458
Bioconjugate Chem. 1991, 2, 458-463
TECHNICAL NOTES Synthesis of Nu-( tert-Butoxycarbonyl)- N - [N-(bromoacetyl)-@-alanyl]L-lysine: Its Use in Peptide Synthesis for Placing a Bromoacetyl Cross-Linking Function at Any Desired Sequence Position John K. Inman,*J Patricia F. Highet,' Nelly Kolodny,: and Frank A. Robeyt Bioorganic Chemistry Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, and the Peptide and Immunochemistry Unit, Laboratory of Cellular Development and Oncology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892. Received July 12, 1991
A new amino acid derivative, N~-(tert-butoxycarbonyl)-N~-[N-(bromoacetyl)-@-alanyl]-~-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a sidechain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-@alanine with Nu-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for Na-Boc amino acid derivatives. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers, alsolinked through thioether bonds. Products made with BBAL peptides may be characterized by amino acid analysis of acid hydrolyzates by quantification of @-alanine,which separates from natural amino acids in suitable analytical systems. Where sulfhydryl groups on coupling partners arise from cysteine residues, S-(carboxymethy1)cysteine in acid hydrolyzates may also be assayed for this purpose. Examples are given of the use of BBAL in preparing peptide polymers and a peptide conjugate with bovine albumin to serve as immunogens or model vaccine components.
INTRODUCTION Conjugates of synthetic peptides with proteins, other peptides, polymers (soluble/insoluble; natural/synthetic), and surfaces of special materials are being employed increasingly in biomedical research and biotechnology. Applications of peptide conjugates include the preparation of immunogens (including synthetic vaccines) for raising antibodies to selected portions of protein antigens (1-4), affinity adsorbents, immunoassay components, and celladhesion surfaces (5, 6). The locus of attachment of a peptide to its conjugate partner may have a major influence on the biological activity or performance of the conjugate (7-9).Strategies for cross-linking a peptide through a single, selected locus are often complicated by the presence of more than one amino or carboxyl group, and the need to protect (then deprotect) amino groups if a carboxyl function is to be activated. Peptides can be more or less selectively derivatized through their N-terminal aminogroups by means of acylation reactions in slightly acidic media and/or with use of certain types of reagents, such as symmetrical anhydrides (10). However, if one wishes to synthesize a peptide in order to prepare a conjugate, planning the synthesis for this purpose can prove very advantageous. For example, some workers have introduced a reactive cysteine residue at the desired position for heteroligation
* Address correspondence to John K. Inman, Ph.D., NIAIDLI, Bldg 10, Room llN311, National Institutes of Health, Bethesda, MD 20892. t National Institute of Allergy and Infectious Diseases. t National Institute of Dental Research. Not subject to U.S. Copyright.
(11, 12);Drijfhout et al. (13) completed a sequence on a solid support with an N-terminal S-acetylmercaptoacetyl group. The deprotected peptide was treated with hydroxylamine to remove the S-acetyl group and then joined through its N-terminal sulfhydryl group to a conjugate partner bearing an SH-selectiveelectrophilic function (e.g., an N-substituted maleimide or an a-haloacetyl moiety) by means of a very stable thioether linkage (12, 13). Strategy considerations may show preference to placing sulfhydryl groups on the conjugate partner (or using those already present) and coupling it to Cys peptides by means of less stable disulfide bonds. The coupling can be effected through an activating group, such as S- [ (3-nitro-2-pyridy1)sulfenyl] (Npys),' previously placed on a cysteine side chain (14-16). A Cys(Npys) residue, introduced in solid-phase peptide synthesis (SPPS), will remain intact during trifluoroacetic acid (TFA) or HF cleavage steps. For an alternative approach, the peptide synthesis program could be modified in order to introduce an SHselective electrophile somewhere in the sequence, again allowing use of stable thioether cross-linkages. Lindner and Robey (17) described the incorporation of N-terminal
'Abbreviations used: Npys, S-[(3-nitro-2-pyridyl)sulfenyl]; SPPS, solid-phase peptide synthesis;TFA, trifluoroaceticacid, BBAL,Na-(tert-butoxycarbonyl)-N~-[N-(bromoacetyl)-&alany1]L-lysine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CMC, S-(carboxymethy1)cysteine; BSA, bovine serum albumin;BuaP, tri-n-butylphosphine;SBAP, succinimidyl 3-(bromoacetamido)propionate;DMF, N,N-dimethylformamide; DCE, 1,2-dichloroethane;HOBt, l-hydroxybenzotriazole; DCC, N,N'-dicyclohexylcarbodiimide.
Published 1991 by American Chemical Society
Technlcal Notes
(chloroacety1)glycylglycyl groups in the last cycle of an automated SPPS. Subsequently, Robey and Fields (18) and Kolodny and Robey (19) described a similar method for introducing the more reactive bromoacetyl group at the N-termini of peptides made by SPPS. Using this approach, these authors have prepared many useful immunogens either as peptide-protein conjugates or as selfpolymers of peptides that contain both a cysteine residue and an N-terminal bromoacetyl group. The main limitation to the above approach is lack of flexibility in choosing the site for an a-haloacetyl group. To our knowledge, there has not yet been reported a method for introducing an SH-selective alkylating function at any desired position in a peptide that is being synthesized sequentially. Accordingly, we designed and synthesized a new Boc-amino acid derivative, Na-(tertbutoxycarbonyl) -Nt-[ N -(bromoacetyl)-@alanyl] lysine (BBAL). With this compound, one can introduce the bromoacetyl cross-linking function at the N- or C-terminus or at any intermediate position of a synthetic peptide that is being prepared by automated SPPSprograms employing temporary Nu-Boc protection and final HF-induced deprotection and cleavage. Coupling of these peptides to thiol-bearing carriers can be readily accomplished by mixing the components in a neutral or slightly alkaline buffered medium. The ensuing peptide-carrier conjugates may be quantitatively characterized by means of the P-alanine liberated upon acid hydrolysis of a sample. If coreactant groups are cysteine sulfhydryls, S-(carboxymethyl)cysteine (CMC) also appears in the hydrolyzate (19).The @-alanineresidue places additional spacing in the crosslink and appears to be a necessary part of the structure of BBAL that results in its being a stable solid that can be conveniently stored, weighed, and dispensed for synthesizer operations. EXPERIMENTAL PROCEDURES
Materials and Methods. Reagent-grade chemicalsand solvents used in the synthesis were obtained from Fisher Scientific (Pittsburgh, PA). 0-Alanine and N-hydroxysuccinimide were purchased from Sigma Chemical Co. (St. Louis, MO). The latter reagent was recrystallized once from ethyl acetate. N,N'-Diisopropylcarbodiimide was obtained from Aldrich Chemical Co. (Milwaukee, WI); bromoacetyl bromide was ordered from Fluka (Ronkonkoma, NY); Na-Boc-L-lysine was obtained from Vega Biochemicals (Tucson, AZ). With the exception of BBAL, all reagents used for the automated synthesis of peptides were purchased from Applied Biosystems, Inc. (Foster City, CA). Sodium dodecy1 sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed on the peptide polymers with the gel electrophoresis system sold by Novex (Encinitas, CAI. This system provided the materials required to run gels by the method of Laemmli (20). Two amino acid analysis systems were used as described by the manufacturers for the analyses of products that were made using BBAL: Amino Quant by Hewlett-Packard, Inc. (Gaithersburg, MD) and Picotag by Waters Associates (Millipore Corp., Milford, MA). CMC and bovine serum albumin (BSA) were from Sigma Chemical Co., and tri-n-butylphosphine (Bu3P) was purchased from Aldrich Chemical Co. NMR spectra for 1H and l3C were obtained on a Varian XL200 spectrometer at 200 and 50 MHz, respectively. Typically, solutions of 10 mM concentration yielded spectra after the collection of 64 free induction decays for 'H (digital resolution of 0.3 Hz) and 60 000 free induction decays for 13C (digital resolution of 1 Hz). Assignments
Bioconlugate Chem., Vol. 2, No. 6, 1991 459
were based on published spectra and known substituent effects (21). N-(Bromoacety1)-&alanine. A solution of 0-alanine (53.5 g, 0.60 mol) in 600 mL of water was cooled to 5 "C with an ice-alcohol bath. Bromoacetyl bromide (60.0 mL as 95 % pure, 0.66 mol) was added under efficient stirring at such a rate as to maintain the temperature below 12 "C. Concurrently, 5 M NaOH was added at a rate needed to keep the pH near 7. These conditions were maintained for 45 min after completing addition of the bromoacetyl bromide. Because the latter reagent is a highly toxic irritant, the above operation was carried out in a hood. The pH of the reaction mixture was then adjusted to 1.92.0 using 48% HBr, and its volume was reduced in a rotary evaporator to ca. 150 mL using a 60 "C bath and aspirator vacuum. The heavy precipitate of NaBr was removed by suction filtration and washed with ca. 15 mL of water. The filtrate was treated with a small volume of water to dissolve newly precipitated NaBr and then shaken once with hexane-ethyl ether 1:l v/v (450mL), once with ethyl ether (450 mL), and four times with ethyl acetate-ethyl ether 1:5 v/v (450 mL each time). The first upper phase (rich in bromoacetic acid) and final lower phase were discarded. The next five upper phases were pooled, filtered through Whatman #1 paper, and rotary evaporated to remove solvent. The residue was dried under vacuum and crystallized from hot ethyl acetate (81 mL) by addition of hexane (about 12 mL) and cooling to 4 "C. The dried product (31.4 g) was similarly recrystallized from ethyl acetate (69 mL) plus hexane (15 mL) and dried under vacuum/CaClz: yield, 30.0 g (23.8%); mp 88.5-91 "C [lit. (22) mp 80-81 "C]; 'H NMR ( D M s 0 - d ~6) 2.43 (t, 2 H, a),3.26 (q, 2 H, p), 3.84 (s, 2 H, a'),8.32 (br, NH), 12.20 (br, COOH); 13C NMR ( D M s 0 - d ~ 6) 29.31 (a'),33.4 (a),35.2 (p), 165.91 (Ac C=O), 172.6 (Ala C=O). Anal. Calcd for C5HsBrN03: C, 28.59; H, 3.84; N, 6.67; Br, 38.05. Found: C, 28.67; H, 3.97; N, 6.64; Br, 38.40. S u c c i n i m i d y l 3-(Bromoacetamido)propionate (SBAP). To a solution of N-(bromoacetyl)-fl-alanine (21.00 g, 100 mmol) and N-hydroxysuccinimide (13.01 g, 113 mmol) in 2-propanol (280 mL) at room temperature was added 1,3-diisopropylcarbodiimide (16.0 mL, 101 mmol). After 8-10 min, oily precipitation of the product began, and the walls of the container were scratched to induce crystallization. The mixture was allowed to stand for 1h at room temperature and overnight a t 4 "C. The crystals were collected, washed with 2-propanol(30 mL), and redissolved in 2-propano1(200 mL brought to reflux). After an overnight stand at 4 "C, the crystals were collected, washed with 2-propanol then hexane, and dried under vacuum/CaClP: yield, 22.9 g (74.6%);mp 107-110.5 "C [lit. (23) mp 104-106 "C]; 'H NMR ( D M s 0 - d ~6) 2.80 (9, 4 H, a"),2.82 (t, 2 H, a),3.64 (q, 2 H, p), 3.82 (s,2 H, a'), 7.03 (br, NH); 13C NMR (CDC13) 6 25.62 (a"),28.67 (a'), 31.39 (a), 35.65 (p),166.20 (AcC=O), 167.30,169.06(CEO, s). Anal. Calcd for CgHllBrN205: C, 35.20; H, 3.61; N, 9.12; Br, 26.02. Found: C, 35.74; H, 3.83; N, 9.20; Br, 26.12. Na-(tert-Butoxycarbony1)-N-[N-( bromoacetyl)-& alanyl]-L-lysine (BBAL). Na-Boc-L-lysine (17.73 g, 72 mmol) was ground to a fine powder and suspended in N,Ndimethylformamide (DMF) (600 mL). SBAP (18.43 g, 60.0 mmol) was added to the suspension in five portions at 10-min intervals. The reaction mixture was stirred for 2 h at room temperature, allowed to stand overnight at 4 "C, filtered, and rotary evaporated to remove DMF (bath 30 "C, pump vacuum). The residue was shaken with a
460
Inman et al.
Bioconju~teChem., Vol. 2, No. 6, 1991
mixture of ethyl acetate (960 mL), l-butanol (240 mL), and aqueous 0.2 M KHSOd (300 mL). The upper phase was shaken twice with 0.2 M KHS04 (300 and 150 mL, respectively), filtered (Whatman #1 paper), and rotary evaporated to remove solvent (32 "C bath, pump vacuum). Vacuum was applied (for a t least 2 h) to purge the last traces of solvent. The oily residue was dissolved in 1,2dichloroethane (DCE) (400 mL) by gentle warming and swirling. The solution was slowly cooled to 15-20 "C during which time the product initially precipitated as an oil but was induced to crystallizeby scratching. After an overnight stand (4 "C), the product was collected, washed (3 X 36mL portions of DCE), and dried in vacuum/CaClz: yield, 17.3 g (65.8%),mp 117-122 "C dec; lH NMR (DMSO-&) 6 1.34 (s, 9 H, a"'), 1.5 (m, 6 H, p, y, 6 ) , 2.22 (t, 2 H, a'), 3.02 (4, imp), 3.50 (m, e, p', imp), 3.82 (s + m, a + a' + imp's), 6.95 (d, Lys-a-NH), 7.82 (t, NH), 8.24 (t,NH); 13C NMR (DMSO-&) 6 22.96 (y), 28.13 (CH3),28.60 (O), 29.40 (a"),30.41 (a'), 34.80 (a), 35.75 (p'),38.17 (e), 53.36 (a), 61.32 (imp, HOCHZCON?),77.90 [OC(CH3)3],155.50(carbamate C=O), 165.81 ((BrCHZC=O), 169.83, 174.08 (C=O's). Anal. Calcd for Cl,jHd3rN301$ C, 43.84; H, 6.44; N, 9.59; Br, 18.23; C1, 0. Found: C, 43.79; H, 6.61; N, 9.51; Br, 18.32; C1, 0.38. Synthesis of BBAL-Containing Peptides. The various BBAL-containing peptides were synthesized using an automated solid-phase peptide synthesizer (Model 430A, Applied Biosystems, Inc., Foster City, CA) that is based on the original Boc/Bzl solid-phase peptide synthesis procedures described in 1963 by Merrifield (24). For introducing BBAL residues into peptides at any desired position along the chain, the same double coupling cycles for asparagine that are preprogrammed into the instrument were found to be most suitable when peptides were synthesized on a 0.5-mmol scale, the larger of the two scales that are preprogrammed for the Model 430A. The reason for this is that BBAL is very soluble in DMF, but sparingly soluble in CHzClz, and the asparagine coupling steps of the Model 430A employ l-hydroxybenzotriazole (HOBt) ester formation in DMF. Briefly, the synthesis first involved the following: A mixture of BBAL and HOBt was made by dissolving 2.0 mmol BBAL in a solution containing 2.0 mmol HOBt in 4.0 mL of DMF and 0.3 mL of CHzClz. The BBAL-HOBt mixture was added to 4.0 mL of 0.5 M N,N'-dicyclohexylcarbodiimide (DCC) in CHzC12. This mixture was agitated by bubbling with N2 over a period of at least 30 min at 25 "C. The dicyclohexylurea byproduct, which precipitated during the formation of the BBAL-OBt ester, was filtered off, and the ester in the filtrate was reacted with a free amine on the PAM resin to give the BBAL PAM-coupled product. t-Boc was removed from the coupled BBAL in the next cycle with TFA in CHpCl2 using the manufacturer's preprogrammed protocol. Following the synthesis, the peptide was deprotected and released from the PAM resin using a standard HF deprotection method as described by Robey and Fields (18)for preparing N-terminally bromoacetylated peptides. The only scavenger used was anisole. As mentioned previously (181, sulfur-containing scavengers such as thiophenol or thioanisole were avoided as a precautionary measure. Synthesis of Peptide Polymers Using BBAL. Peptide polymers prepared with the use of BBAL have been synthesized as candidate immunogens (F. A. Robey, unpublished results). These polymerized peptides were all assembled by following the same procedure detailed previously for making peptide polymers coupled head-
Scheme I
?
+
Br-CH2-C-Br
?
H2N-CH2-CHe-C-OH
t
0
?
P
+
Br-CHg-c-NH-CH2-CH2-C-OH
HO-N
BrAc-pAla-0H
I
+
3
0
(CH&CH-NmCmN-CH(CHJp
0
9 Br-CH2-C-NH-CH2-CH2-C-O-N SBAP (BrAc-pAlaQSu)
to-tail with cysteine at the peptide's C-terminus and bromoacetyl at the N-terminus (18). For polymers involving the use of synthetic peptides containing BBAL, the peptide is designed to contain cysteine at any desired position and BBAL at any other position. The BBAL, Cyscontaining peptide was dissolved in 0.5 M phosphate buffer, pH 7.2, at a concentration of 20 mg/mL. The polymer was formed generally within 3 h at ambient temperatures, but we routinely allowed the mixture to stir overnight. The mixture was then dialyzed and lyophilized as detailed previously (18). Coupling BBAL-Containing Peptides to Bovine Serum Albumin (BSA). Synthetic peptides were coupled to BSA following the same procedure outlined by Kolodny and Robey (19) for coupling N-terminal bromoacetyl peptides to BSA To 30 mg of BSA dissolved in 5 mL of 0.1 M NaHC03 was added 0.2 mL of a 0.7 M solution of Bu3P in 2-propanol, and the reaction mixture was stirred for 30 min at room temperature. Then, 30 mg of the BBAL-containing peptide was added, and the reaction mixture was continuously stirred for 1 h. The entire 6 mL of the conjugation reaction mixture was dialyzed for 1 2 h at 4 "C against a 6-L batch of 0.1 M NH4HC03 and then against three changes of the same solution over a 2-day period. The conjugates were then obtained in powder form by lyophilization. RESULTS
The synthesis of BBAL was accomplished in three stages as shown in Schemes I and 11. First, N4bromoacetyl)@-alaninewas prepared by a procedure similar to the ones reported by Yamada et al. (22) and Zaitsu et al. (23) (Scheme I, first reaction). The product was adequately separated from bromoacetic acid, resulting from hydrolysis of excess bromoacetyl bromide, by a series of extractions that obviated the need for an adsorption chromatography step. The product was purified by two crystallizations from ethyl acetate-hexane, rather than from tetrahydrofuran-isopropylether, and gave a melting point about 10 "C higher than that reported by Yamada et al. (22). This acid was converted to its N-hydroxysuccinimide active ester, SBAP (Scheme I, second reaction), by a technically facile approach whereby the product itself crystallized from the reaction mixture rather than a urea byproduct: Coupling was accomplished with N,"-diisopropylcarbodiimide in 2-propanol instead of with DCC in a less polar solvent as is commonly done (23). The synthesis of BBAL was carried out conveniently and
Bioconlugate Chem., Vol. 2, No. 6, 1991 461
Technical Notes
Scheme I1
A
B
42.7kD 97.4kD 66.2kD
31.0kD
?
-
+ Y
1
SBAP
0
DMF
0
0 H3C-$-O-C-NH-CH
-&OH
CH3 BBAL ~Lys(BrAo-f3-Ah)-OH]
cleanly with SBAP and commercially available Nu-BocL-lysine (Scheme 11). During the course of developing the above synthesis, alternative reagents and solvents were tried. Preliminary examination of products by NMR or MS often revealed significant exchange of Br with C1 in the bromoacetyl moiety whenever C1 was present in the system (data not given). This problem was especially severe when bromoacetylchloride was used and extractions were conducted over aqueous NaC1, suggesting an ionic mechanism for the halogen exchange. All C1-containing reagents were then eliminated from use except 1,2-dichloroethane used to crystallize BBAL, since it and dichloromethane were the only solvents that we found from which the final solidification and purification of BBAL could be achieved. The C1 content of BBAL, crystallized once from dichloroethane, was 0.38% (4.7 mol %), but it increased to 1.1% (13.6 mol 76) following recrystallization from the same solvent. A simpler analogue of BBAL, Na-( tert-butoxycarbony1)N'-(bromoacetyI)-L-lysine, was prepared by three different routes, each leading to a noncrystallizable, vitreous product (data not given). The method deemed most satisfactory for yielding a pure product was the one that paralleled the BBAL synthesis reported above wherein SBAP was substituted with N-succinimidyl bromoacetate, prepared as described by Bernatowicz and Matsueda (12). BBAL can be used effectively in strategies for polymerizing synthetic peptides. For example, the peptides can be polymerized "tail-to tail" if both cysteine and BBAL have been placed in the C-terminal region of the peptide monomers and if they are subjected to conditions that favor reaction of a thiol group of one molecule with a bromoacetyl moiety on another one, etc. The monomer peptides are thus joined through stable thioether linkages. The result of one such experiment is shown by the SDSPAGE run presented in Figure 1. Molecular masses as high as 30 kDa are observable in the mixture of polymer sizes. Other peptides have been polymerized in like
Figure 1. SDS-PAGE run of a peptide polymer obtained by intermolecular cross-linkingof a BBAL-containingpeptide with itself through a coexisting cysteine residue. Lane A shows the molecular weight standards;lane B shows the synthetic peptide polymer having the monomer sequence Lys-Ser-Ile-Arg-Ile-GlnArg-Gly-Pro-Gly-Arg-Val-Ile-Tyr-Cys-BBAL-NH2. The gel was stained with Coomassie Brilliant Blue R250.
manner to yield polymer components with molecular masses exceeding 30 kDa (results not shown). In general, we have found that the size distribution of the peptide polymers depends in part on the solubility and size of the peptide monomers; the greater the solubility, the higher is the degree of polymerization. Although we have not investigated cyclization in detail, there appears to be a tendency for peptides to cyclize that have four to six amino acid residues between the bromoacetyl-bearing and Cys residues (unpublished observations). Therefore, the recommendation for optimal polymerization (greatest number of repeat units) is to use peptide monomers with more than six interstitial residues, preferably ones with 15-20 such residues. The sequence of the peptide used in the experiment illustrated by Figure 1 is given in the caption. Synthetic peptides containing BBAL, but not cysteine, are readily conjugated with carriers bearing reactive sulfhydryl groups. Peptide-carrier conjugates are commonly used as immunogens or test antigens. We have employed BSA as a carrier following reduction of some of its cystine disulfide bonds with Bu3P (25)in order to release multiple thiol groups as conjugation sites (see the Experimental Procedures and ref 19). Two systems of amino acid analysis were used in this study to evaluate the number of BBAL peptides conjugated to modified BSA. The Picotag chromatogramof Figure 2 clearly shows a measurable amount of CMC from the hydrolysis of Gly-Arg-Gly-GluPro-Thr-BBAL linked to reduced BSA. The amino acid standard chromatogram containing CMC in the Picotag system and the calculation to quantify the CMC were presented previously (19). The Picotag amino acid analysis system could not be used to quantify the @-alaninethat is also formed from the acid hydrolysis of BBAL-peptide-derived structures. @-Alanineelutes a t the same position as histidine (ca. 4.24.5 min), and thus the two components cannot be distinguished. A value for @-alaninecould be obtained by Picotag if the sample undergoing analysis contained no histidine. The second amino acid analysis system that we employed was Amino Quant. The 1-nmol standard chromatogram, containing 1nmol of @-alanine,is shown in Figure 3. It is clear from examining this chromatogram that the @-alanine peak (designated Beta-ALA) is completely resolved from the nearest amino acids (threonine and alanine). Shown in Figure 4 is the Amino Quant chromatogram of
462
Inman et al.
Bioconjqte Chem., Vol. 2, No. 6, 1991
:q
0.30
0.25
2
3
120
090 0.15
60
0.10
40
0.05
20
0.00 0
2
4
a
6
lo
12
TIME (mid
Figure 2. A Picotag chromatogram of the acid hydrolyzate of the conjugate (Gly-Arg-Gly-Glu-Pro-Thr-BBAL),,-BSA. The conjugate was formed by reaction of the BBAL peptide with BusP-reduced BSA as detailed under Experimental Procedures and in ref 19. CMC is cleanly resolved, allowing the measurement of BBAL peptide covalently coupled to BSA. @-Alanine,which also results from hydrolysis of the conjugate, overlaps the histidine peak and therefore cannot be assayed using the standard conditions for analysis in the Picotag system.
7 300
100 0
0
2
4
6
8
10
12
TIME Imin)
Figure 3. Chromatogram for a 1-nmol amino acid standard spiked with @-alanine,using the Amino Quant system. @-Alanine is cleanly resolved from the adjacent amino acids, Thr and Ala, and thus, is used to quantitate directlythe amount of BBALcontaining peptide conjugated to a protein. The naturally occurring amino acids from the carrier protein are likely to mask all other amino acids in the synthetic peptide. CMC, readily detected with the Picotag system, cannot be measured from a typical Amino Quant run because it elutes with the acidic amino acids (not shown). the acid hydrolyzate of the same peptide-BSA conjugate described in Figure 2. Again, the @-alaninepeak is cleanly baseline-resolved from the other amino acids. Thus, Figures 2 and 4 demonstrate the unique versatility of conjugating peptides to cysteine residues of a protein carrier; more than one amino acid marker can be used to quantify the amount of peptide coupled to the carrier protein. DISCUSSION We have described in this paper the synthesis and characterization of a new compound, BBAL, that provides the peptide chemist with a novel means for building peptides that contain a residue bearing a thiol-reactive side chain at any desired location in the sequence. With this capability, he/she is able to prepare specific peptide components of biological response modifiers that can be readily attached to other molecular species or specialized surfaces through stable thioether bonds. The locus of attachment of peptide epitopes and thus their orientation and biological activity will be uniform and controlled by design. Furthermore, peptides synthesized with both BBAL and Cys residues may be cyclized or self-polymerized.
0
2
4
6
8
10
12
TIME (mid
Figure 4. Amino Quant analysis of the hydrolyzate from the same peptide-BSA conjugate described in the Figure 2 caption. The @-alaninepeak is clearly displayed in this system. The first two stages of the synthesis of BBAL yielded a reagent, N-succinimidyl 3-(bromoacetamido)propionate (SBAP) (Scheme I) that provided a direct and convenient means for preparing BBAL from commercially available Na-Boc-L-lysine (Scheme 11). The conditions for the synthesis of SBAP and BBAL were developed with the goal of allowing easy scale up. It was pointed out by Zaitsu et al. (23)that SBAP itself has the properties of a generally useful heterobifunctional cross-linking reagent. The method for synthesis of this compound that has been detailed in this report should prove easier to carry out on lab or pilot scale than the one previously described (22, 23). Attempts to prepare other functionally equivalent analogues of BBAL [e.g., Boc-Lys(BrAc)-OH and BocOrn(BrAc-@-Ala)-OH]resulted in products that could not be obtained in solid form, placing a practical constraint on their use and rendering them more difficult to purify on a large scale. The intended characteristics of our reagent were (1) possession of three functional groups, bromoacetyl, Boc-amine, and free carboxyl, and (2) that it be a stable, crystalline solid. BBAL met these demands, and the @-alanineresidue incorporated into its structure provided four atoms of additional spacing in the sidechain linkage function plus an acid-stable marker (@-alanine) for analysis of conjugate composition. BBAL has been successfully employed with a standard solid-phase synthesis program based onNa-Boc temporary protection, benzyl-type side-chain protection and final H F deprotection/cleavage. In this type of system, there is the possibility of some nucleophilic attack at the a-bromo carbon atom of BBAL side chains by HOBt or freshly liberated a-amino groups following TFA cleavage and alkalinization steps. We have observed minor amounts of byproducts that may have resulted from this side reaction; however, it would be feasible to remove these impurities if they should enter into and impair functioning of the end product. Although we have not tried using BBAL with methods based on Na-Fmoc protection, it seems to us that its use might not be feasible anywhere except at the N-terminal position because of the expected reaction of bromoacetyl groups with the nucleophilic Fmoc cleavage reagent (e.g., piperidine). The bromoacetyl group, like the somewhat less reactive chloroacetyl moiety, survives H F cleavage conditions (17, 18), while the iodoacetamido function does not (F. A. Robey, unpublished observation). The selectivity of bromoacetamido groups for sulfhydryl anions in neutral or slightly alkaline media is comparable to that of iodoacetamido and may be significantly greater than that of maleimido groups (12). Alarge number of practical applications can be envisaged
Technical Notes
wherein a substance such as BBAL, incorporated into synthetic peptides, could play a critical role. In the case of synthetic peptide vaccine candidates, linear polymerization of peptides appears to enhance immunogenicity (17,18,26). BBAL, Cys peptide polymerizations may be directed in "tail-to-tail", "head-to-tail", or "head-to-head" modes by virtue of the independence allowed in positioning the functional residues in the monomer epitope. Used as immunogens, the resulting polymers gave rise to significantly different immune responses (F. A. Robey, unpublished observations). Cyclic peptides may be favored by autoreaction of BBAL, Cys peptides in very dilute solution. These looped peptides may form the basis of epitopes that better imitate those of the native protein (27).Potentially useful peptide conjugates with many types of molecules, polymers, or surfaces may be made from BBAL peptides. For example, conjugates can be prepared with oriented peptide epitopes attached to immunogen carriers, adsorbents, immunoassay materials, or surfaces of medical prostheses. These products should have clearly defined biological responses and distinct properties made possible by the flexible and uniform positioning of cross-linking loci. ACKNOWLEDGMENT
We express our gratitude to Dr. Robert J. Highet of the National Heart Lung and Blood Institute, National Institutes of Health, for the NMR analyses and interpretation of the spectra. We also thank Allison Murphey for valuable technical assistance and recognizethe excellent service of Galbraith Laboratories, Inc., Knoxville, TN, where the elemental analyses were performed. LITERATURE CITED
(1) Lerner, R. A. (1982)Tapping the immunological repertoire to produce antibodies of predetermined specificity. Nature 299,592-596. (2) Zavala, F.,Tam, J. P., Hollingdale, M. R., Cochrane, A. H., Quakyi, I., Nussenzweig, R. S., and Nussenzweig, V. (1985) Rationale for development of asynthetic vaccine against Plasmodium falciparum malaria. Science 228, 1436-1440. (3)Tam, J.P.(1988)Synthetic peptide vaccine design: Synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. U.S.A. 85,5409-5413. (4) Milich, D.R. (1989)Synthetic T and B cell recognition sites. Adu. Immunol. 45, 195-282. (5) Massia, S. P., and Hubbell, J. A. (1990)Covalent surface immobilization of Arg-Gly-Asp- and Tyr-Ile-Gly-Ser-Argcontaining peptides to obtain well-defined cell-adhesive substrates. Anal. Biochem. 187,292-301. (6) Brandley, B. K., and Schnaar, R. L. (1988) Covalent attachment of an Arg-Gly-Asp sequence to derivatizable polyacrylamide surfaces: Support of fibroblast adhesion and longterm growth. Anal. Biochem. 172,210-278. (7)Dyrberg, T., and Oldstone, M. B. A. (1986)Peptides as antigens. Importance of orientation. J.Exp. Med. 164,13441349. (8) Schaaper, W. M. M., Lankhof, H., Puijk, W. C., and Meloen, R. H. (1989)Manipulation of antipeptide immune response by varying the coupling of the peptide with the carrier protein. Mol. Immunol. 26,81-85. (9) Golvano, J., Lasarte, J. J., Sarobe, P., Gullbn, A., Prieto, J., and BorrBs-Cuesta, F. (1990)Polarity of immunogens: Implications for vaccine design. Eur. J. Immunol. 20, 23632366.
Bioconjugate Chem., Vol. 2, No. 6, 1991 463
(10) Wetzel, R., Halualani, R., Stults, J. T., and Quan, C. (1990) A general method for highly selective cross-linking of unprotected polypeptides via pH-controlled modification of N-terminal a-amino groups. Bioconjugate Chem. 1 , 114-122. (11) Green, N., Alexander, H., Olson, A., Sutcliffe, J. G., and Lerner, R. A. (1982)Immunogenic structure of the influenza virus hemagglutinin. Cell 28,477-487. (12)Bernatowicz, M. S.,and Matsueda, G. R. (1986)Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifundional crosslinkingreagent. Anal. Biochem. 155,95-102. (13)Drijfhout, J. W., Bloemhoff, W., Poolman, J. T., and Hoogerhout, P. (1990)Solid-phase synthesis and applications ofN-(Sacetylmercaptoacetyl) peptides. Anal. Biochem. 187, 349354. (14) Bernatowicz, M. S.,Matsueda, R., and Matsueda, G. R. (1986)Preparation of Boc- [S-(3-nitro-2-pyridinesulfenyl)]-cysteine and its use for unsymmetrical disulfide bond formation. Int. J. Pept. Protein Res. 28, 107-112. (15)Drijfhout, J. W., Perdijk, E. W., Weijer, W. J., and Bloemhoff, W. (1988)Controlled peptide-protein conjugation by means of 3-nitro-2-pyridinesulfenylprotection-activation. Int. J. Pept. Protein Res. 32, 161-166. (16) Ponsati, B., Giralt, E., and Andreu, D. (1989)A synthetic strategy for simultaneous purification-conjugation of antigenic peptides. Anal. Biochem. 181,389-395. (17) Lindner, W., and Robey, F. A. (1987)Automated synthesis and use of N-chloroacetyl-modified peptides for the preparation of synthetic peptide polymers and peptide-protein immunogens. Int. J. Pept. Protein Res. 30,794-800. (18) Robey, F. A., and Fields, R. L. (1989)Automated synthesis of N-bromoacetyl-modified peptides for the preparation of synthetic peptide polymers, peptide-protein conjugates, and cyclic peptides. Anal. Biochem. 177,373-377. (19) Kolodny, N., and Robey, F. A. (1990) Conjugation of synthetic peptides to proteins: Quantitation from S-carboxymethylcysteine released upon acid hydrolysis. Anal. Biochem. 187,136-140. (20)Laemmli, U. K. (1970)Cleavage of structuralproteinsduring the assembly of the head of bacteriophage T4. Nature 227, 680-685. (21) Kalinowski, H.-O., Berger, S., and Braun, S. (1988)Carbon13 NMR Spectroscopy, John Wiley & Sons, New York. (22) Yamada, H., Uozumi,F., Ishikawa, A., and Imoto, T. (1984) Nature of the binding site around and reactivity of histidine15 in lysozyme. J. Biochem. 95,503-510. (23) Zaitsu, K., Ohnishi, M., Hosoya, H., Sugimoto, H., and Ohkura, Y. (1987)New heterobifunctional cross-linking reagents for protein conjugation, N-(bromoacetamiden-alkanoy1oxy)succinimides. Chem. Pharm. Bull. 35,1991-1997. (24) Merrifield, R. B. (1963)Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J.Am. Chem. SOC. 85,21492154. (25) Ruegg, U.Th., and Rudinger, J. (1977)Reductive cleavage of cystine disulfides with tributylphosphine. Methods Enzymol. 47, 111-116. (26)Borras-Cuesta,F., Fedon, Y., and Petit-Camurdan, A. (1988) Enhancement of peptide immunogenicity by linear polymerization. Eur. J. Immunol. 18,199-202. (27) Jemmerson, R., and Hutchinson, R. M. (1990)Fine manipulation of antibody affinity for synthetic epitopes by altering peptide structure: antibody binding to looped peptides. Eur. J. Immunol. 20,579-585.
