Bioconjugate Chem. 2008, 19, 153–157
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Attachment of Peptide Building Blocks to Proteins Through Tyrosine Bioconjugation Dante W. Romanini† and Matthew B. Francis*,‡ Department of Chemistry, University of California, Berkeley, California 94720-1460, and Materials Sciences Division, Lawrence Berkeley National Labs, Berkeley, California 94720-1460. Received June 21, 2007; Revised Manuscript Received October 4, 2007
Recent efforts have yielded a number of short peptide sequences with useful binding, sensing, and cellular uptake properties. In order to attach these sequences to tyrosine residues on intact proteins, a three-component Mannichtype strategy is reported. Two solid phase synthetic routes were developed to access peptides up to 20 residues in length with anilines at either the N- or C-termini. In the presence of 20 mM formaldehyde, these functional groups were coupled to tyrosine residues on proteins under mild reaction conditions. The identities of the resulting bioconjugates were confirmed using mass spectrometry and immunoblot analysis. Screening experiments have demonstrated that the method is compatible with substrates containing all of the amino acids, including lysine and cysteine residues. Importantly, tyrosine residues on proteins exhibit much faster reaction rates, allowing short peptides containing this residue to be coupled without cross reactions.
INTRODUCTION Structural studies on naturally occurring proteins have identified a number of short peptide sequences with functions ranging from ion sensing to cellular uptake enhancement (1–4). In terms of de noVo strategies, techniques such as phage display (5) and combinatorial synthesis (6) have yielded numerous peptides with specific target binding capabilities. These building blocks allow new functions to be added to virtually any protein of interest, provided that a convenient strategy is available for their incorporation into the full sequence. While genetic methods often suffice for the introduction of desired sequences at the Nand C-termini or in flexible loops, such additions are limited to proteins that are expressed in exogenous hosts and are difficult to use for the introduction of peptides containing unnatural amino acids. To expand the available options, several chemical strategies have been developed to incorporate synthetic peptides into fullsized proteins. In particular, the native chemical ligation has emerged as a powerful tool for the total or semisynthesis of proteins through the coupling of C-terminal thioesters to N-terminal cysteine residues (7, 8). To introduce peptides at locations other than the termini, a chemoselective Staudinger ligation has been used to attach peptide-phosphine conjugates to azides displayed on carbohydrate moieties (9, 10) and unnatural amino acids (11–13). More recently, a Cu(I)-catalyzed cycloaddition reaction has been reported for the attachment of peptides to azides and alkynes previously introduced using NHS-ester coupling reactions (14). To add to these methods, we have applied a one-step Mannich-type coupling reaction for the conjugation of synthetic aniline-containing peptides (1) to native tyrosine residues on proteins (eq 1) (15). We have performed this reaction with peptides containing all the amino acids without cross reactivity, including the target side chain of tyrosine, in contrast to methods such as cysteine alkylation or lysine acylation. Furthermore, we * Corresponding author. Phone: 510-643-9915. Fax: 510-643-3079. E-mail:
[email protected]. † University of California. ‡ Lawrence Berkeley National Labs.
have devised a simple synthetic route toward the reagents to allow the attachment of peptides via their N- or C-termini.
EXPERIMENTAL PROCEDURES General Procedures and Materials. Unless otherwise noted, all chemicals and reagents were obtained from commercial sources and used without further purification. Protected amino acids were obtained from Novabiochem (San Diego, CA, USA). Water used in biological procedures or as a reaction or chromatography solvent was deionized using a NANOpure purification system (Barnstead, USA). Acetonitrile was HPLCgrade and was used without purification. Dry dimethylformamide was obtained from Acros Organics (USA). Chymotrypsinogen A, Hen Egg White Lysozyme, and RNase A were obtained from Sigma (USA) and used without purification. Enhanced Green Fluorescent Protein was obtained from Aaron Esser-Kahn (16). Instrumentation and Sample Analysis Preparations. UV– vis spectroscopic measurements were conducted on a Tidas-II benchtop spectrophotometer (J & M, Germany). Centrifugations were conducted with the following: (1) Allegra 64R Tabletop Centrifuge (Beckman Coulter, Inc., USA); (2) Sorvall RC5C refrigerated high-speed centrifuge (Sorvall, USA); or (3) 5415D Benchtop Centrifuge (Eppendorf, Germany). Desalting and removal of other small molecules of protein samples was achieved using Microcon YM-10 (10,000 MWCO) centrifugal concentrators (Millipore, USA). 1 H and 13C spectra were measured with either a Bruker AVB400 (400 MHz) or DRX-500 (500 MHz) spectrometer, as indicated. Proton chemical shifts are reported as δ in units of parts per million (ppm) relative to chloroform-d (δ 7.26, s) or deuterium oxide (δ 4.79, s). Multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), and br (broadened). Coupling constants are reported as a J value in Hertz (Hz). The number
10.1021/bc700231v CCC: $40.75 2008 American Chemical Society Published on Web 12/11/2007
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of protons (n) for a given resonance is indicated as nH and is based on spectral integration values. 13C NMR spectra are reported as δ in units of parts per million (ppm) relative to chloroform-d (δ 77.23, t). HPLC was performed on an Agilent 1100 series HPLC system (Agilent Technologies, USA). The mobile phases used were (A) ddH2O + 0.1% TFA; (B) MeCN + 0.1% TFA. Sample analysis for all HPLC experiments was achieved with an inline diode array detector (DAD) and an inline fluorescence detector (FLD). Analytical chromatography was performed using a Jupiter 5 µm C18 300 Å reversed phase column (2.0 mm × 150 mm) (Phenomenex, USA). Preparative HPLC was performed on a Zorbax 300SB-C18 300 Å reversed phase column (9.4 mm × 250 mm) (Agilent). Fast atom bombardment (FAB) mass spectra were obtained at the UC Berkeley Mass Spectrometry facility. Matrix assisted laser desoprtion-ionization time of flight (MALDI-TOF) mass spectra were obtained on Voyager DE-PRO (Applied Biosystems, USA). MALDI matrices were prepared daily as solutions (generally 10 mg/mL). For protein and peptide analysis, R-cyano-4-hydroxycinnamic acid (CHCA) or sinapinic acid in 3:2 MeCN/H2O (0.1% TFA) were used. Electrospray ionization (ESI) mass spectra were obtained on a Bruker Apex II FT-ICR mass spectrometer (Bruker, Germany) or an API 150EX system (Applied Biosystems, USA) equipped with a Turbospray ion source and an Agilent 1100 series LC pump. Peptide chromatography was performed using a Jupiter 5 µm C18 300 Å reversed phase column (2.0 mm × 150 mm) (Phenomenex, USA). Protein chromatography was performed using a Jupiter 5 µm C5 300 Å reversed phase column (2.0 mm × 150 mm) (Phenomenex). A MeCN/ddH2O gradient mobile phase containing 0.1% formic acid (250 µL/min) was used for all LC/MS. Protein mass reconstruction was performed on the charge ladder with Analyst software (version 1.3.1, Applied Biosystems). For protein analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was accomplished on a MiniProtean apparatus (Bio-Rad, USA). Commercially available markers (Bio-Rad, USA) were applied to one lane of each gel for the calculation of apparent molecular weights. Visualization of protein bands was accomplished by staining with Coomassie Brilliant Blue R-250 (Fisher, USA). Gel imaging was performed on an EpiChem3 Darkroom system (UVP, USA). Relative protein amounts were determined from densitometry of gel bands with ImageJ software (NIH, USA). 3-(4-Azidophenyl)propionic Acid (9). To a solution of 3-(4aminophenyl)propionic acid (200 mg, 1.21 mmol) in 20% sulfuric acid (20 mL) at 0 °C was added sodium nitrite (92 mg, 1.3 mmol) dissolved in ddH2O (3 mL). After stirring for 15 min, a solution of sodium azide (157 mg, 2.42 mmol) in ddH2O (3 mL) was added. Following continued stirring at 0 °C for 1 h, a white precipitate formed. The precipitate was collected by filtration, and the filtrate was placed at 4 °C for 2 h. Following this, a new crop of precipitate was collected. The combined white solids, 146 mg (64%), were used without further purification. 1H NMR (500 MHz, CDCl3): δ 7.20 (d, 2H, J ) 8.5 Hz), 6.96 (d, 2H, J ) 8 Hz), 2.94 (t, 2H, J ) 7.5 Hz), 2.67 (t, 2H, J ) 7.5 Hz). 13C NMR (125 MHz, CDCl3): δ 178.1, 138.1, 136.8, 129.6, 119.1, 35.4, 29.9. HRMS (FAB) calcd for C9H9N3O2 (M+) 191.0695, found 191.0692. 2-(4-Azidophenyl)ethylamine, Tosylate Salt (10). To a stirring solution of 2-(4-aminophenyl)ethylamine (800 mg, 5.87 mmol) and p-toluenesulfonic acid monohydrate (3.91 g, 20.5 mmol) in ddH2O (60 mL) at 0 °C was added sodium nitrite (425 mg, 6.16 mmol) dissolved in ddH2O (3 mL). Upon the addition of sodium nitrite, the solution turned a slight orange-brown color. After stirring for 15 min, a solution of sodium azide (954 mg, 14.7 mmol) in ddH2O (3 mL) was added.
Romanini and Francis
A thick white precipitate immediately formed. The precipitate was collected by filtration, and the filtrate was placed at 4 °C for 2 h. Following this, a new crop of precipitate was collected. The combined off-white solids, 1453 mg (74%), were used without further purification. 1H NMR (500 MHz, D2O): δ 7.64 (d, 2H, J ) 8), 7.23 (d, 2H, J ) 8 Hz), 7.28 (d, 2H, J ) 8 Hz), 7.06 (d, 2H, J ) 8 Hz), 3.21 (t, 2H, J ) 7.5 Hz), 2.93 (t, 2H, J ) 7.5 Hz), 2.35 (s, 3H). 13C NMR (125 MHz, D2O): δ 142.3, 139.2, 138.6, 133.1, 130.2, 129.3, 125.2, 119.2, 40.4, 32.1, 20.3. HRMS (FAB) calcd for C8H11N4 (M+) 163.0984, found 163.0982. General Procedures for Peptide Synthesis. Peptides were synthesized using standard Fmoc-based chemistry. The side chain protecting groups used were as follows: Asn(Trt), Asp(tBu), Arg(Pbf), Cys(Trt), Gln(Trt), Glu(tBu), His(Trt), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc), and Tyr(tBu). Synthesis was accomplished through one of the following methods: (1) manually, using 5 equiv of amino acid in dimethylformamide with O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) (5 equiv) as the coupling reagent with 1-hydroxybenzotriazole (HOBT) (5 equiv) and N,N-diisopropylethylamine (DIPEA) (10 equiv) as additives; (2) on an Apex 396 Peptide Synthesizer (AAPPTech, USA) using 10 equiv of amino acid in N-methylpyrrolidone with diisopropylcarbodiimide (10 equiv) as the coupling reagent with HOBT (10 equiv) as an additive; and (3) by the Howard Hughes Mass Spectrometry and Peptide Synthesis facility at the University of California, Berkeley, on a model 433 peptide synthesizer (Applied Biosystems, USA). All peptides were cleaved from resin using a cocktail of 94% trifluoroacetic acid, 5% H2O, and 1% triisopropylsilane and precipitated in cold tert-butylmethylether. In the cases of peptides containing cysteine, ethanedithiol was added at 1% v/v to the cleavage cocktail. Crude peptides were purified using preparative reversed-phase HPLC and subsequently lyophilized before use. Synthesis of C-terminal Anilines though Backbone Amide Linker: Reductive Amination to Form 7. To a 50 mL round-bottom flask equipped with a Teflon stirbar was added (4-formyl-3-methoxyphenoxy)butyryl AM resin (200 mg, 0.18 mmol aldehyde) (Novabiochem, USA) and dimethylformamide (5 mL). The resin was stirred and allowed to swell for 30 min. N,N-Diisopropylethylamine (219 mg, 1.69 mmol) was added, followed by a solution of 10 (595 mg, 1.78 mmol) in 99:1 DMF/ AcOH (5 mL). Sodium cyanoborohydride (112 mg, 1.78 mmol) was added as a solid, and any residual solids were washed into the suspension with DMF. The mixture was stirred at room temperature overnight. The resin was then transferred to a fritted peptide synthesis tube (Econopak tubes, BioRad, USA) and washed with CH2Cl2 (5 mL), DMF (5 mL), 9:1 DMF/DIPEA (5 mL), DMF (2 × 5 mL), and CH2Cl2 (3 × 5 mL). Acylation of 7. The resin was swelled in dry DMF (3 mL) for 15 min with gentle end-over-end agitation and then filtered. In a separate vessel were combined N-Fmoc-glycine (265 mg, 0.89 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU, 312 mg, 0.89 mmol), and dry DMF (3 mL). After the addition of DIPEA (230 mg, 1.78 mmol), this solution was immediately added to the resin, and agitation was continued at room temperature for 6 h. The resin was then filtered and washed with DMF (3 × 5 mL), CH2Cl2 (3 × 5 mL), and MeOH (1 × 5 mL). UV quantitation of dibenzofulvene released during Fmoc removal generally showed between 60 and 90% yield over two steps. Following these steps, peptides were synthesized according to standard methods.
Attachment of Peptides to Proteins at Tyrosines
Bioconjugate Chem., Vol. 19, No. 1, 2008 155
Scheme 1. Synthesis of Peptides with N- and C-Terminal Anilinesa
Figure 1. Chymotrypsinogen A (10) after incubation with 4 mM peptide aniline and 20 mM formaldehyde at 37 °C for 20 h. Higher molecularweight bands indicate Mannich products. In the absence of formaldehyde (lane 1) or peptide (lane 2), no addition was observed.
a
Reagents: (a) 3-(4-Azidophenyl)propionic acid, HBTU, HOBT, DIPEA, and DMF. (b) (NH4)2S, H2O/DMF. (c) TFA, H2O, TIPS. (d) NaCNBH3, DMF. (e) N-Fmoc-AA, HATU, DIPEA, DMF, then Fmoc SPPS.
General Procedure for the Reduction of Aryl Azides on Solid Support. Dry resin containing the protected peptide was placed in a disposable peptide synthesis tube. Dimethylformamide (1800 uL) was added, and the resin was incubated with gentle end-over-end agitation for 15 min. Ammonium sulfide (aqueous solution, 21.2% w/v, 200 µL) was added to the resin suspension, which immediately turned green. Agitation was continued for 2 h at room temperature. The resin was then filtered and washed with DMF (4 × 5 mL), CH2Cl2 (3 × 5 mL), MeOH (5 mL), CH2Cl2 (5 mL), and MeOH (2 × 5 mL), and dried. General Procedure for Protein Labeling Reactions. In a 0.6 mL conical centrifuge tube were combined solutions of protein (1 µL of 1 mM in pH 6.5 phosphate buffer), peptide (8 µL of 10 mM in pH 6.5 phosphate buffer), formaldehyde (3.3 µL of 150 mM in pH 6.5 phosphate buffer), and 100 mM phosphate buffer (pH 6.5) to a final volume of 20 µL. Final concentrations were 50 µM in protein, 4 mM in peptide, and 25 mM in formaldehyde. For peptides with reduced cysteines, tris(carboxyethyl)phosphine (TCEP) was added to a final concentration of 10 mM. Reactions were incubated at 37 °C in a heated incubator for 20–24 h. This procedure was preferable to incubation in a water bath, which led to solvent evaporation and condensation on the caps of the tubes and concomitant precipitation of protein. Reactions were quenched with the addition of a 1 M solution of hydroxylamine HCl (pH adjusted to 6 with 5 N NaOH) to a final concentration of 100 mM. Samples were purified using centrifugal dialysis or sizeexclusion chromatography. General Procedure for Immunoblots. Protein samples were separated by SDS-PAGE according to the procedure developed by Laemmli. Proteins were then transferred to nitrocellulose using a Mini Trans-Blot assembly (BioRad) in 13 mM NaHCO3 buffer at pH 9.9 (with 20% MeOH) at 350 mA for 1 h. The nitrocellulose membrane was blocked with 5% dry milk in TBSTween20 at room temperature overnight. Primary antibody (mouse R-FLAG conjugated to horseradish peroxidase, Sigma, USA) was added at 1:1000 to 1:5000 dilution in 5% milk/TBS-T and was incubated at RT for 1–2 h. Membrane was washed 4 × 5 min in TBS-T and visualized by treatment with an ECL detection kit (Amersham Biosciences, UK) and exposing BioMax MR Film (Kodak, USA).
RESULTS AND DISCUSSION To access aniline-bearing peptides, two similar solid-phase strategies were developed (Scheme 1). In the first, a peptide containing the Myc epitope (17) (EQKLISEEDL) was synthe-
sized on acid-cleavable Wang resin (18) to yield 3. The terminal amino group was then acylated with 3-(4-azidophenyl)propionic acid using HBTU as a coupling agent. Reduction of the aryl azide with ammonium sulfide in DMF/H2O proved to be the most effective method for unmasking the aniline group, affording quantitative conversion in less than 2 h. Peptide-aniline 4 was obtained following resin cleavage and protecting group removal using trifluoroacetic acid (TFA). A second synthetic route was developed to prepare peptides bearing an aniline at the C-terminus. Following the attachment of amine 6 to 4-formyl-3-methoxyphenoxybutyryl AM resin (19) (5) via reductive amination with NaCNBH3, the resulting secondary amine (7) was acylated with N-Fmoc-glycine and HATU. Standard Fmoc solid-phase peptide synthesis was then carried out to produce a resin-bound protected peptide of the desired sequence (8). After the reduction of the azide with ammonium sulfide, peptide 9 was cleaved from the resin and deprotected using TFA. The peptides prepared using both methods were purified using HPLC and characterized via ESIMS (Table 1). Following the synthesis and purification of the peptides, tyrosine modification was achieved by exposing a 50 µM solution of R-chymotrypsinogen A (10) to 2–4 mM peptideaniline and 20 mM formaldehyde in phosphate buffer (pH 6.5) at 37 °C for 24 h. The reactions were then treated with a solution of hydroxylamine (previously adjusted to pH 6.0), a step we have found to be necessary to cleave any remaining formaldehyde imines. This quenching step was found to be less effective at pH values below 6. Following the removal of the small molecules using ultrafiltration, protein modification could be observed through the appearance of a new higher molecular weight band in a Coomassie-stained SDS-PAGE gel (Figure 1, Lanes 3–8). Mass spectral analysis of these samples indicated an increase in mass corresponding to the peptide plus 1 equiv of formaldehyde (Figure 2a). No new bands were observed when either formaldehyde (lane 1) or the peptide (lane 2) was withheld from the reaction mixture. We have found that this amount of formaldehyde does not lead to protein cross-linking (protocols for this purpose typically use concentrations at least 10 times higher). It is notable that these concentrations of reactants are lower than our previously reported amounts necessary for adequate labeling using this method (15). In the case of these anilines, we have successfully performed the reaction with concentrations up to an order of magnitude lower. In addition to the Myc tag, several other useful peptide anilines were synthesized and coupled to 10 (Table 1). This set included the FLAG epitope (13) (20), an HIV-Tat derived peptide (14) (2), a sequence that nucleates gallium arsenide nanocrystals (15) (21), and a sequence that binds terbium(III) ions (16) (22). In each case, 34% to 84% conversion was achieved, as determined using densitometry measurements after Coomassie staining (Table 1). In general, an increase in the number of flexible glycine residues between the aniline and the R-branched amino acids led to an increase in reactivity,
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Table 1. Peptide Anilines Screened in the Mannich Reactiona peptide
sequence
function
massb
MW (calcd)
MW (found)
+0
+1
+2
ref
11 12 13 14 15 16 17c
AnEQKLISEEDL AnGGGEQKLISEEDL DYKDDDDKGGGGGAn AnGGGRKKRRQRRR AQNPSDNNTHTHGGGAn ACADYNKDGWYEELECAGGGAn
Myc Myc FLAG Tat GaAs LBT LBT
(M+H)+ (M+H)+ M+ (M+H)+ (M+H)+ M+ M+
1350.7 1521.7 1415.6 1658.0 1624.7 2267.9 2266.0
1351.3 1522.4 1415.6 1658.7 1625.3 2267.9 2266.0
47 26 35 17 66 66 100
42 49 45 64 34 34 0
11 25 21 20 0 0 0
17 17 20 2 21 22 22
ACADYNKDGWYEELECAGGGAn
a
An indicates the site of the aniline, either 3-(4-aminophenyl)propionic acid (N-terminal anilines, i.e., 4) or 2-(4-aminophenyl)ethylamine (C-terminal anilines, i.e., 9). Coupling yields (expressed as percents, +n where n is the number of modifications) were determined through densitometry analysis of the Coomassie-stained gel in Figure 1. b Calculated peptide masses are monoisotopic. Spectra for multiply charged species were deconvoluted to the parent mass. c Peptide 17 was oxidized to form a cyclic disulfide.
ibility of tyrosine-containing peptides with the Mannich-type reaction, which is a potential advantage of this method compared to lysine- and cysteine-targeting strategies. While it is likely that cross-reactivity could occur in some situations, these results suggest that a wide range of sequences may be compatible with the Mannich-type coupling. Consistent with previous observations of this reaction, eGFP (24) (which has several exposed tyrosines on its surface when viewed as a crystal structure) participates readily, while myoglobin demonstrates poor levels of reactivity (Figure 2d). Clearly, the local environments of tyrosine residues have a significant impact on their reactivity, a feature that is anticipated to yield significant advantages for site selectivity as these effects are elucidated.
CONCLUSIONS
Figure 2. (a) Typical ESI-MS spectrum of chymotrypsinogen (10) modified with peptide-aniline 13. (b) Immunoblot and Coomassie stained gel of 10 modified with 13. Antibody binding was observed only when both peptide-aniline and formaldehyde are present. (c) MALDI-TOF MS of 13 after incubation with formaldehyde for 20 h at 37 °C. (d) Coomassie-stained gels of proteins following reaction with 11. Proteins are eGFP (Lanes 1 and 2) and Myoglobin (Lanes 3 and 4).
suggesting that the steric environment around the aniline was crucial to its ability to undergo the Mannich reaction. Both Nand C-terminal peptide-anilines provided similar coupling results. Notably, a peptide containing free cysteine residues (16) participated in the reaction, but upon oxidation to form disulfide 17, reactivity was abrogated. To confirm that the new species observed in the gels contained desired peptide, a sample of chymotrypsinogen A modified with FLAG sequence 13 was transferred to nitrocellulose and probed with HRP-conjugated anti-FLAG antibodies. The presence of the FLAG epitope was only detected in reactions containing both peptide-aniline and formaldehyde (Figure 2b). Trypsin digests of Chymotrypsinogen labeled with 14 indicated the modification of a peptide fragment containing Y171. The small amount of double labeling is presumed to occur at Y146, which has been observed to participate in other coupling reactions (15, 23); however, neither this nor other modified fragments could be identified in the digested samples. In previous studies (15), we have observed that this coupling reaction proceeds much more rapidly on proteins than on peptide and small molecule substrates. Presumably, this is due to hydrophobic interactions between the reactive partners that serve to increase the effective concentration. This reactivity difference suggested that tyrosine-containing peptides could also be used as substrates in the reaction. To examine this possiblity, peptides 13, 16, and 17 were incubated with formaldehyde under reaction conditions in the presence and absence of protein. No peptide cross-linking was observed with MALDI-TOF mass spectrometry (Figure 2c). These experiments demonstrate the compat-
We have demonstrated the utility of a previously reported three-component tyrosine bioconjugation reaction for the coupling of peptides to preexisting proteins. Such ligations are challenging propositions for which few methods exist. This method should find use in cellular uptake and trafficking studies, protein purification, and materials applications. It also provides a simple way to add peptides containing unnatural amino acids to full-size proteins. We are currently investigating these peptides for use in other bioconjugation reactions involving anilines (25), and we are continuing to examine the mechanisms of bioconjugation reactions to improve and expand the options available to protein chemists and biochemists.
ACKNOWLEDGMENT We gratefully acknowledge the NIH (GM072700-01) and the DOE Nanoscale Science, Engineering, and Technology Program (NSET) for funding. We also thank David King and Jason Rush for lending their peptide synthesis knowledge and Arnold Falick for his mass spectrometry expertise. Supporting Information Available: Procedures and spectra for protein digests and specific control experiments. This material is available free of charge via the Internet at http:// pubs.acs.org.
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