Reversible Hydrogen Transfer between Cysteine Thiyl Radical and

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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 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 oneelectron 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

CysSH + H2O2 f CysSOH + H2O

(1)

CysSH + HO• f CysS• + H2O

(2)

CysSSCys + e- + H+ f CysS• + CysSH

(3)

CysSSCys + hV f 2CysS•

(4)

For long, CysS• radicals have been considered as rather unreactive toward biological substrates. Hence, the recombination 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: [email protected]. † University of Kansas. ‡ Angen Inc.

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

Time-resolved kinetic measurements on reversible unimolecular 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-AcCys-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-toD-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

10.1021/jp101508b  2010 American Chemical Society Published on Web 04/23/2010

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SCHEME 1: Reaction Scheme for the Covalent H/D Exchange between a CysS• Radical and an rC-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 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, GGCGGL, and LGACAGL were obtained by reduction of the disulfidelinked 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

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. Immediately 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 (Waters 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 (ESITOF-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

Hydrogen Transfer between CysS•, Gly, Ala data for 3 s per cycle. Time to mass calibration was made with CsI cluster ions acquired under the same conditions. 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-pyrrol1-yl)-9-methoxy-3-oxo, methyl ester-3H-naphtol[2,1-b]pyrans-carboxylic acid; Calbiochem, EMD Chemicals, Gibbstown,

J. Phys. Chem. B, Vol. 114, No. 19, 2010 6753 NJ) was used for the quantification of free thiols.35 A spectrofluorometer (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. 2.7. 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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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 distribution 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. 2.9. Quantum Yield. The quantum yield (Φ) for the formation 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 photoirradiation. 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-

Mozziconacci et al. 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 hydrolyzed 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 Fractionation of FMOC-N-Ala Amino Acids. After hydrolysis, the amino acids were derivatized with FMOC-Cl (9-fluorenylmethyloxycarbonyl 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 FMOCN-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

SCHEME 2: Reaction Scheme for the Formation of the Major Products Generated after UV Irradiation of Disulfide-Containing Peptides in Ar-Saturated Aqueous Solution

Hydrogen Transfer between CysS•, Gly, Ala

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Figure 1. HPLC chromatogram of UV-irradiated, Ar-saturated aqueous solutions containing (LGACAGL)2 at 100 µM. The structures of the products are described in Scheme 2.

by a Varian 9012 pump system. The isocratic solvent consisted of H2O, acetic acid (pH 4.5)/methanol (MeOH)/ACN/tetrahydrofuran at a ratio of 49/26/21/4 (v:v:v:v). The position of FMOC-N-L/D-Ala was calibrated with an authentic standard of FMOC-derivatized L/D-Ala. The FMOC-N-L/D-Ala fraction obtained from the photoirradiated solutions and control sample were collected, and the solvents were removed under vacuum by rotary evaporation. 2.10.3. Separation of FMOC-N-L-Ala and FMOC-N-D-Ala on a Chiral Column. FMOC-N-L-Ala and FMOC-N-D-Ala were separated on a Chirobiotic T column (15 cm ×4.6 mm, 5um; Supelco, Bellefonte, PA) by isocratic elution with a solvent consisting of 0.1% TEA (pH 4.0)/MeOH/H2O at a ratio of 10/25/ 65 (v:v:v).39 The 0.1% TEA stock solution was prepared by mixing equal volumes of TEA and acetic acid, followed by a 100-fold dilution into H2O. 3. Results 3.1. UV Irradiation of Disulfide-Linked Peptides in H2O. 3.1.1. Photolysis of Peptide 1a. (i) Products 2a and 2a* (m/z 604.3). Product 2a (Scheme 2) elutes with tR ) 13.2 min (Figure 1). The MS/MS fragmentation of 2a after derivatization with NEM displays the expected fragments b2-b6, and y4-y6 (Supporting Information Figure S1). LC-MS analysis also reveals the presence of a product 2a*, a second peak eluting with tR ) 13.7 min (Figure 1), that is isobaric to 2a. The MS/ MS fragmentation of 2a* shows exactly the same fragments as those observed for product 2a. Therefore product 2a* appears to be an isomer of product 2a (see below). (ii) Product 4a (m/z 586.3). Product 4a (Scheme 2) elutes with tR ) 11.7 min (Figure 1). The MS/MS fragmentation of 4a displays the expected fragments b3-b6, and y2-y6 (Supporting Information Figure S2). The fragments b4 and y4 display a difference of 18 Da between product 4a and 2a, consistent with a replacement of thiol by aldehyde (Supporting Information Figures S1 and S2). 3.1.2. Photolysis of Peptide 1b. The photoproducts obtained after irradiation at 253.7 nm of peptide 1b are similar to those observed after irradiation at the same wavelength with peptide 1a (Scheme 2). 3.1.3. Photolysis of Peptide 1c. A detailed description of the photoproducts generated after photoirradiation of peptide 1c is presented elsewhere.31 3.1.4. Photolysis of Peptide 1d. The photolysis of peptide 1d at 253.7 nm yields the thiol AACAA, product 2d. The MS/ MS fragmentation of 2d displays the expected fragments b3, b4, and y2-y4 (Supporting Information Figure S3).

Figure 2. Amino acid analysis of UV-irradiated solution containing (LGACAGL)2. The FMOC-N-L,D-Ala residues were collected on a regular C18 column before separation on a Chirobiotic column. Nonirradiated peptide (dashed line), irradiated peptide (solid line). Fluorescence detection: λex ) 240 nm; λem ) 313 nm.

3.1.5. Epimerization at Ala during the Photoirradiation of Peptides 1a and 1d. Photoirradiation of peptide 1a at pH 3.5 at 253.7 nm for up to 10 min leads to the formation of D-Ala, detected according to the protocol described in section 2.9. In contrast, D-Ala is clearly absent in the nonirradiated control of peptide 1a (Figure 2), indicating that D-Ala formation is not the result of sample preparation. In these chromatograms, we can quantitatively compare the peak areas for L-Ala and D-Ala in an individual analytical run; however, a quantitative comparison of peak areas between separate runs is not possible because the protocol described in section 2.10 was not optimized to allow such comparison. The variation of the fluorescence intensity for the peak of L-Ala between the nonirradiated (control) and the irradiated sample can be caused by multiple factors, including derivatization efficiencies and sample recovery during the fractionation steps. Integration of the peak areas of FMOC-N-L-Ala and FMOC-N-D-Ala reveals that a 10 min photoirradiation leads to epimerization of peptide 1a to an extent of ∼35% (Figure 2). Photoirradiation of peptide 1d under the same conditions reveals significantly lower epimerization (yield < 10%). 3.1.6. Quantification of Thiol Products 2a, 2b, 2d. Products 2a, 2b, and 2d were quantified by fluorescence spectroscopy after derivatization with ThioGlo1. A 10 min photoirradiation of 1a, 1b, and 1d generated 4 µM of 2d (AACAA), 12 µM of 2a (LGACAGL), and 52 µM of 2b (LGGCGGL), respectively. The yields of 2d, 2a, and 2b represent 4%, 12% and 52% relative to the starting concentrations of the native disulfidecontaining peptides. Hence, it appears that the presence of an increasing number of Ala residues in the peptide lowers the yield of thiol formation, possibly due to a higher efficiency of thiyl radical-thiyl radical recombination of the Ala-containing peptides (see below).

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Figure 3. Mass spectrometry analysis of a UV-irradiated, Ar-saturated solution of 100 µM LGGCGGL in acetone/water (1:4, v:v).

3.2. UV Irradiation of Cys-Containing Peptides in Acetone/ H2O. The 253.7 nm photolysis of acetone at room temperature leads to the formation of methyl (H3C•) and acetyl radicals (CH3CO•) (reaction 10).40 H3C• radicals abstract hydrogen atoms from thiols to yield thiyl radicals (reaction 11, k11 ) 7.4 × 107 M-1 s-1).41 In contrast, the rate constants for the abstraction of hydrogen atoms from C-H bonds are significantly lower (k < 3 × 103 M-1 s-1).42,43 The reactivity of acyl radicals is comparable to that of alkyl radicals.44 In fact, theoretical calculations show comparable rate constants for the reaction of thiol with H3C• (4.3 × 107 M-1 s-1, T ) 310 K) and CH3CO• (2.15 × 107 M-1 s-1, T ) 310 K).45 Thus, acetyl radicals originating from the photolysis of acetone are expected to generate thiyl radicals according to reaction 12. The rate constant for the dissociation of CH3CO• into H3C• and CO in the gas phase has been determined to log k(s-1) ) 13.5-72 (kJ/mol)/2.3 RT.46 Hence, we expect this dissociation to occur in solution with k < 7.3 s-1 (T ) 298 K),

suggesting that CH3CO• radicals are sufficiently stable to enter reaction 12.

hV

H3CCOCH3 98 H3C• + H3CC•O

(10)

RSH + H3C• f RS• + CH4

(11)

RSH + H3CC•O f RS• + H3CCHO

(12)

RS• + H3C• /H3CC•O f RS - CH3 /RS - CO - CH3 R,β • Cpeptide

R,β • Cpeptide

+ H3C• f

R,β

+ H3CCO• f

R,β

Cpeptide - CH3 Cpeptide - COCH3

SCHEME 3: Reaction Scheme Illustrating Multiple Alkyl Incorporation into a Peptide during UV Irradiation of Ar-Saturated Acetone/H2O Containing either LGGCGGL or GGCGGLa

a

An analogous mechanism operates for acetyl radical incorporation.

(13) (14) (15)

Hydrogen Transfer between CysS•, Gly, Ala CysS• radicals will be involved in reversible hydrogen transfer reactions as outlined in Scheme 1. Subsequently, peptide radicals will have the chance to recombine with excess H3C•/CH3CO•, as indicated in the general reactions 13-15. Here, R,βC• refers to carbon-centered radicals generated through hydrogen abstraction from RC-H and βC-H bonds. 3.2.1. Photolysis of LGGCGGL (2b) in Acetone-h6/Water. The cysteine-containing peptide 2b (LGGCGGL) (100 µM) was photoirradiated at λ ) 253.7 nm in an Ar-saturated solution of acetone/water (1:4 v:v), the solvents were removed under vacuum, and the dry sample was redissolved in 100 µL of H2O prior to mass spectrometry analysis (Figure 3). Five products were identified: (i) the disulfide-containing peptide 1b (LGGCGGL)2 (m/z 575.3; [M + 2H]2+), most likely generated via the intermolecular recombination of two thiyl radicals, and (ii) a series of recombination products between peptide radicals and either H3C• or CH3CO•, with m/z 590.3 ([M + H]+, trace), 604.3 ([M + H]+), 618.3 ([M + H]+), and 632.3 ([M + H]+). They all differ by multiples of 14 Da from the native peptide LGGCGGL (Figure 3), indicating either multiple incorporations of H3C, a combination of H3C• and CH3CO•, or both. For example, the product with m/z 590.3 indicates the reaction of

J. Phys. Chem. B, Vol. 114, No. 19, 2010 6757 one H3C• radical with a radical of the peptide LGGCGGL. The product with m/z 604.3 is isobaric to peptide 2a (LGACAGL, Scheme 2), indicating the net addition of two H3C• radicals to radicals originating from LGGCGGL. We expect that the net addition of more than one H3C•/CH3CO• radical to a peptide radical will occur consecutively; that is, it will involve the formation of a peptide radical, followed by the addition of H3C•/ CH3CO•, followed by a second formation of the peptide radical, again followed by the addition of H3C•/CH3CO•, etc. For a general scheme of these consecutive radical formation and radical-radical recombination reactions, see Scheme 3. The products with m/z 618.3 and 632.4 may correspond to different combinations of H3C• and CH3CO• added to radicals LGGCGGL. In fact, the addition of three methyl groups to radicals from LGGCGGL (∆ ) 42 amu) would give a product with the same mass as that resulting from the reaction of one acetyl radical (∆ ) 42 amu). Thus, the product with m/z 618.3 corresponds either to the reaction of three methyl radicals or of one acetyl radical. The product with m/z 632.4 represents a combination reaction of four methyl radicals or one methyl and one acetyl radical with radicals of the peptide LGGCGGL. MS/ MS fragmentation confirms that the product with m/z 618.4

TABLE 1: CID Mass Spectrum of a Product with m/z 508.2 Obtained by Means of a Q-TOF Mass Spectrometer, Generated by UV Irradiation of Ar-Saturated Acetone-d6/H2O Containing 100 µM GGCGGLa

a

Two isobaric products (I and II) explain the MS/MS fragmentation pattern. The localization of the incorporation of the deuterated acyl radical is based on discriminated fragments, which are indicated in bold.

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TABLE 2: CID Mass Spectrum of a Product with m/z 553.2 Obtained by Means of a Q-TOF Mass Spectrometer, Generated by UV Irradiation of Ar-Saturated Acetone-d6/H2O Containing 100 µM GGCGGLa

a

Two isobaric products (III and IV) explain the MS/MS fragmentation pattern. The localization of the incorporation of the deuterated acyl radical is based on discriminated fragments, which are indicated in bold.

results from the reaction of one acetyl radical with a radical on the cysteine residue of the peptide LGGCGGL (data not shown). The photoirradiation of a peptide lacking Cys, hexaglycine (Gly6), in an Ar-saturated solution of acetone/water (1:4, v:v) does not reveal any photoproduct (Supporting Information Figure S4). This control experiment confirms that the hydrogen abstractions at the RC (and possibly βC) positions of our Cyscontaining peptides are consecutive to the formation of the CysS• radical through reactions 11 and 12 under our experimental conditions. In addition, the absence of photoproducts after the photoirradiation of Gly6 in an Ar-saturated solution of acetone/ water is consistent with the low reactivity of H3C•/CH3O• with C-H bonds. Earlier publications had reported on the formation of carbon-centered radicals in Gly-containing peptides through acetone photolysis for g22 h;47 instead, the strong catalytic effect of Cys (and the Cys• radical) in our experiments is evident after photolysis for 10 min. To further address the question of the reaction of H3C• vs CH3CO•, experiments were performed with acetone-d6, as described below.

3.2.2. Photolysis of GGCGGL in Acetone-d6/Water. After photoirradiation at 253.7 nm of 100 µM of GGCGGL (1c) in Ar-saturated acetone-d6/water (1:4, v:v) we observed several reaction products indicating various stoichiometries of the reactions of CD3CO• and D3C• with radicals from GGCGGL. The most important products are given in Tables 1 and 2. Product I (m/z 508.2) represents a structure generated through the reaction of CD3CO• with GGCGGL. MS/MS fragmentation clearly shows that CD3CO• is preferentially incorporated into the original Cys residue (Table 1, I). Product I is likely the product of a reaction of CD3CO• with a thiyl radical generated from GGCGGL. Another option would be reaction with an R-mercapto-substituted βC• radical of the original Cys residue, which could form via 1,2-H-shift of the original thiyl radical.18,37 To test for this possibility, we reacted product I with N-ethylmaleimide (NEM). The reaction of NEM with a free thiol of product I is expected to generate a product with m/z 633.2 amu ([M + H]+); however, only a trace of such product

Hydrogen Transfer between CysS•, Gly, Ala was detected, indicating that the structure for I shown in Table 1 is the predominant product. A product isobaric to I, referred to as II in Table 1, was assigned to a structure formed via the reaction of CD3CO• with a Gly(RC•) radical of GGCGGL. Only the difference of the masses of the fragments b3 and y3 between the products I and II can demonstrate that CD3CO• is incorporated into the first neighboring Gly residue to the cysteine. The variation of mass of the fragment b3 between products I and II is -45 amu. The variation of mass of the fragment y3 between product I and II is +45 amu. These variations of mass in the b3/y3 fragments confirm that in product II, CD3CO• is attached to the first C-terminal Gly residue adjacent to the cysteine moiety. A product with m/z 553.2 ([M + H]+) was separated chromatographically from products with m/z 508.2. The difference of mass between 553.2 and 508.3 is 45 amu. This difference of mass corresponds to an additional reaction of CD3CO• with a peptide radical (i.e., the consecutive formation of two radicals on the peptide and their reaction with CD3CO•). The MS/MS analysis of the product with m/z 553.2 shows the presence of two isobaric products (Table 2, compounds III and IV). The fragments b3, y3, and y4 of product III and the fragments b3 and b4 of product IV confirm the recombination of a Cys-derived radical with CD3CO•. By analogy to products I and II, products III and IV are most likely due to the reaction of one CD3CO• with a thiyl radical and of one CD3CO• with a carbon-centered radical of the first Gly residue adjacent to Cys. The acylation of the Gly residues confirms the intermediary formation of an RC• radical of Gly. This is consistent with covalent H/D-exchange during the photolysis of 1c in D2O reported earlier.31 To separately confirm the formation of Gly-CR• and Ala-CR•, we generated CysS• radicals in Gly- and Ala-containing peptides through both disulfide photolysis of peptides 1a/1b and the reaction of H3C•/CH3CO• with the Cys-containing peptide 2a. 3.3. Covalent H/D Exchange during the Direct Photolysis of Disulfide-Containing Peptides (LGACAGL)2 and (LGGCGGL)2. (LGACAGL)2 (1a) was photoirradiated at 253.7 nm in Ar-saturated H2O or D2O. The MS spectra of the photoproducts obtained after photoirradiation in either H2O or D2O were overlaid to highlight covalent deuterium incorporation as described in Scheme 1. The photoproducts 2a and 4a show deuterium incorporation with a ∆BPI ) 15% and 20%, respectively (Figure 4B, C). In addition, the disulfide-containing peptide 1a shows deuterium incorporation, but only after photoirradiation (BPI ) 30%), suggesting that the MS spectra of the disulfide obtained after UV irradiation represent an average of those of the unreacted native peptide (Figure 4A) and disulfides resulting from the recombination of thiyl radicals (Figure 4D; Scheme 2, reaction 9) after they had gone through at least one H/D exchange according to equilibrium 6a-6c (Scheme 1). An analogous experiment with peptide 1b showed negligible incorporation of D into the disulfide but ∼2-3-fold higher incorporation of D into product 2b as compared to 2a. 3.4. Covalent H/D Exchange in a Thiol-Containing Peptide, LGACAGL during Photolysis in Acetone/Water. The Cys-containing peptide 2a (LGACAGL) (m/z 604.4) was photoirradiated at 253.7 nm in Ar-saturated acetone-h6/H2O (1: 4, v:v) or acetone-h6/D2O (1:4, v:v). The products observed after photoirradiation of LGACAGL in H2O/acetone-h6 are analogous to those observed after photoirradiation of GGCGGL in H2O/ acetone-h6 as described above: the major product corresponds to the recombination of a Cys thiyl radical with CH3CO• (Table


Figure 4. Covalent deuterium incorporation observed in the photoproducts generated after UV irradiation of Ar-saturated solutions containing peptide 1a (LGACAGL)2 in H2O and D2O. The black area highlights the deuterium incorporation after overlaying the MS scans of the photoproducts generated either in H2O or in D2O. (A) Nonirradiated peptide, (B) product 2a, (C) product 4a, and (D) mixture of the original disulfide and the disulfide resulting from the recombination of the thiyl radicals.

3, product V). The overlay of the MS spectra of photoproduct V obtained after photoirradiation of 2a in acetone-h6/H2O and

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TABLE 3: CID Mass Spectrum of a Product with m/z 646.3 Obtained by Means of a Q-TOF Mass Spectrometer, Generated by UV Irradiation of Ar-Saturated Acetone-h6/H2O Containing 100 µM LGACAGL

acetone-h6/D2O shows efficient deuterium incorporation (Figure 5). To localize the deuterium incorporation, the MS/MS spectra of the photoproduct V (Figure 5) obtained after photoirradiation in either acetone-h6/H2O or acetone-h6/D2O were overlaid to highlight covalent deuterium incorporation into the b fragment ions. Fragment b1 is not displayed because it is not observed after MS/MS fragmentation. The fragment b2 shows ∆BPI < 5%, and the ∆BPI of fragment b3 is almost zero. These data suggest that the first three N-terminal amino acid residues do not covalently incorporate any deuterium atoms. The variation in the isotopic distribution becomes significant for fragments b4 (∆BPI ) 21%) and b5 (∆BPI ) 43%), suggesting that most of the deuterium incorporation in the peptide occurs in the sequence -CAG-. In addition, the fragment b6 shows a ∆BPI ) 75% which is close to that observed in the parent ion (∆BPI ) 76%), suggesting that the C-terminal Leu residue does not incorporate a significant amount of deuterium atoms. A quantitative analysis suggests that deuterium is incorporated to an extent of 21% into Cys,4 22% into Ala,5 and 32% into Gly.6 The incorporation of deuterium into Cys and Gly is consistent with our earlier data31 in which the Cys thiyl radical of GGCGGL was generated during the photolysis of the disulfide 1c. The incorporation of deuterium into Ala is consistent with the formation of an Ala-RC• radical, intermediates in the L-to-D epimerization, which is described above. The deuterium incorporation is more efficient after

generating the CysS• radical in LGACAGL (2a) in acetone/ D2O solution than after generating CysS• radicals through the homolytic cleavage of the disulfide bond of (LGACAGL)2 (1a). This latter observation may be rationalized by an efficient recombination/disproportionation of CysS• radicals in the solvent cage. 4. Discussion 4.1. Formation and Reactions of Thiyl Radicals: Covalent H/D Exchange and L-Ala-to-D-Ala Conversion. In cystinecontaining peptides, the photolytic generation of thiyl radical pairs occurs via direct photodissociation of the disulfide bond (Scheme 1, reaction 6). Disproportionation represents one important bimolecular reaction of these thiyl radical pairs, evidenced by the formation of thiol (Scheme 2, 2a) and thioaldehyde (Scheme 2, 3a, reaction 7). The thioaldehyde product can be detected, but in water, it rapidly transforms into aldehyde 4a. Analogous products were observed during the photodissociation of other small disulfide-containing peptides in solution31 and of disulfides in the gas phase.48 The distinct intermediacy of thiyl radicals in this process was indicated by the occurrence of intramolecular hydrogen transfer reactions, evidenced by covalent H/D exchange when reactions are performed in D2O.31 The structures of 2a and 4a were confirmed through MS/MS analysis (Supporting Information Figures S1 and S2). A product

Hydrogen Transfer between CysS•, Gly, Ala


Figure 5. Covalent deuterium incorporation observed in photoproduct V, generated after UV irradiation of an Ar-saturated solution containing LGACAGL in acetone-h6/H2O and acetone-h6/D2O (1:4, v:v). The black area highlights the deuterium incorporation after overlaying the MS scans of the photoproducts generated in either H2O or D2O. Overlay of the MS/MS spectra are presented for the b fragments.

isobaric to 2a, referred to as 2a*, elutes 0.5 min after 2a (Figure 1). The MS/MS spectrum of 2a* is identical to that of product 2a, suggesting that 2a* may be an epimer of 2a (likely containing D-Ala). Importantly, the HPLC chromatograms of photoirradiated (LGGCGGL)2 and (GGCGGL)2 do not show such isobaric products, eluting after the photoproducts LGGCGGL or GGCGGL, respectively (data not shown), which is consistent with the potential of epimerization at Ala but not at Gly. This was separately confirmed through relative quantification of L-Ala and D-Ala after photoirradiation of (LGACAGL)2 (Figure 2). The L-Ala-to-D-Ala epimerization in photoproduct 2a is rationalized by reversible intramolecular hydrogen transfer between Ala-RC-H and CysS•.31,32 Such intramolecular reaction is expected to generate RC• radicals at the Ala residue. Reverse hydrogen transfer between RC• and the thiol generates either L-Ala or D-Ala. The relative yields of both products will depend on the rate constant for the actual hydrogen transfer and the flexibility of the peptide, which will control the efficiency by which the Cys residue can approach the Ala(RC•) radical from either side. In addition, an important factor controlling the relative yields of L-Ala and D-Ala will be how the peptide sequence energetically affects the orientation of Ala(RC•) for the respective transition states prior to hydrogen transfer. The introduction of D-amino acids into all L-amino acid peptides leads to considerable conformational changes, and especially, the L-amino acid in position i + 1 relative to the position where the D-amino acid is introduced has a great effect on the energetics of the resulting sequence.49 In proteins, L-to-D isomerization of amino acids will most likely be possibly only in relatively flexible sequences. The presence of Ala residues in the sequence of a peptide increases its tendency for helicity.50–52 The amount of thiol generated after photoirradiation of the peptides (LGGCGGL)2,

(LGACAGL)2, and (AACAA)2 decreases with an increasing number of Ala residues present in the peptide. In addition, we observed that the epimerization of the Ala residues after photoirradiation of (AACAA)2 is less efficient (D-Ala/L-Ala < 10%) than in (LGACAGL)2 (D-Ala/L-Ala ) 35%). These preliminary observations suggest that if a Cys thiyl radical is formed in a structured environment, the recombination of the CysS• radicals may be faster than both the disproportionation reaction between CysS• radicals and the intramolecular hydrogen transfer reaction between CysS• and RC-H. 4.2. Carbon-carbon Bond Formation of rC• Radicals. In previous publications, we reported on an intramolecular hydrogen transfer between CysS• and RC-H of Gly bonds during the photolysis of cystine-containing peptides and proteins.31,32 For the photoirradiation of (GGCGGL)2, we observed by covalent H/D exchange that hydrogen transfer occurred predominantly between Cys and Gly residues, whereas no significant hydrogen transfer was detected for Leu residues.31 In the present paper, we designed three complementary strategies to confirm the intermediacy of RC• radicals, covalent H/D exchange, the conversion of L-Ala to D-Ala, and radical-radical recombination products specifically formed during the reaction of Cys-containing peptides with H3C•/CH3CO• radicals in acetone/water mixtures. The primary radicals generated upon the photolysis of acetone, CH3CO• and H3C•, will react exclusively with the Cys residue present in the target peptides. Hence, any RC• peptide radical formed must originate through subsequent reaction of the CysS• radical. In addition to the formation of RC•, we need to consider the possibility of a 1,2-hydrogen shift of the CysS• radical, generating a βC• radical on the Cys residue. Recombination can then occur between CH3CO• and H3C• and (i) CysS•, (ii) RC•, and (iii) βC•. On the basis of a slightly (∼2-fold) higher reactivity of Cys with H3C• as compared with CH3CO•, we anticipate a

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slightly higher steady-state concentration of CH3CO• available for recombination with the peptide radical, and this is reflected in the actual yields of the recombination products. In addition, the spontaneous dissociation of CH3CO• into carbon monoxide (CO) and H3C• radical is not favorable in aqueous solution. Indeed, the low quantum yield of carbon monoxide (CO) (Φ ) 10-4 at 25 °C) resulting from the spontaneous dissociation of the excited CH3CO• into H3C• and CO suggests that the excited acetyl radical is deactivated in aqueous solution.38 In addition, the few molecules of CO resulting from some dissociation of acetyl radicals can react with H3C• radicals (k ) (2.0 ( 0.3) × 106 M-1 s-1) to regenerate CH3CO• radicals.53 All these features suggest a higher steady state concentration of acetyl radicals as compared to methyl radicals. Thus, the most abundant radical recombination products should originate from the reactions of the peptide CysS• radical with CH3CO• (Table 1, I). Of significantly lower abundance is product II (Table 1), displaying the recombination of RC• with CH3CO•. The incorporation of methyl groups into Gly residues was indicated by mass spectrometry analysis, but the MS/MS fragmentation could not be performed because of the low yield of these products (Figure 3, m/z 604, two H3C• incorporated into LGGCGGL). However, we demonstrated the incorporation of an acetyl group into a Gly residue (Table 2, two CH3CO• incorporated into GGCGGL). 5. Conclusion Our data unambiguously demonstrate an intramolecular reaction of CysS• thiyl radical with C-H bonds in peptides. For amino acids different from Gly (e.g., Ala), these processes can lead to L-to-D amino acid conversion. Acknowledgment. We are grateful to Amgen Inc. for financial support. Supporting Information Available: Figures S1-S4 as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Jones, D. P. Am. J. Physiol. Cell Physiol. 2008, 295, C849. (2) Stamler, J. S.; Hausladen, A. Nat. Struct. Biol. 1998, 5, 247. (3) Scho¨neich, C. Chem. Res. Toxicol. 2008, 21, 1175. (4) Stubbe, J.; van Der Donk, W. A. Chem. ReV. 1998, 98, 705. (5) Wardman, P. Thiyl Radicals in Biology: Their Role As Molecular Switch Central to Cellular Oxidative Stress. In S-Centered Radicals; Alfassi, Z. B., Ed.; Wiley: New-York, 1999; pp 289. (6) Wang, W.; Singh, S.; Zeng, D. L.; King, K.; Nema, S. J. Pharm. Sci. 2007, 96, 1. (7) Volkin, D. B.; Mach, H.; Middaugh, C. R. Mol. Biotechnol. 1997, 8, 105. (8) Dayong, L.; Scott, W. S.; Anderson, B. D. J. Pharm. Sci. 2005, 94, 304. (9) Bonifacˇic´, M.; Asmus, K. D. J. Phys. Chem. 1976, 80, 2426. (10) Bonifacˇic´, M.; Asmus, K. D. Int. J. Radiat. Biol. 1984, 46, 35. (11) Bonifacˇic´, M.; Schaefer, K.; Moeckel, H.; Asmus, K. D. J. Phys. Chem. 1975, 79, 1496. (12) Moeckel, H.; Bonifacˇic´, M.; Asmus, K. D. J. Phys. Chem. 1974, 78, 282.

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Reversible Hydrogen Transfer between Cysteine Thiyl Radical and

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