Bioconjugate Chem. 1995, 6,323-326
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A Convenient Route to Thiol Terminated Peptides for Conjugation and Surface Functionalization Strategies T. M. Winger,? P. J. Ludovice,+ and E. L. ChaikoPS'J School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, and Department of Surgery, Emory University School of Medicine, Atlanta, Georgia 30322. Received December 12, 1994@
The derivatization of poly@-(chloromethyl)styrene-co-divinylbenzene)(Merrifield resin) with N-(tertbutoxycarbonyl)-2-aminoethanethiolis presented a s a convenient route for the generation of thiol terminated peptides using a solid phase methodology. Maximum resin substitution reached 92% (773 pmollg) after 24 h. However, a t 30 min, yields exceeded 400 pmollg, above which the resin is suitable for solid phase peptide synthesis. Thiol terminated peptides are well-suited for subsequent chemical conjugation reactions or for the formation of organic monolayers on metal substrates.
INTRODUCTION
EXPERIMENTAL PROCEDURES
Self-assembled organic monolayers of various functionality have been formed on solid substrates on the basis of the coordination of thiol or disulfide groups with gold, silver, and platinum (1). In this regard, most strategies directed a t the creation of a peptide monolayer have used the presence of cysteine residues in synthesized or genetically engineered peptides or proteins to mediate surface binding ( 2 ) . An alternative method has been recently reported by Whitesell and Chang. Following the amination of a gold surface with an aminotrithiol, a peptide monolayer was produced by in situ polymerization (3). The inability to precisely control peptide chain growth led to significant variability in ellipsometric measurements and appears to be a limitation of this approach. Although thiol groups act as a n effective binding residue to gold and other metal substrates, the chemical structure of the entire molecule will affect both packing density and chain orientation of any self-assembled monolayer ( 4 ) . As noted above, most peptide monolayers have been bound to gold substrates by a ,!?-carboxylatebearing thiol, like cysteine. Since the supramolecular structure of a thin film may significantly alter its physical and chemical properties, we began to explore the synthesis of less hindered thiol-terminated peptides. We present an effective solid phase procedure for the creation of w-thiol oligopeptides. This approach is based on the modification and extension of earlier studies which examined the derivatization of chloromethylated polystyrene resins through a nucleophilic substitution reaction in the presence of a thiol (5). The advantages of terminal thiols produced in this fashion for the formation of peptide conjugates were not evaluated. Nonetheless, we believe that peptides produced by this technique may be effectively used in chemical conjugation schemes, if so desired.
Materials. The Merrifield resin (0.84 mequiv/g, 1% cross-linked with divinylbenzene, Fluka) was washed with methanol before use, and fine floating beads were discarded. 1,4-Dioxane (Aldrich) was treated with basic alumina to remove peroxides. Trifluoroacetic acid, tetrabutylammonium hydroxide (TBAH; 1 M in MeOH), cystamine dihydrochloride, and methylamine (MeNH2; 2 M in MeOH) were purchased from Aldrich. All other chemicals used for resin derivatization were reagent or spectrophotometric grade. Peptides were prepared on a fully automated Applied Biosystems Model 430A peptide synthesizer (0.5-mmol scale). The apparatus used for hydrogen fluoride cleavage was from Peninsula Laboratories. All solvents were of synthetic grade and were supplied by Burdick and Jackson. Highest grade (tert-buty1oxy)carbonyl (Boc)protected amino acids were purchased from commercial sources (Applied Biosystems, Inc. (Foster City, CA), Bachem Biosciences (King of Prussia, PA), and Advanced Chemtech (Louisville, KY)). The side-chain protecting groups were [(2-bromobenzyl)oxylcarbonyl(2-BrZ) for tyrosine and mesitylenesulfonyl (Mts) for arginine (Bachem Biosciences). Analytic Methods. Thin layer chromatography (TLC) analysis was performed with iodine vapor for nonspecific detection of all compounds, and the ninhydrin spray test was used for the detection of free amines. The reagents (monitors 1, 2, and 3) used in the ninhydrin assay ( 6 ) were obtained from Applied Biosystems. The 250-pm layer thickness silica plates used for TLC were purchased from Whatman. IH nuclear magnetic resonance (NMR) was performed on a General Electric QE 300 Plus spectrometer a t 300 MHz in deuterated chloroform (CDC13). Chemical shifts (6) were measured with respect to tetramethylsilane (TMS), which was used as the 0 ppm internal reference. Fast atom bombardment (FAB)mass spectrometry was measured on a VG Instruments 70SE mass spectrometer. Synthesis of NJV-Bis(tert-butoxycarbony1)cystamine (2). The synthetic approach for 2 and its subsequent reduction to 3, a s described below, is similar to those in the literature (5, 7-9). An aqueous/organic biphasic mixture of 2 g (225.20 g/mol, 8.88 mmol) of cystamine dihydrochloride (l),40 mL ofp-dioxane, 10 mL of water, 7.8 mL (6.3 equiv) of triethylamine, and 4.3 g (218.25 g/mol, 2.2 equiv) of di-tert-butoxycarbonate was stirred for 18 h in a n open 100-mL round-bottom flask
* Author to whom correspondence should be addressed: Elliot L. Chaikof, M.D., Ph.D., Department of Surgery, Emory University Hospital, Box M-11, 1364 Clifton Rd, N.E., Atlanta, GA 30322. (404) 727-8413 phone, (404) 727-3660 fax, echaikdeagle. cc.emory.edu. ' Georgia Institute of Technology. t Emory University School of Medicine. Abstract published in Advance A C S Abstracts, May 1,1995. @
1043-1802/95/2906-0323$09.00/0 0 1995 American Chemical Society
Winger et al.
324 Bioconjugate Chem., Vol. 6,No. 3, 1995
a t room temperature. The reaction mixture was then concentrated to a final volume of 10 mL and poured into 280 mL of water, upon which the bis-protected disulfide 2 precipitated. Precipitation was complete after continuous overnight stirring at room temperature, followed by a 30-min sojourn a t 0 "C. Filtration and high vacuum drying in a desiccator over NaOH yielded 3 g (96%) of crude (BocNHCH~CH~S)~, which was subsequently allowed to crystallize from toluenehexane (113 v/v) for 2 days in the refrigerator. Vacuum drying afforded 2.9 g (8.23 mmol, 93%) of product in the form of white needles. Purity was checked by TLC in CHCldMeOH (80/20 v/v). The Rfvalues for the starting material 1 and the bisBoc-protected product 2 were 0.00 and 0.76, respectively. Synthesis of N-(tert-Butoxycarbonyl)-2-aminoethanethiol (3). A mixture of 2.9 g (8.23 mmol) of 2, 1.1mL of NjV-diethylethanolamine,1.28 g of dithiothreito1 (DTT), and 80 mL of dichloromethane was stirred a t room temperature for 24 h in a 250-mL round-bottom flask. The reaction mixture was then washed twice with 80 mL of a 0.1 N HC1 solution. The organic phase was separated and washed four times with 80 mL of water, dried over MgS04, filtered, and evaporated to dryness. The resulting oily residue was subsequently dissolved in 20 mL of hexane and left a t room temperature for 2 days in order to allow the remaining starting material to crystallize. Subsequent filtration and evaporation to dryness under nitrogen afforded 2.5 g (85%)of 3 (95+% pure). Purity was confirmed by TLC in hexane/ethyl acetate (EtOAc)(80/20 v/v). The Rfvalues for the starting material 2 and the product 3 were 0.40 and 0.62, respectively. 'H NMR (CDC13) data were as follows: 6 1.36 (t, l H , -SH), 6 1.45 (s, 9H, 3 x CH3), 6 2.64 (9, 2H, -NHCH,-), 6 3.32 (q,2H, -CH2CH2SH), 6 4.98 (broad s, l H , -CONH-). Derivatization of the Merrifield Resin. A screwcap glass flask (100-mL capacity, Gibco) was loaded with 5.14 g of Merrifield resin 4 (4.11 mmol of active sites), 22 mL ofp-dioxane, 15 mL of MeOH, 1.17 g (1.6 equiv) of 3,and 5.75 mL of a 1M solution of TBAH in MeOH. The reactor was sparged with nitrogen for 5 min, capped and sealed with Teflon, and vigorously shaken for 20 h a t 65 "C. The supernatant was then discarded, and the resin was washed twice with 100 mL p-dioxane/MeOH (1/1) (polarity index (p.i.1 = 171, twice with 100 mL of iPrOWwater (1/1) (p.i. x 50), and again twice with 100 mL of p-dioxane/MeOH (1/1) ( p i . = 17). All solvent compositions given are volumetric (v/v). Low polarity index washes caused the resin to swell. At high pi., the polystyrene beads shrank. Cyclic low p.i./high p.i. washes therefore improved the rinsing of the resin. End-capping was performed in the presence of a 25-fold molar excess of methylamine by continuous shaking of the resin in 50 mL of a 2 M solution of methylamine in methanol at 65 "C for 6 h. The liquid was subsequently discarded and the resin washed twice with 100 mL ofp-dioxane/MeOH ( l / l ) , twice with 100 mL of iPrOWwater ( l / l ) , and twice with 100 mL of chlorobutane (BuC1) (p.i. = 14). The resin was deprotected by reaction with 100 mL of BuCl/trifluoroacetic acid (TFA) (2/1 v/v) added in portions of 10 mL a t 0 "C for 5.5 h a t room temperature. The resin was then washed twice with 100 mL of BuC1, twice with 100 mL of p-dioxane, twice with 100 mL of iPrOW water (6/1), and twice with 100 mL of MeOH. It was then predried on a rotary evaporator a t 50 "C. Drying was completed in high vacuum (0.1 mmHg) for 4 h at room temperature. This afforded 4.89 g of amine-functionalized resin 6. The substitution of the functionalized Merrifield resin was determined by a ninhydrin test adapted from the method developed by Sarin (6). In
order to determine the optimal period for maximal substitution, the resin substitution reaction was evaluated after reaction times of 0.5, 1.5, and 24 h. At the indicated time, the resin was end-capped, deprotected, and worked up as previously stated. The ninhydrin test was performed in triplicate for each reactor.
Solid Phase Peptide Synthesis and Purification. The synthesis of a decapeptide (SFLLRN(PA)~Y(CHZ)ZSH) was performed in routine fashion. The first amino acid to be attached to the previously thiolamino-derivatized Merrifield resin was activated with dicyclohexylcarbodiimide (DCC) and reacted with 1-hydroxybenzotriazole (HOBt) to form the HOBt ester. Mixing a t basic pH readily yielded the protected amino acid-functionalized resin through nucleophilic inactivation of the ester. Acidic deblocking in the presence of trifluoroacetic acid yielded the free amino terminus. The subsequent three cycles consisted of similar coupling/deprotection sequences using DCC-activated p-alanine as the electrophile. The remaining six amino acids were coupled in routine fashion. The peptide was cleaved from the resin and the side-chain protecting groups removed by treatment with 1.3 mL of anisole and 26 mL of liquid hydrogen fluoride (HF) for 90 min a t 0 "C. After removal of the HF a t 0 "C with a stream of nitrogen, the excess anisole was removed by four washes, each with 30 mL of anhydrous ether. The peptide was subsequently extracted using an aqueous acetic acid solution (50% v/v), and the resulting mixture was freeze-dried in water. Purification of the peptide was achieved by reversephase high performance liquid chromatography (RPHPLC) using a Waters Delta Prep 3000 preparative system and a Waters 3000 systems controller equipped with a Waters 740 data module. The column used was a n Aquapore RP-300 CIS silica column (1 x 10 cm, Applied Biosystems). Elution was performed with a gradient of acetonitrile in water using two buffers: a 0.1% aqueous TFA solution for the first (buffer A) and a mixture of CH~CN/water(80/20 v/v) containing 0.1% TFA for the second (buffer B). The fractions of interest were combined, concentrated on SpeedVac a t room temperature, lyophilized, and stored under nitrogen a t -20 "C. Purity of the fractions was checked by microbore RPHPLC on a reverse-phase CISsilica column (1 x 250 mm, Applied Biosystems). The final sample was analyzed by mass spectrometry for molecular weight confirmation. RESULTS AND DISCUSSION
Synthetic Scheme. The route taken for the synthesis of thiol terminated peptides is summarized in Scheme 1. Precursor synthesis involves the nucleophilic attack of the free amines of neutralized cystamine 1 onto either one of the two carbonyl groups of di-tert-butoxycarbonate (BoczO). The Boc-protected cystamine 2 thereby obtained is subsequently cleaved into its two N-(tert-butoxycarbonyl)-2-aminoethanethiolmonomers 3 through a disulfide reduction reaction in the presence of DTT (5). The derivatization of poly(p-(chloromethyl)styrene-co-divinylbenzene) (Merrifield resin) involves a simple nucleophilic substitution (SN2) of a resin-borne chlorine atom 4 by a thiol. Since the nucleophile is a negatively charged thiolate, resin functionalization requires the presence of an organic base. By working in basic conditions, one also prevents undesirable acidolysis of the Boc group. Such a reaction would deprotect the amino group of compound 3, allowing potential substitution of the chlorine atom leading to a misoriented product. The reactivity of a thiolate (pK, =8.3) is 1000 times greater than that of a primary amine (pK, = 9.5). Thus, the above-mentioned side reaction would only occur to a limited extent,
Bioconjugafe Chem., Vol. 6,No. 3, 1995 325
Technical Notes
Table 1. Ninhydrin Test Results for the Determination of the Degree of Substitutionof the Merrifield Resin vs Reaction Time reaction time (h) resin wt (mg) av absorbance at 570 nm substitution“ &mol of NHdg) a e‘ = 0.012 & 0.003 based on the actual
0.5
1
5
24
5.45 f 0.05 0.139 f 0.002 532 f 156
5.54 f 0.05 0.193 f 0.002 726 f 210
5.41 & 0.05 0.199 f 0.002 767 f 222
5.42 f 0.05 0.201 i 0.002 773 i 223
weight of pure peptide recovered from a given solid phase synthesis.
Scheme 1. Preparation of Precursors and Derivatization of the Merrifield Resin (HCI.H2NCH>CH,S),
1
m I
4
I
--4 Drovided the DH is keDt between 8 and 10. Unreacted chloromethyl kmctioniwere end-capped by a similar s ~ 2 type reaction with methylamine. Acidolysis of the Boc groups at the surface of the derivatized resin yields the amine-functionalized resin suited for solid phase peptide synthesis. Following peptide synthesis, para-substituted benzyl thioethers generate a free thiol upon cleavage with hydrogen fluoride (10). Chemical and Physical Characterizationof Resin Substitution. Resins that exhibit optimal characteristics for solid phase peptide synthesis have a typical substitution of 400-800 pmollg. In our case, approximately 20 min of reaction time was required for a small scale batch (150 mg of resin) to reach the 400 pmollg threshold, above which the resin becomes well-suited for solid phase peptide synthesis (Table 1). Maximum resin substitution reached 92% (773 pmollg) after 24 h. For scaled-up batches of 5 g of resin, a longer reaction time of 20 h was used to ensure maximal conversion. Subsequent analysis with a ninhydrin assay indicated a substitution of 354 f 170 pmol/g. Incidentally, we noted a simple correlation between bead density and its degree of substitution. Indeed, preliminary resin substitution experiments yielded a binodal bead distribution. A small fraction of the resin was substituted a t 200 pmollg, while the remainder represented a high-substitution population starting a t 400 pmollg. The low-substitution beads floated on BuCll
TFA (2/1 v/v) (which has a density of approximately 1.08 g/mL) but settled in BuCl(0.89 g/mL). Hence, the density of the 200 pmollg beads lay between 0.89 and 1.08 g/mL. The high-substitution beads, on the contrary, settled in BuCVTFA (2/1) (1.08 g/mL) but stayed afloat on CHzClz (1.32 g/mL), indicating that their density lay between those two values. Thiol Terminated Peptide Synthesis. The peptide synthesized for the purpose of the present study was a human thrombin receptor activating sequence, Ser-PheLeu-Leu-Arg-Asn-(,Mla)3-Tyr-NHCHzCHzSH. Initially, 554 mg (88%)of crude peptide was obtained. After HPLC purification, the overall yield was approximately 60% (97% purity). The peptide was characterized by TLC in hexane/chloroform/2-propanol/glacial acetic acid/water (15/5/60/3/17 v/v). The TLC spot (R,P 0.51) turned bright yellow upon application of a few drops of a 1 mg/mL aqueous solution of Ellman’s reagent (5,5’-dithiobis(2nitrobenzoic acid)) (11). As expected, ninhydrin testing confirmed the presence of free amino groups. The respective disulfide had an Rf of 0.35 and stained positive with ninhydrin but not with Ellman’s reagent. FAB mass spectrometry confirmed the expected molecular weight (1184.4 g/mol). Solid phase methodology was introduced by Merrifield in 1963 (12)and has since become an invaluable tool for routine peptide synthesis. In this method, a growing polypeptide chain is covalently anchored, usually by its C-terminus, to a n insoluble solid support such as beads of polystyrene resin, and the appropriately blocked amino acids and reagents are added in the proper sequence. Our results demonstrate the successful preparation of a thiolterminated oligopeptide through the use of a (chlorobenzy1)polystyrene resin. Derivatization with a n aminoethanethiol anchor yielded the thiol terminated peptide directly, without the addition of a cysteine residue or the use of other postsynthesis derivatization steps. ACKNOWLEDGMENT
The authors acknowledge the support of Emory/ Georgia Tech Biomedical Technology Research Center and the American College of Surgeons Faculty Fellowship Award. LITERATURE CITED (1) Bain, C. D., Troughton, E. B., Tao, Y.-T., Evall, J., Whitesides, G. M., and Nuzzo, R. G. (1989) Formation of monolayer
films by the spontaneous assembly of organic thiols from solution onto gold. J . Am. Chem. SOC.121, 321-335. (2) Amador, S. M., Pachence, J. M., Fischetti, R., and McCauley, J. P., Jr. (1993) Use of self-assembled monolayers to covalently tether protein monolayers to the surface of solid substrates. Langmuir 9, 812-817. (3) Whitesell, J. K., and Chang, H. K. (1993) Directionally aligned helical peptides on surfaces. Science 261, 73-76. (4)Ulman, A. (1991) An Introduction to Ultrathin Organic Films. From Langmuir-Blodgett to Self-Assembly, pp 237301, Academic Press, San Diego. ( 5 ) Inman, J. K., Dubois, G. C., and Appella, E. (1977) Synthesis of macroporous polystyrene derivatives for supports in sequential degradation of peptides. Solid Phase Methods i n
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326 Bioconjugafe Chem., Vol. 6,No. 3, 1995 Protein Sequence Analysis, INSERM Symposium, pp 81-94, Elseviermorth-Holland Biomedical Press, New York. (6) Sarin, V. K., Kent, S. B. H., Tam, J. P. and Merrifield, R. B. (1981) Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction. Anal. Biochem. 117, 147157. ( 7 ) Moroder, L., Hallet, A., Wunsch, E., Keller, 0. and Wersin, G. (1976) Di-tert-butyl-dicarbonate, a useful tert-butyloxycarbonylating reagent. Hoppe-Seylers 2.Physiol. Chem. 357, 1651-1653. (8) Van den Broek, L. A. G. M., Fennis, P. J., Arevalo, M. A., Lazaro, E., Ballesta, J. P. G., Lelieveld, P., and Ottenheijm, H. C. J. (1989) The role of hydroxymethyl function on the
biological activity of the antitumor antibiotic sparsomycin. Eur. J . Med. Chem. 24,503-510. (9) Stindl, A., and Keller, U. (1993) The initiation of peptide formation in the biosynthesis of actinomycin. J . Biol. Chem. 268, 10612-10620. (10) Greene, T. W. (1981) Protective Groups in Organic Syntheses, pp 193-217, Wiley-Interscience Press, New York. (11) Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77. (12) Merrifield, R. B. (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J . Am. Chem. SOC.85, 2149-2154. BC9500220
