were either generated through photochemical cleavage of
disulfide bonds or through the reaction of Cys thiol with •CH3 or CH3C•O radicals, or both, generated through
photolysis of acetone. In D2O, the reversible hydrogen transfer leads to covalent H/D exchange, indicative of
the location of intermediary carbon-centered radicals. In addition, the reversible formation of RC• radicals on
Ala leads to the conversion of L-Ala to D-Ala, where the efficiency of this conversion depends on the primary
sequence of the Ala-containing peptide. When Cys thiyl radicals are generated through the reaction of Cys
•
thiol with CH3 or CH3C•O radicals, various recombination products between these initiating radicals and
peptide thiyl and carbon-centered radicals provide further evidence for the location of intermediary radicals
within the peptide sequence.
1. Introduction
potential of thiyl radicals to induce biological damage became
apparent after a series of studies established hydrogen transfer
reactions with alcohols,19 ethers,20,21 carbohydrates,22 amines,23
and polyunsaturated fatty acids,24 as well as reversible addition
reactions to unsaturated fatty acids, resulting in cis-trans
isomerization.25–28 Subsequently, kinetic NMR experiments
demonstrated the reaction of thiyl radicals with predominantly
the RC-H bonds of amino acids in a series of model peptides.29
Protein cysteine (Cys) residues represent primary targets for
oxidative modification through a variety of oxidizing agents
relevant for biological conditions of oxidative stress1–5 and the
production and formulation of biotechnology products.6,7 Here,
the two-electron oxidation, (e.g., through H2O2) of Cys directly
yields sulfenic acid (CysSOH) (reaction 1),8 whereas the one-
electron oxidation (e.g., through hydroxyl radical (HO•)) results
in the formation of a thiyl radical, CysS• (reaction 2).9–12 CysS•
radicals are also easily generated through the one-electron
reduction (reaction 3)13,14 or photolysis (reaction 4) of cystine.15,16
Especially the photolysis of disulfides presents an important
problem for the production and long-term stabilization of protein
pharmaceuticals.17
Time-resolved kinetic measurements on reversible unimo-
lecular hydrogen transfer reactions established absolute rate
constants on the order of k5 ≈ 105 s-1 and k-5 ≈ 106 s-1 for
thiyl radicals in the model peptides N-Ac-Cys-Gly6 and N-Ac-
Cys-Gly2-Asp-Gly3 and k5 ≈ 104 s-1 and k-5 ≈ 105 s-1 for thiyl
radicals in the model peptide N-Ac-Cys-Ala2-Asp-Ala3.30 The
occurrence of such reversible hydrogen transfer reactions was
confirmed in a separate series of experiments in which a small
cystine-containing model peptide, (GGCGGL)2, was photolyzed
in D2O, and the formation of C-D bonds according to reactions
6-8 (Scheme 1) was monitored by mass spectrometry.31 Such
covalent H/D exchange was subsequently documented for the
photolysis of insulin, a protein that contains three disulfide
bonds.32
On the basis of reactions 6-8, presented in Scheme 1, any
reversible hydrogen transfer with amino acids other than Gly
could potentially lead to epimerization of peptides through L-to-
D-amino acid conversion. Such reactions are not unexpected,
on the basis of known propensity of thiyl radicals to racemize
amines.33,34 Therefore, we have generated Cys thiyl radicals
(CysS•) in a series of Gly- and Ala-containing model peptides
and evaluated the propensity of thiyl radicals for L-to-D-amino
CysSH + H2O2 f CysSOH + H2O
CysSH + HO• f CysS• + H2O
(1)
(2)
CysSSCys + e- + H+ f CysS• + CysSH
CysSSCys + hV f 2CysS•
(3)
(4)
For long, CysS• radicals have been considered as rather
unreactive toward biological substrates. Hence, the recombina-
tion to disulfide was considered a prominent pathway of thiyl
radicals in the absence of oxygen, whereas reversible oxygen
addition,18 ultimately yielding sulfonic acid, was considered a
prominent reaction under aerobic reaction conditions. The
* Corresponding author. Fax: (785) 864-5736. E-mail: schoneic@ku.edu.
† University of Kansas.
‡ Angen Inc.
10.1021/jp101508b  2010 American Chemical Society
Published on Web 04/23/2010

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6752 J. Phys. Chem. B, Vol. 114, No. 19, 2010
Mozziconacci et al.
r
SCHEME 1: Reaction Scheme for the Covalent H/D Exchange between a CysS• Radical and an C-H Bond during the
Photolysis of a Cystine-Containing Peptide in D2O
acid conversion and for promoting covalent H/D exchange in
these peptides. For these studies, we decided to generate thiyl
radicals via two independent pathways; that is, (i) disulfide
photolysis and (ii) the reaction of Cys-containing peptides with
•CH3/CH3CO• radicals generated via the photolysis of acetone.
Specifically, the second method of thiyl radical generation
represents an oxidative pathway that not only is of great
biological significance but also serves to validate some of our
previous experiments,31,32 in which thiyl radicals were generated
through disulfide homolysis. Our new data will demonstrate that
thiyl radical-dependent formation of carbon-centered radicals
will not only result in covalent H/D exchange but can also be
monitored through characteristic recombination products of
mixtures, each stock solution (acetone, water, peptide-containing
solutions) was gently flushed for several minutes independently
with argon prior to mixing the different solutions in an argon
presaturated quartz tube. The solutions were irradiated for up
to 10 min with four UV lamps (Southern England, RMA-500)
emitting light of λ ) 253.7 nm light in a Rayonet photochemical
reactor (Southern England, MA).
2.2. Sample Preparation for UPLC-MS Analysis. Im-
mediately after photoirradiation, the samples were injected onto
a Vydac column (25 cm ×1 mm C18, 3.5 µm), and eluted with
a linear gradient delivered at the rate of 90 µL min-1 by an
AcquityUltra-Performance Liquid Chromatography system (Wa-
ters Corporation, Milford, MA). Mobile phases consisted of
water/acetonitrile (ACN)/formic acid at a ratio of 99%, 1%,
0.08% (v:v:v) for solvent A and a ratio of 1%, 99%, 0.06%
(v:v:v) for solvent B. The following linear gradients were set:
1% of solvent B for 1 min, 1-75% of solvent B within 35 min.
The samples photoirradiated in the presence of acetone were
dried in a speedvac (4 h spin at 35 °C under vacuum). The
individual samples were then resolubilized in 100 µL of water
prior to injection onto the capillary column.
2.3. Nano-Electrospray Ionization Time-of-Flight (ESI-
TOF-MS) Analysis. ESI-MS spectra were acquired on a
Q-TOF-2 (Micromass Ltd., Manchester, U.K.) hybrid mass
spectrometer operated in the MS1 mode and acquiring data with
the time-of-flight analyzer. The instrument was operated for
maximum resolution with all lenses optimized on the [M +
2H]2+ ion from the cyclic peptide Gramicidin S. The cone
voltage was 35 eV, and Ar was admitted to the collision cell at
a pressure that attenuates the beam to about 20%, and the cell
was operated at 12 eV (maximum transmission). Spectra were
acquired at 16 129 Hz pusher frequency covering the mass range
350-2000 amu (amu ) atomic mass unit) and accumulating
•
peptide radicals with CH3 and CH3CO• derived from acetone
photolysis.
2. Experimental Section
2.1. Materials and Reactions. The disulfide-linked peptides
(LGACAGL)2, (LGGCGGL)2, and (AACAA)2 (Chart 1) were
synthesized by the Biochemical Resource Service Laboratory
(BRSL) at the University of Kansas and purified to a purity
level of >95%. The disulfide-linked peptide (GGCGGL)2 was
provided by Amgen. The reduced peptides LGGCGGL, GGCG-
GL, and LGACAGL were obtained by reduction of the disulfide-
linked peptides (LGGCGGL)2, (GGCGGL)2, and (LGACAGL)2
with dithiothreitol (DTT) at pH 8.5. The reduced peptides were
purified by HPLC. Triethylamine (TEA) and acetone were
purchased from Sigma-Aldrich (St Louis, MO) at the highest
available purity grade. Deuterated acetone (acetone-d6) was
purchased from Cambridge Isotope Laboratories at the highest
purity grade. The peptides were dissolved in H2O (MilliporeQ)
or in acetone/H2O (1:4, v:v) at a concentration of 100 µM. Prior
to UV irradiation, a 200 µL aliquot of each solution was placed
in a quartz tube and saturated with Ar. For acetone/H2O

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Hydrogen Transfer between CysS•, Gly, Ala
J. Phys. Chem. B, Vol. 114, No. 19, 2010 6753
data for 3 s per cycle. Time to mass calibration was made with
CsI cluster ions acquired under the same conditions.
NJ) was used for the quantification of free thiols.35 A spectro-
fluorometer (RF 5000U, Shimadzu) was set at λex ) 379 nm
and λem ) 513 nm and was calibrated with different known
concentrations of reduced glutathione in the range of 0-2 µM.
The derivatization with TG (15 µM) was carried out in 1 mL
of a solution of ammonium bicarbonate buffer (50 mM, pH 7.3)
containing 10 uL of the irradiated sample. The fluorescence
intensity was measured after 1 h of incubation with TG.
2.6. MS/MS Analysis. CID spectra were acquired by setting
the MS1 quadrupole to transmit a precursor mass window of
(1.5 amu centered on the most abundant isotopomer. Ar was
the collision gas admitted at a density that attenuates the beam
to 20%; this corresponds to 16 psi on the supply regulator or
5.3 × 10-5 mbar on a penning gauge near the collision cell.
The collision energy varied between 20 and 45 eV. Spectra were
acquired for 2-3 min in 5 s cycles as the peptides were eluted
off a desalting column.
2.4. Gas Chromatography/Mass Spectrometric (GC/MS)
Analysis. The gas chromatography/mass spectrometric data were
collected on an Agilent 6890N gas chromatograph interfaced
with a mass analyzer (Quattro Micro GC, Waters Corp., Milford,
MA). A 5% phenyl, methyl silicone stationary phase (HP-5MS)
was used (l ) 15 m, 0.25 in. i.d.). The carrier gas was helium,
and the constant flow mode was used to maintain 1.5 mL/min.
Injections of 10.0 µL were made into the injector port heated
to 240 °C, and a split ratio of 10 was used. The GC thermal
gradient was as follows: an initial temperature of 35 °C was
held for 1 min, after which the temperature was increased 50
°C/min to a final temperature of 300 °C and held for 2 min.
Ionization was by electron impact at 70 eV.
2.5. Quantification of Free Thiols. The fluorogenic thiol
reagent ThioGlo1 (TG; 10-(2,5-dihydro-2,5-dioxo-1H-pyrrol-
1-yl)-9-methoxy-3-oxo, methyl ester-3H-naphtol[2,1-b]pyran-
s-carboxylic acid; Calbiochem, EMD Chemicals, Gibbstown,
2.7. Covalent H/D Exchange and Isotopic Correction. The
deuterium composition of peptide ions and their fragments was
CHART 1: Representation of the Cystine-Containing Peptide Models

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6754 J. Phys. Chem. B, Vol. 114, No. 19, 2010
Mozziconacci et al.
determined from the differences between the average mass of
a covalently deuterated peptide and the average mass of the
corresponding fully protonated peptide. The average masses
were calculated from centroided isotopic distributions. The
distribution of deuterium incorporation was obtained after
isotopic correction by subtracting the isotope abundance dis-
tribution in the product formed during UV irradiation in H2O
from the isotope abundance distribution of the same product
generated in D2O. This variation of the isotopic distribution
between the experiments performed in deuterium oxide solution
and water is given by the variation of the percent base peak
intensity (% ∆BPI).
2.8. Actinometry. The chemical actinometer KI/KIO3 was
used to calculate the fluence (UV dose) of our photoreactor
according to a published procedure.36 On exposure to UV light
(253.7 nm), the KI/KIO3 system forms triiodide, which was
determined spectrophotochemically and used to determine the
UV fluence (I0) using a quantum yield (QY) at 254 nm of 0.73.36
I0 was equal to 2.96 × 10-6 einstein/min.
diate the sample. The GC/MS instrument was calibrated with
different mixtures of acetone/water. According to this protocol,
Φ (-acetone) ) 8 × 10-4. Such a low quantum yield of
photochemical acetone decomposition in aqueous solution can
be explained by either a deactivation of the excited state of
acetone in the liquid phase38 or an efficient recombination
process in the solvent cage.38
2.10. Separation and Relative Quantification of L-Ala and
D-Ala. 2.10.1. Hydrolysis of the Peptides. Peptides 1a and 1d
(Chart 1) were photoirradiated at 253.7 nm for up to 10 min at
pH 3.5 (adjusted with HCl). All peptide products were hydro-
lyzed by incubation under vacuum for 10 h at 115 °C in 6 N
HCl solution (1:1, v:v). A control sample containing only the
native, nonirradiated, peptide 1a or 1d underwent the same
treatment. After hydrolysis, the solvents were removed in a
SpeedVac under vacuum. The samples were reconstituted in
0.1 M borate buffer at pH 8.0.
2.10.2. FMOC DeriWatization of Amino Acids and Frac-
tionation of FMOC-N-Ala Amino Acids. After hydrolysis, the
amino acids were derivatized with FMOC-Cl (9-fluorenylm-
ethyloxycarbonyl chloride) to yield FMOC-N-amino acids. A
solution of (1.5 mg/mL) FMOC-Cl was prepared in acetone. A
200 µL portion of the FMOC-Cl solution was added to 200 µL
of the borate buffer solution containing the amino acids. The
mixture was immediately vortexed for 3 min. The reaction was
terminated by extraction of excess FMOC-Cl and acetone with
500 µL of pentane. After vortexing for a short period (1 min),
the upper layer was discarded. After three additional extractions,
the solutions containing the derivatized FMOC-N-amino acids
were fractionated by reverse-phase HPLC and monitored by
fluorescence detection (λex, 240 nm; λem, 313 nm). Separation
of FMOC-N-Leu, FMOC-N-Gly, FMOC-N-Cys, and FMOC-
N-Ala was achieved on a Zorbax Rx-C18 column (15 cm ×4.6
mm), eluted isocratically at a flow rate of 1 mL min-1, delivered
2.9. Quantum Yield. The quantum yield (Φ) for the forma-
tion of H3C•/CH3CO• in an acetone/water mixture (1:4, v:v) was
measured at 253.7 nm with a band pass of 2 nm with an arc
lamp (model 68806; Oriel Instrument) using an integrating
sphere (Labsphere, Inc., North Sutton, NH) coupled to a
monochromator (model 77250; Oriel Instruments). The device
is described elsewhere.37 The acetone/water mixture was placed
in a 1 mL quartz cuvette that had been designed to fit the
integrating sphere. The lower intensity of incident light of
the integrating sphere necessitated irradiation of up to 5 h. The
irradiated mixture was analyzed by GC/MS to determine the
number of moles of acetone consumed during the photoirra-
diation. Φ was calculated as follows: the number of moles of
acetone lost during the irradiation, determined by GC/MS, was
divided by the number of moles of photons used to photoirra-
SCHEME 2: Reaction Scheme for the Formation of the Major Products Generated after UV Irradiation of
Disulfide-Containing Peptides in Ar-Saturated Aqueous Solution