Bioconjugate Chem. 2001, 12, 1051−1056
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Systematic Modulation of Michael-Type Reactivity of Thiols through the Use of Charged Amino Acids M. P. Lutolf,† N. Tirelli,†,* S. Cerritelli,‡ L. Cavalli,† and J. A. Hubbell† Department of Materials and Institute for Biomedical Engineering, ETH Zurich and the University of Zurich, Zurich, Switzerland and Department of Chemistry, Chemical Engineering and Materials, University of L’Aquila, L’Aquila, Italy. Received June 22, 2001; Revised Manuscript Received August 7, 2001
A quantitative structure-reactivity relationship for the Michael-type addition of thiols onto acrylates was determined. Several thiol-containing peptides were investigated by examining the correlation between the second-order rate constant of their addition onto PEG-diacrylate and the pKa of the thiols within a peptide. By introducing charged amino acids in close proximity to a cysteine, the pKa of the thiol was systematically modulated by electrostatic interactions. Positive charges from the amino acid arginine decreased the pKa of the thiol and accelerated the reaction with acrylates while negative charges from aspartic acids showed the opposite effect. A linear correlation between thiolate concentrations and kinetic constants was found, confirming the role of thiolates as the reactive species in this Michael-type reaction. The relevant factors influencing the reactivity were the sign and the number of the neighboring charges, while the position of these charges had little effect on reactivity. These results provide a basis for the rational design of peptides, where the kinetics and thus selectivity of protein/peptide conjugation with polymeric structures via Michael-type addition reactions can be controlled.
INTRODUCTION
Conjugation with poly(ethylene glycol) (PEG), also termed PEGylation, is a common method for the modification of proteins and peptides, as well as other drugs, with synthetic polymers. As a result of the favorable properties of PEG, such as protein repellence, high hydrophilicity, and low degradability by mammalian enzymes, PEGylation can dramatically extend the stability and improve the pharmacology of a biomolecule in vivo (1, 2). One common coupling strategy involves the modification of terminal hydroxyl groups with electrophilic functionalities, such as the N-hydroxysuccinimide esters of PEG carboxylic acids (PEG-NHS) (3-5), PEGoxycarbonylimidazole (CDI-PEG) (6), or PEG nitrophenyl carbonate (7, 8). However, since these functionalized PEGs react unselectively with both thiols and amines, a distribution of products can be obtained, resulting in grafting at multiple sites of the biomacromolecule, additionally with an uncontrolled PEGylation degree (due to the limited stability of thioester linkages). To achieve a better coupling selectivity, two classes of functional groups have been utilized. The first one is based on substitution onto groups such as iodo-, bromo-, and chloroacetamides and -acetates (9, 10); the second class on the conjugate addition (Michael-type) onto unsaturated groups such as maleimides (10), vinyl sulfones (11, 12), acrylamides (13), and acrylates (14). Our recent interests have focused on the development of PEG-based synthetic hydrogels in contact with pro* To whom correspondence should be addressed at the Institute for Biomedical Engineering, ETH Zurich and University of Zurich, Moussonstrasse 18, CH-8044 Zurich, Switzerland, Tel: +41 1 634 26 17, Fax: +41 1 632 12 14. e-mail: tirelli@ biomed.mat.ethz.ch. † ETH Zurich and the University of Zurich. ‡ University of L’Aquila.
teins (15), isolated cells, and tissues via the use of in situ cross-linking reactions, e.g., by reacting a multifunctional PEG with a correspondingly reactive multifunctional PEG or peptide. A satisfactory exploitation of this scheme depends on the possibility of obtaining quantitative conversions in biologically or surgically relevant time scales and with adequate selectivity. Furthermore, the same scheme could be applied to PEGylate biologically active molecules, so as to graft them into the above matrixes. Among the other reactions, the Michael-type addition of thiols onto unsaturated esters is an ideal candidate for such processes, due to its high speed, the ease of preparation of the unsaturated compound, the negligible influence of competing reactions with other nucleophiles (at physiological pH, amines react generally at least 1 order of magnitude slower than thiols (16)), and the absence of any byproducts. Preliminary experiments (17) have shown acrylates to react considerably faster than other unsaturated esters (methacrylates, crotonates, 2,2-dimethylacrylates, cynnamates, cyclopropancarbonylates) and were therefore chosen as standard conjugated unsaturated partner. To better meet the requirements for bioconjugation and gel formation and to optimally exploit the selectivity of the Michael-type addition reaction, knowledge of the thiol structure-reactivity relationship is crucial. Thiols are key players in determining the structure and function of many proteins, for example, as active sites of enzymes. The protonation state of the active site of many enzymes is determined by its pKa which in turn strongly influences protein catalytic activity (18, 19). For example, thioredoxin (20) and thioltranferase (21) have highly reactive thiols with very low pKa’s, which increases their activity toward reducing disulfide bonds. Many examples in the literature reveal that pKa shifts of thiols can be induced by electrostatic interactions with neighboring ionizable amino acids (22) and/or polar residues (e.g. amides
10.1021/bc015519e CCC: $20.00 © 2001 American Chemical Society Published on Web 09/21/2001
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Figure 1. Synthesis of and Michael-type reaction on PEG diacrylate. Table 1. Series of Peptides with Ionizable and/or Neutral Amino Acids in Close Proximity to a Cysteine Residue to Alter Its Protonation State (pKa) by Electrostatic Interaction peptide
GCRRG
GRCRG
GCRG
GRCDG
GCRDG
GCDRG
GCDG
GDCDG
GCDDG
abreviation net charge
crr +2
rcr +2
cr +1
rcd 0
crd 0
cdr 0
cd -1
dcd -2
cdd -2
dipoles (23)) or surfactants (19). For instance, changes in enzymatic activity have been observed for thioredoxin (22) and 4-oxalocrotonoate tautomerase (24) as a result of pKa shifts in the active site due to mutations on charged residues close to the active thiol. It was stated that charge not only influences pKa shifts, but can also change the transition state energy (19) influencing the reaction kinetics. Since thiolates rather than thiols are reported to be the reactive species also in Michael-type reactions (9 ,16, 25), the pKa value of the sulfhydryl-group is the design parameter of choice for this study. The present work outlines a quantitative relationship between the chemical structure and the Michael-type reactivity for a model system constituted of PEG-diacrylate and peptide thiols (see Figure 1). A homologous series of peptides with charged and/or neutral amino acids in close proximity to a cysteine residue was designed in order to confirm the role of thiolates in the Michael addition and quantitatively show how the charge of the neighboring groups of a thiol can influence its pKa. In this system, pKa-shiftssindependent of any other structural factorsslinearly correlate with a change in reaction kinetics. This knowledge will aid in the rational design of peptides and proteins conjugated to polymeric structures with controlled kinetics and selectivity. EXPERIMENTAL SECTION
Materials. Poly(ethylene glycol) mol wt 3400 (PEG3400) and triethylamine were purchased from Aldrich (Buchs, Switzerland) and were used without purification. Acryloyl chloride was purchased from Aldrich (Buchs, Switzerland) and was used freshly after distillation over quinoline. All standard chemicals used in peptide synthesis were analytical grade or better and were purchased from Novabiochem (La¨ufelfingen, Switzerland). 1H NMR spectra were recorded on a 300 MHz Bruker spectrometer. FT-IR spectra were recorded in ATR mode on a Spectrum One Perkin-Elmer spectrometer. UV-Vis spectra were recorded on Perkin-Elmer Lambda 20 spectrometer Synthesis of PEG Diacrylate. PEG diol with 3400 MW (10 g, 2.94 mmol) was dissolved under argon in 300 mL of toluene and dried azeotropically. After cooling to
5 °C, 50 mL of dichloromethane was added. After the addition of triethylamine (1.64 mL, 2.0 equiv), acryloyl chloride (0.72 mL, 1.5 equiv) was added dropwise, and the reaction proceeded overnight, in the dark, at room temperature. The resulting solution was filtered through a neutral alumina bed. Sodium carbonate was added to the toluene solution and removed by filtration, and the volume of the solution was reduced by rotary evaporation. The polymeric material was precipitated twice in with diethyl ether and dried in vacuo to yield a white powder. Yield: 88%; conversion of OH to acrylate: 100% (from 1 H NMR analysis). 1H NMR (CDCl3): 3.6 (156H, PEG chain protons), 4.3 (t, 2H, CH2CH2OCOCHdCH2), 5.8 (dd, 1H, CH2dCHCOO), 6.1 and 6.4 (dd, 1H, CH2dCHCOO) ppm. FT-IR (film on KBr plate): 2990-2790 (ν C-H), 1724 (ν CdO), 1460 (δs CH2), 1344, 1281, 1242, 1097 (νas C-O-C), 952, 842 (νs C-O-C) cm-1. Peptide Synthesis and Purification. A series of cysteine-containing peptides was synthesized, derived from four amino acid building blocks: a positively charged (at neutral pH) (arginine, R), a negatively charged (aspartic acid, D), a neutral (glycine, G), and a thiol-bearing amino acid (cysteine, C). The peptide sequences and their abbreviations used throughout the paper are listed in Table 1. Synthesis was performed by solid-state chemistry on resin using an automated peptide synthesizer (Perceptive Biosystems, Farmington, MA), with standard 9-fluorenylmethyloxycarbonyl chemistry (26). Peptides were purified by C18 chromatography (Perceptive Biosystems Biocad 700E) and analyzed by MALDI-TOF mass spectrometry. All peptides were synthesized bearing a Nacetylglycine at the amino terminus, to suppress reaction of acrylate-groups with the R-amine. Validation of an Analytical Method To Determine Reaction Kinetics. The Michael-type reactions on two model compounds, namely cysteine and PEGDA, was followed monitoring the absorbance at 233 nm, where a pH-dependent absorption peak is shown by thiols (associated to the thiolate form) (22, 27) and where unsaturated esters absorb too. The method was validated using reaction mixtures prepared by keeping the overall number of reacting groups (the sum of acrylates and thiols) constant, but varying their relative quantities. In this
Michael-Type Reactivity of Thiols
Bioconjugate Chem., Vol. 12, No. 6, 2001 1053
way, the absorption could be divided by the number of reacting groups, yielding an averaged extinction coefficient, , which is a linear combination of the extinction coefficients of the absorbing species. Determination of Michael Addition Kinetics for Thiol-Containing Model Peptides on PEGDA. Peptides (Table 1) were dissolved in 100 mM HEPES [4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid] buffered saline and reacted with PEGDA (pH 7.4 and 37 °C) at different molar ratios. The overall concentration of both reacting groups was kept constant at 2.5 mM. The absorption data at 233 nm were fitted with single exponential decays of the form y(t) ) y0 + A1(exp - t/τ). For a second-order reaction, the equation
ln([PEGDA]/[peptide]) ) ln([PEGDA0]/[peptide 0]) + kefft (1) with keff ) k([PEGDA 0] - [peptide0]), implies a linear time dependence for ln([PEGDA]/[peptide]). pKa Determination. The pKa of all thiols was determined spectrophotometrically using a method related to the one desribed above for measurement of reaction kinetics. The absorption at 233 nm was measured for each peptide at various pH for solutions having identical concentrations. Solutions of peptides (0.1 mM) were prepared in a buffer system (depending on the pH regime), composed of two components: a 0.01 M acetate-, phosphate-, tris- or borate-buffer (depending on the pH), and 0.1 M NaCl (stable ionic strength). Freshly prepared solutions were immediately measured in the UV region with a scan from 190 to 300 nm. Statistics. Kinetic studies were conducted in triplicate for every peptide. Mean values and standard error of the mean are shown. RESULTS AND DISCUSSION
Validation of an Analytical Method To Determine Reaction Kinetics. In a buffered environment, the intensity of the 233 nm peak is proportional to the concentration of unreacted acrylates and thiolates; compared to the Michael-type addition, the proton transfer is instantaneous, and from the equilibrium [S-] ) [SH]Ka/[H+] one can assume [S-] to be constantly proportional to the overall thiol concentration Cthiol ) [S-] + [SH] ) [S-](1 + [H+]/Ka). Thus, whether the active group is the thiol, the thiolate, or both of them, the time evolution of this peak allows one to monitor on-line the extent of every thiol-consuming reaction. Saturated esters and thioethers (in the reaction product) have a negligible absorption at that wavelength. If the change of the 233 nm absorption depends only on the reaction extent, this is indicative of a 1:1 reaction, and if disulfide formation plays a negligible role during the reaction (at least for cysteine), then a graph of vs the equivalent fraction of PEGDA (number of acrylates divided by the number of reacting groups; the same applies to cysteine) at time ∞ (complete reaction) should show a minimum at 0.5, corresponding to of the pure product. Moreover, the time dependence of should follow a single-exponential decay, because thiols and acrylates would be consumed at the same rate. These assumptions were all indeed confirmed, as shown by the reaction diagram in Figure 2A and by the time dependence of the absorption in Figure 2B. Thus, this method was used for all further kinetic experiments. Influence of the Primary Structure of the Peptide on the Michael Addition Reactivity. According
Figure 2. (A) Reaction diagram for PEGDA and cysteine as model compounds obtained by measuring absorbance at 233 nm for various PEGDA/Cys molar ratios. The overall concentration c of both reacting groups was kept constant at 2.5 mM. The absorbance divided by c is depicted on the ordinate as . The ratio between the concentrations of reacting groups was drawn on the abscissa as fraction of acrylic groups. (B) Absorbance of a PEGDA-cysteine solution over time (c ) 2.5 mM). The reaction is complete after approximately 30 min under these conditions (pH 7.4, 37 °C), yielding a minimum of corresponding to the pure product.
to eq 1, true second order kinetics would imply a linear time dependence for ln([PEGDA]/[peptide]). This was indeed shown by all peptides investigated. In Figure 3, the kinetic data for some peptides upon reaction with PEGDA at 80:20 acrylate over thiol ratio are shown. Positive charges close to the SH-groups were observed to speed the reaction considerably, while the opposite effect was shown by nearby groups of negative charge. The effect did not depend on the precise location of the charges: the rate constant for crr and rcr or rcd, crd, and cdr were not significantly different from each other. The sequence cdd possessed a considerably higher kvalue than dcd, but this may be due to the low precision in the dcd measurement. Michael-type addition was generally faster in the presence of excess of acrylic groups, particularly when positive charges were close to the thiol, as depicted in Figure 4 for some peptides and cysteine. In our interpretation, however, this difference derives from an incorrect estimation of the concentration of initial thiols in
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Table 2. k and pKa Values of the Cysteine-Containing Peptides. Both pKa and k Respond to Changes in the Primary Peptide Structure peptide
crr
rcr
cr
cys
rcd
crd
cdr
cd
dcd
cdd
ka [L/mol‚min] SDb pKac
136.1 20.49 8.12
124.0 7.82 8.22
107.1 11.09 8.31
88.5 9.11 8.49
58.3 10.11 8.50
58.9 7.91 8.51
58.9 5.70 8.43
43.9 13.01 8.57
40.4 17.61 8.85
21.9 6.14 8.93
a Averaged over three to four measurements. b SD: standard deviation. c Averaged over two or three measurements, standard deviation ( 0.05 (estimated max.).
cysteine, generally assumed to dimerize at a rate 2-4 times lower than other peptides (10), did not show this asymmetry. Relationship between Primary Structure of the Thiol and Its pKa. A typical titration curve followed spectrophotometrically at 233 nm is shown in Figure 5A for the peptide GCDDG (cdd). Since at high pH, thiols are completely deprotonated, the absorbance Amax was assumed equal to S-Cthiol, where S- is the absorption coefficient of the thiolate, and Cthiol is the peptide concentration. The pKa was determined by observing, that for a measurement i, the following holds:
-log([SH]i/[S-]i) ) pHi - pKa
(2)
and [SH]i/[S-]i ) (Cthiol - [S-]i)/[S-]i ) (Amax - Ai)/Ai (3) Figure 3. ln([PEGDA]/[peptide]) over time for several peptides (as examples) reacted with PEGDA (here at 80:20 acrylate over thiol ratio). The linearity indicates true second-order kinetics according to eq 1.
Figure 4. τ values from single-exponential fit from secondorder reaction for various PEGDA/peptide ratios. Error bars are standard error of the mean, n ) 3.
reduced form. We assume the disulfide concentration to be roughly quadratically dependent on the peptide concentration. Thus, in high excess of peptide, disulfide formation could reduce the effective thiol concentration and hence also the calculated reaction rate: e.g., changing a theoretical 80:20 to a an actual 75:25 molar ratio. This assumption can be confirmed by the asymmetry in the τ plot of Figure 4. The slowest reacting mixture should be the one having acrylic and thiol groups in stoichiometric amounts. However, an interpolation of the graphic revealed that this was exhibited with samples having a peptide excess of ca. 5-10%. Furthermore,
As required by eq 3, the intercept with the abscissa in a graphical representation of -ln[(Amax - Ai)/Ai] vs pH yielded the pKa value (Figure 5B). The pKa of cysteine agrees with the commonly accepted value of 8.4-8.5. The presence of charges close to the thiols strongly influenced the pKa (Table 2), hindering the deprotonation in case of negative charges and facilitating it for positive ones. In contrast to the results obtained for the second-order rate constants, pKa values generally responded to the charge location. The pKa of crr and cdd could be distinguished from rcr and dcd, respectively. A stronger charge effect was found for peptides bearing vicinal charges (crr, cdd). For the zwitterionic peptides rcd, crd, and cdr, as well as for cysteine (which is zwitterionic itself), similar values were measured. Quantitative Relationship between Thiol pKa and Michael Addition Reactivity. Positive charges were shown to decrease the pKa of thiols, that is, at constant pH, to increase the concentration of thiolates. Along with the protonation state, an increase in Michael-type reactivity was observed (as described above). The parallel change of both parameters would confirm the observation of other groups (25), that thiolates are the reactive moiety in this reaction. Under this assumption, the second-order kinetic equation v ) kCthiolCacrylate can be expressed as
v ) kreal[S-]Cacrylate
(4)
thus, k ) kreal[1/(1 + [H+]/Ka)] ) krealKa/(Ka + [H+]) (5) According to eq 5, a linear relationship should be seen between Ka/(Ka + [H+]) and k. Whereas cysteine did not precisely follow this correlation, a good agreement was shown for all peptides (Figure 6). The behavior of cysteine is likely due to the shorter distance of the ionic groups from the thiol and to the different structure of the molecule (no peptide bond).
Michael-Type Reactivity of Thiols
Bioconjugate Chem., Vol. 12, No. 6, 2001 1055
Figure 5. (A) Absorbance at 233 nm as a function of pH for the peptide GCDDG. (B) log[(Amax - Ai)/Ai] vs pH. The pKa value corresponds to the intercept with the abscissa. The linear approximation was only used in the buffer region (in this case, the five central points). CONCLUSION
Figure 6. Correlation of Ka/(Ka + [H+]) and k. Peptides (O), cysteine (9). Error bars indicate standard error of the mean, n ) 3.
A linear fit of the k values for the all investigated peptides gave a kreal value of 1040 ( 40 L/(mol×e1‚min).
Due to its selectivity and favorable kinetics in physiological conditions, the Michael-type addition of sulfhydryl-containing biomolecules onto unsaturated groups is an ideal method for many bioconjugation purposes, both in the formation of cross-linked gels and in the PEGylation of therapeutic proteins. Obtaining high self-selectivity in the coupling and tailoring the reaction kinetics to the needs of an application can be crucial in many applications, e.g., in the preparation of polymer-peptide conjugates for in vivo cross-linking materials. In the present work, this issue was approached by designing a homologous series of peptides with charged amino acids flanking a cysteine, to elaborate the relationship between structure of the reaction partners and Michael-type reactivity. The influence of the peptide primary structure on thiol reactivity was significant: charged amino acids close to the SH groups considerably altered the secondorder reaction kinetics, positive charges increasing and negative charges decreasing the reaction rate. In terms of changes in pKa, a decrease of approximately 0.2 or an increase of approximately 0.2 units per positive or negative charge, respectively, was observed. The good correlation between the variations in pKa and reaction rate confirmed the thiolates to be the only active group
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and that the effect of thiolate concentration on reaction kinetics most likely overwhelms any other structural factor. These results provide the basis for the rational design of peptides or proteins for optimal use in conjugation schemes with polymeric structures, where selectivity and controlled reaction kinetics are desired. ACKNOWLEDGMENT
We would like to thank Dr. Roberta Marcone for preliminary work on difunctional PEGs (other than acrylates). We also would like to thank Dr. Jason C. Schense for helpful discussions and review of the manuscript. LITERATURE CITED (1) Abuchowski, A., McCoy, J. R., Palczuk, N. C., van Es, T., and Davis, F. F. (1977) Effect of covalent attachment of poly(ethylene glycol) on immunogenicity and circulating life of bovine liver catalase. J. Biol. Chem. 252, 3582-6. (2) Harris, J. M., Ed. (1992) Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications, Plenum Press, New York. (3) Belcheva, N., Woodrow-Mumford, K., Mahoney, M. J., and Saltzman, W. M. (1999) Synthesis and biological activity of poly(ethylene glycol)-mouse nerve growth factor conjugate. Bioconjugate Chem. 10, 932-937. (4) Belcheva, N., Baldwin, S. P., and Saltzman, W. M. (1998) Synthesis and characterization of polymer-(multi)-peptide conjugates for control of specific cell aggregation. J. Biomater. Sci. Polym. Ed. 9, 207-226. (5) Veronese, F. M., Sacca, B., Polverino de Laureto, P., Sergi, M., Caliceti, P., Schiavon, O., and Orsolini, P. (2001) New PEGs for peptide and protein modification, suitable for identification of the PEGylation site. Bioconjugate Chem. 12, 62-70. (6) Beauchamp, C. O., Gonias, S. L., Menapace, D. P., and Pizzo, S. V. (1983) A new procedure for the synthesis of poly(ethylene glycol)-protein adducts, e!ects on function, receptor recognition and clearance of superoxide dismutase, lactoferrin and a2 -macroglubulin. Anal. Biochem. 131, 25-33. (7) Veronese, F. M., Largajolli, R., Boccu, E., Benassi, C. A., and Schiavon, O. (1985) Surface modification of proteins Activation of monomerthoxy-poly(ethylene glycol)s by phenylchloroformates and modification of ribonuclease and superoxide-dismutase. Appl. Biochem. Biotechnol. 11, 141-152. (8) Demers, N. Agostinelli, E., Averill-Bates, D. A., and Fortier, G. (2001) Immobilization of native and poly(ethylene glycol)treated (‘PEGylated’) bovine serum amine oxidase into a biocompatible hydrogel. Biotechnol. Appl. Biochem. 33, 201207. (9) Lindley, H. (1959) A study of the Kinetics of the Reaction between Thiol Compounds and Chloroacetamide. Biochemistry 74, 577-584. (10) Schelte´, P., Boeckler, C., Frisch, B., and Schuber, F. (2000) Differential reactivity of maleimide and bromoacetyl functions with thiols: application to the preparation of liposomal diepitope constructs. Bioconjugate Chem. 11, 118-123. (11) Masri, M. S., and Friedman, M. (1988) Protein reactions with methyl and ethyl vinyl sulfones. J. Protein Chem. 7, 4954.
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BC015519E
