Synthetic Approaches to Multivalent Lipopeptide Dendrimers

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Bioconjugate Chem. 2003, 14, 144−152

Synthetic Approaches to Multivalent Lipopeptide Dendrimers Containing Cyclic Disulfide Epitopes of Foot-and-Mouth Disease Virus Eliandre de Oliveira,† Judit Villeń,† Ernest Giralt, and David Andreu*,† Department of Organic Chemistry, University of Barcelona, Barcelona, Spain. Received July 17, 2002; Revised Manuscript Received November 5, 2002

The synthesis of a multiantigenic peptide dendrimer incorporating four copies of a cyclic disulfide epitope has been undertaken. Since standard chemoselective ligation procedures involving thioether formation are inadvisable in the presence of a preformed disulfide, conjugation through a peptide bond between the lipidated branched lysine scaffold and a suitably protected version of the cyclic disulfide has been used instead. Several synthetic approaches to the partially protected cyclic disulfide peptide have been explored. The most effective involves building a minimally protected version of the peptide by Boc solid phase synthesis, using fluorenyl-based anchorings and cysteine protecting groups. Peptide-resin cleavage and cysteine deprotection/oxidation are performed simultaneously by basepromoted elimination. The cyclic disulfide epitope is readily obtained in sufficient amounts by this procedure and subsequently incorporated to the lipidated lysine core by peptide bond formation in solution. A final acid deprotection step in anhydrous HF yields a peptide construction containing a maximum of three copies of the cyclic disulfide epitope, the lower substitution being attributable to steric constraints. This immunogen has been successfully used in an experimental vaccination trial against foot-and-mouth disease virus.

INTRODUCTION

Interest in peptide-based vaccines remains high despite the limited number of practical applications reported to date (1-4). A particular concern in the design and production of peptide vaccines is defining an appropriate carrier/adjuvant system that enhances the generally reduced immunogenicity of free peptides (5). Conjugation of the peptide to a carrier protein (6) is an effective solution but suffers from drawbacks such as poor chemical definition, unwanted peptide or carrier modification during the coupling steps, or undesired response to the carrier (7). Other alternatives, such as encapsulation of the peptide antigen into liposomes (8) or conjugation to poly-lysine-based synthetic carriers (9), have been proposed. A particularly fruitful strategy has been the multiple antigenic peptide (MAP)1 system (10), a dendrimeric construction that displays four or eight copies of peptide on a branched lysine scaffold. The MAP concept has been further expanded by combining in a single molecular entity the antigen multiplicity provided by the MAP with the adjuvant effect of lipid moieties (11-13). This type of constructs are regarded as promising candidates for a new generation of fully synthetic, peptidebased vaccines. MAP immunogens have been prepared by either stepwise or convergent synthetic approaches. In the original procedure (10), the construct was synthesized entirely in the solid phase: the four (or eight) amino groups of a tri- (or hepta-) lysine core were used as starting points * Corresponding author. Address: Department of Experimental and Health Sciences, Pompeu Fabra University, Doctor Aiguader 80, E-08003 Barcelona, Spain. Phone/Fax: (34) 935 422 934. E-mail: [email protected]. † Present address: Department of Experimental and Health Sciences, Pompeu Fabra University, Doctor Aiguader 80, 08003 Barcelona, Spain.

for assembly of the immunogenic peptide sequence. The potential advantages of this approach, namely, simplicity and expediency, are usually offset by the difficulties of achieving quantitative couplings within a sterically crowded dendrimer network (14), which result in heterogeneus products. More recently, chemoselective ligation (15) strategies have been shown as convenient alternatives to this procedure. A typical approach involves suitable functionalization (e.g., haloacetylation) of the lysine core followed by conjugation in solution, e.g., SN2 reaction with the Cys thiol group of an independently synthesized, well characterized peptide (16-18), to give four (eight) copies of immunogen conjugated to the lysine core through thioether bonds. The serious economical impact of foot-and-mouth disease (19) has been acutely highlighted by the 2001 outbreak in the U.K. (20). Disadvantadges of the use of conventional vaccines against foot-and-mouth disease virus (FMDV), as well as considerable hurdles for new vaccine design, have been described (21). As a result, new 1 Abbreviations: AAA, amino acid analysis; Acm, acetamidomethyl; Ada, 2-aminodecanoic acid; DCM, dichoromethane; DIC, diisopropylcarbodiimide; DIEA, N,N′-diisopropylethylamine; DMAP, 4-(dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; EDT, 1,2-ethanedithiol; ES, electrospray; Fm, fluorenylmethyl; FMDV, foot-andmouth disease virus; HMFS, N-[(9-oxymethyl)-2-fluorenyl]succinamic acid; HMPB, 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid; HOBt, 1-hydroxybenzotriazole; HOOBt, 3,4dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine; MALDI-TOF, matrix-assisted laser desorption with time-of-flight detection; MAP, multiple antigenic peptide; MBHA, 4-methylbenzhydrylamine; MeCN, acetonitrile; MeOH, methanol; MS, mass spectrometry; NMP, N-methylpyrrolidone; PEG-PS, poly(ethylene glycol)polystyrene; TBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TEA, triethylamine; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TIS, triisopropylsilane; Trt, trityl; WSC, 1-ethyl-3(3′-dimethylaminopropyl)carbodiimide.

10.1021/bc025577f CCC: $25.00 © 2003 American Chemical Society Published on Web 12/04/2002

Multivalent Lipopeptide Dendrimers

Figure 1. Schematic view of multiantigenic peptide system consisting of four cyclic disulfide peptide epitopes (depicted as ribbons) conjugated to a lipidated core of (Lys)2-Lys-Ada2NH2.

efforts to develop FMDV vaccines requiring no handling or administration of virus particles are under way. Use of synthetic peptides reproducing relevant antigenic sites (22) is particularly attractive in the case of FMDV, given the relevance of the humoral response in the control of the disease (23). We have recently shown (24) that cyclic disulfide versions of the main antigenic site of FMDV (site A) provide excellent mimicry of the bioactive conformation, with a substantial (10-100-fold) improvement in antigenic recognition over the consensus sequence (YTASARGDLAHLTTT, residues 136-150 of viral protein 1 (VP1), isolate C-S8c1). We are seeking to develop these cyclic peptide antigens into practical vaccine candidates by combining them with the known immunogenic properties of lipo-MAP systems. Our target, a lipidated lysine core displaying four copies of the cyclic disulfide peptide (Figure 1), is substantially more complex from the synthetic point of view than any of the MAP constructs reported so far. In this case, the thioether-based ligation strategy described above is not advisable due to the incompatibility of a free thiol and an internal disulfide within the same molecule. In view of these difficulties, we have opted for a more elaborate approach, involving the conjugation, through a peptide bond, of the lipo-MAP core to a protected version of the cyclic disulfide peptide epitope. In this paper we analyze several approaches to this challenging synthetic goal and propose a feasible solution to the problem. EXPERIMENTAL SECTION

General. 2′-Chlorotrityl chloride resin and p-methylbenzhydrylamine resin were from Novabiochem. Poly(ethylene glycol)-polystyrene resin (0.31 mmol/g) was from Perseptive Biosystems (Framingham, MA). Protected (Boc and Fmoc) amino acids were from Bachem (Bubendorf, Switzerland), Neosystem (Strasbourg, France), or Novabiochem (Laüfelfingen, Switzerland). In the

Bioconjugate Chem., Vol. 14, No. 1, 2003 145

Fmoc-based synthesis, the side chain protections were Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Trt), Cys(Acm), Gln(Trt), Lys(Boc), and Ser(tBu). The Boc-based synthetic runs used Asp(OcHex), Cys(Fm), Lys(ClZ), and Ser(Bn). Peptide-synthesis grade DCM and DMF, and HPLCgrade acetonitrile were from Scharlau (Barcelona, Spain). 2-Aminodecanoic acid (Ada) was a gift from Prof. William Gibbons (School of Pharmacy, University of London). All other chemicals for peptide synthesis were of the highest quality available from Sigma-Aldrich (Madrid, Spain). Analytical HPLC was performed on Nucleosil C18 or C4 reverse-phase columns (4.6 × 250 mm, 5 µm particle size) on a Shimadzu system. Preparative HPLC runs were done on Vydac C8 column (20 × 250 mm, 10 µm particle size) columns in a Waters Delta Prep 4000 system. Semipreparative HPLC runs were performed on Vydac C4 (25 × 300 mm, 15-20 µm particle size). Amino acid analyses (AAA) of peptide hydrolysates (6 N HCl, 150 °C, 3 h) were run in a Beckmann 6300 autoanalyzer. MALDI-TOF and ES mass spectra were recorded in Voyager DE-RP (Applied Biosystems, Foster City, CA) and VG-Quattro (Micromass, U.K.) instruments, respectively. Lipidated Lysine Core. The branched Lys2-(Lys)Ada2-NH2 scaffold was synthesized manually by standard Boc chemistry protocols (25) on 0.2 mmol (286 mg) of MBHA resin. The two residues of Boc-Ada (0.4 mmol, 115 mg for each coupling, 2× molar excess) were followed by one, then (after deprotection of both R and amino groups) two residues of Boc-Lys(Boc) [0.6 mmol (208 mg) per residue, 3× molar excess], to give the tetravalent dendrimer core. Couplings were mediated by TBTU (equimolar to amino acid) and DIEA (2× molar excess relative to TBTU) in DMF for 1.5 h. The peptide-resin was deprotected at the N-termini with 40% TFA in DCM and then treated with HF/anisole (9:1 v/v, 0 °C, 1 h) to release the lipidated Lys core, which was eluted in 10% HOAc and lyophilized. Purification by preparative HPLC using a linear gradient of 10-50% MeCN in water (+0.05% TFA) over 180 min at a flow rate of 3 mL/min afforded 32.3 mg (22% yield) of the branched lipopeptide. Since two residues of racemic Ada had been used, four clustered peaks were observed by analytical HPLC, corresponding to the possible diastereomers of the product, each of them with the correct mass by MALDI-TOF MS (theoretical: 740.27 Da, found: 740.67 Da). Partially Protected Cyclic Disulfide Version of Site A Epitope Peptide. Four different synthetic approaches (Scheme 1) were attempted, as follows: Route A. The initial substitution (1-1.5 mmol/g) of 300 mg of commercial 2′-chlorotrityl chloride resin was lowered by coupling of a substoichiometrical amount of Fmoc-Ala (0.18 mmol, 56 mg, 0.5 equiv) in the presence of DIEA (1.8 mmol, 308.3 µL) in DCM for 1 h, followed by capping with MeOH (400 µL, 10 min). A new substitution of 0.38 mmol/g was determined by AAA. The remaining amino acids in the target sequence were incorporated by standard Fmoc protocols (26) in a Milligen 9050 synthesizer. Deprotections were done with 20% (v/v) piperidine in DMF (6 min, continuous flow), and couplings with 5 equiv (1.9 mmol) of Fmoc-amino acid and TBTU and 10 equiv of DIEA (3.8 mmol) in DMF for 60 min. After the target sequence was assembled, formation of the internal disulfide was attempted by two different procedures: (i) peptide-resin cleavage and selective deprotection of the two Cys(Trt) residues with simultaneous oxidation and (ii) cleavage and Cys(Trt) deprotection followed by oxidative treatment. In procedure i, 20 mg peptide-resin (ca. 2.5 µmol) were treated

146 Bioconjugate Chem., Vol. 14, No. 1, 2003

de Oliveira et al.

Scheme 1. Synthetic Approaches to Protected Cyclic Disulfide Epitope Peptidesa

a (i) I (10 eq) in DCM/TFE/HOAc (7:2:1); (ii) DCM/TFE/HOAc (7:2:1); (iii) I (10 eq); (iv) 0.1 M Hg(OAc) in DMF; (v) 2 2 2 β-mercaptoethanol/DMF (1:9); (vi) DCM/TFA (98:2); (vii) 0.1 M NH4HCO3, pH 8/DMSO (1:1); (viii) DMSO/NMP (2:8) or 20 mM TEA/ 20 mM CCl4 in NMP; (ix) piperidine/DMF (1:1).

with 500 µL of a DCM/TFE/HOAc (7:2:1) solution containing 25 µmol iodine (27) for 10 min at 25 °C. The resin was filtered and washed twice with 500 µL DCM/TFE/ HOAc (7:2:1), and the combined filtrates were stirred into 1.5 mL of a 0.1% solution of Na2S2O3‚5H2O in water until complete disappearance of the brownish color. The aqueous phase was washed with DCM, and the combined organic phases were briefly dried with anhydrous MgSO4 and evaporated to dryness under vacuum. In procedure ii, the same amount of peptide-resin was stirred with 500 µL of DCM/TFE/HOAc (7:2:1) for 15 min at 25 °C, filtered, and washed with 500 µL of the same reagent. The combined filtrates were then added dropwise to a solution of 25 µmol I2 in 1 mL DCM/TFE/HOAc (7:2:1), stirred for 10 min, and worked up as in procedure i.

Route B. Fmoc-Ala (0.75 mmol, 5 equivalent) was manually coupled (3 × 30 min) in the presence of DMAP (0.075 mmol, 0.5 equiv) and TBTU (0.75 mmol, 5 equiv) to a PEG-PS resin (0.22 mmol/g) functionalized with the HMPB (28) handle. A substitution of 0.13 mmol/g was determined by AAA. The remaining residues in the target sequence were incorporated by standard Fmoc protocols in a Milligen 9050 synthesizer as in approach A, except that Cys(Acm) (29) instead of Cys(Trt) was used. Recouplings of Arg138,145, Asn139,143, and Leu144 were performed. After chain assembly was completed, the Cys(Acm) residues were selectively deprotected by treatment with a 0.1 M solution of Hg(OAc)2 in DMF for 3 h in the dark (30). The resin was then washed with DMF (6 × 1 min) and treated with β-mercaptoethanol/DMF (1:9 v/v, 6 × 1



Figure 2. MALDI-TOF mass spectrum (A) and analytical HPLC (B) of the lipidated lysine core. The peak at 740.61 Da, which fits with the theoretical mass of Lys2-Lys-Ada2-NH2 (740.27 Da), is accompanied by its sodium (762.6 Da) and potassium (778.56 Da) adducts. The unusually broad HPLC peak is due to the diastereomeric mixture resulting from use of racemic Ada (see text). HPLC was performed on a C4 column eluted with a linear gradient of 10-60% B in 30 min, flow rate 1.0 mL/min.

Figure 3. Analytical HPLC of a fully deprotected version of the 21-residue peptide epitope prepared by route A. HPLC was performed on a C18 column eluted with a linear gradient of 10 to 60% B in 30 min, flow rate 1.0 mL/min.

min and overnight) to remove the mercuric salts from the peptide-resin and release the free thiols, which gave a positive (orange) Ellman (31) test. The protected peptide (in free dithiol form) was then cleaved from the resin by treatment with TFA/DCM (1:99 v/v, 4 ×10 mL, 30 s). The filtrates from each treatment were poured over 40 mL H2O, combined, concentrated on a rotary evaporator and lyophilized. The residue was dissolved in a 1:1 (v/v) solution of DMSO in 0.1 M NH4HCO3 (1:1), pH 8, to 49 µM peptide concentration, and the progress of the oxidation reaction was monitored by both HPLC and a qualitative Ellman test. For HPLC analysis, 100 µL aliquots of the oxidation solution were neutralized with HOAc, injected into a C4 column, and eluted with a linear 60-100% gradient of MeCN (+0.036% TFA) into water (+0.045% TFA) over 30 min at 1 mL/min flow rate. Oxidation was judged to be complete after 128 h. Route C. Peptide synthesis and Cys deprotection were performed as in approach B. After the removal of the mercuric salts, the oxidation step was carried out in the solid phase by treating the peptide-resin with either DMSO/NMP (2:8 v/v) or 20 mM carbon tetrachloridetriethylamine (CCl4/TEA) in NMP. In both cases, the peptide-resin gave a negative (faint yellow) qualitative

Ellman test after 24 h. The protected peptide was cleaved from the resin by treatment with TFA/DCM (2:98 v/v, 4 × 1 min) and analyzed by HPLC. The identity of the cyclic peptide was further confirmed by AAA and by ESMS after complete deprotection in TFA/H2O/TIS(95:2.5: 2.5; v/v/v) (theoretical, 2255.16 Da; found, 2253.35 Da). Route D. The substitution of 1 g of commercial MBHA resin (0.7 mmol/g) was reduced to 0.25 mmol/g by coupling of Boc-Gly (0.3 mmol, 52 mg) with DIC for 2 h in DCM. Next, the HMFS handle (32) (0.375 mmol, 79 mg) was coupled with DIC/HOBt (1:1:1 molar ratio) in DMF (overnight + 2 h recoupling).The first amino acid in the target sequence (Ala) was esterified to the polymer using two 60 min couplings with 5 eq each of Boc-Ala (1.25 mmol, 237 mg) in the presence of DIC (1.25 mmol) and DMAP (0.125 mmol) in DCM. The remaining residues in the target sequence were incorporated by a manual protocol that included (i) Boc deprotection (neat TFA, 1 + 10 min), (ii) washes (DCM, 6 × 1 min), (iii) neutralization (5% DIEA/DCM, 3 × 1 min), washes (DCM, 6 × 1 min), and (iv) single couplings with 5 eqiv of both Boc-amino acid and DIC in DCM; for Asn, Arg, and Gln residues that were incorporated without side chain protection, HOBt (5 equiv) was added to the coupling mixture and DCM/DMF (1:1 v/v) was used as solvent. Coupling reactions were run for 30-40 min, until a negative Kaiser ninhydrin test was obtained. Upon completion of chain assembly, the peptide resin was treated with 500 mL piperidine/DMF (1:1) to selectively deprotect/oxidize (33) the Cys(Fm) residues at positions 136 and 155 and simultaneously cleave the peptide from the resin. The oxidation/cyclization reaction was monitored by analytical HPLC of the filtrates and judged to be complete after 3 h. The filtrate was then cooled in an ice bath, acidified to pH 4 by dropwise glacial HOAc addition, and concentrated in a rotary evaporator to induce precipitation of salts, which were removed by filtration. The resulting solution was loaded onto a Vydac preparative C8 HPLC column (20 × 250 mm) equilibrated with 0.1% TFA in water (solvent A) in a Waters Delta Prep 4000 system. Purification was done by a linear gradient of 0-20% solvent B (0.1% TFA in MeCN) into A over 5 min followed by another linear gradient of 2070% B into A over 100 min, at a 25 mL/min flow rate.


de Oliveira et al.

0 °C, 1 h). After evaporation, the residue was dissolved in 1 M HOAc and lyophilized, and then filtered through a Sephadex G 25 column (300 × 10 mm) eluted with 0.1 M HOAc at 20 mL/h. Fractions shown by MALDI-TOF MS to contain the conjugation product were pooled and lyophilized. The resulting product was characterized by HPLC, AAA, and MALDI-TOF. RESULTS AND DISCUSSION

The goal of this work was to develop an efficient synthetic approach to a multivalent format of the previously described, antigenically optimized cyclic disulfide version of site A peptide from FMDV (24). For serotype O1BFS, the specific epitope was a 21-residue cyclic Figure 4. HPLC analysis of epitope peptide prepared according to route B, (A) with protected side chains but Cys in free dithiol form and (B) after oxidative treatment with DMSO in pH 8 buffer. HPLC was performed on a C4 column eluted with a linear gradient of 60-100% B in 30 min, flow rate 1.0 mL/min.

Figure 5. HPLC analysis of epitope peptide prepared according to route C, with protected side chains and free Cys (dithiol form) (A), and after solid-phase oxidation with either DMSO/NMP (2: 8) (B) or 20 mM CCl4/TEA in NMP (C). HPLC was performed on a C4 column eluted with a linear gradient of 60-100% B in 30 min, flow rate 1.0 mL/min.

Fractions judged to be homogeneous by analytical HPLC were pooled and lyophilized. The purified partially protected peptide was satisfactorily characterized by MALDI-TOF (theoretical, 2650.19 Da; found, 2644.42 Da) and AAA. Synthesis of the Branched Multiple Antigenic Peptide. The lipidated lysine core (0.05 µmol), the partially protected cyclic disulfide site A peptide (prepared by route D, 0.6 µmol, 12 equ), WSC, and HOOBt (0.6 µmol each) were dissolved in 100 µL of DMF and allowed to react for 48 h at 25 °C. The reaction mixture was diluted with water (10 mL) and lyophilized and the lipopeptide fully deprotected with HF/anisole (90:10,

disulfide (CSRNAVPNLRGDLQVLAQKCA) displayed 4-fold on a lipidated branched lysine core (Figure 1). Although this type of structure is conceptually identical to other MAP constructions, the need to incorporate several copies of a large cyclic disulfide (predictably bulkier and less flexible than a linear peptide chain of similar length) substantially increases its synthetic difficulty. To synthesize MAP type structures, the most reliable approaches involve ligation strategies, i.e., the lysine core and the epitope peptide, are synthesized independently and, after adequate purification and characterization, conjugated in solution. Such an approach has been shown to provide much better products fully stepwise solid phase schemes, where sterical crowding in the dendrimeric structure increases the risk of incomplete couplings and consequently heterogeneous products (34, 35). Thioetherbased ligation, a widely used approach for this type of constructs, is clearly unadvisable in the present case, since the integrity of any disulfide (four in our target structure) cannot be guaranteed in the presence of the free thiol required for thioether formation. This drawback forced us to consider as an alternative approach the ligation of the cyclic disulfide epitope to the lipidated MAP core by means of a peptide bond, i.e., linking a suitably protected version of the cyclic disulfide peptide through its C-terminus to the lipo-MAP scaffold. This relatively elaborated approach to disulfide-containing MAP constructions has, to our knowledge, not been previously attempted. In the present work, we have explored several potentially useful synthetic routes and settled for one which in our hands has turned out to be most efficient. A. Lipidated Lysine Core. The carrier structure chosen for our conjugate was a branched lysine scaffold bearing two residues of 2-aminodecanoic acid (Ada) (36) at its C-terminus. The synthesis of this lipo-MAP core was straightforward by standard Boc solid-phase methods. Since the lipoamino acid was used in racemic form, the final product obtained after HF cleavage was an almost equimolar mixture of the four possible diastereomers. We found it expedient to work with this mixture, after ensuring that each of the four peaks observed by HPLC (Figure 2) had a MALDI-TOF spectrum consistent with the desired structure. The amphipathic character of the MAP facilitated its purification by reverse phase HPLC using a simple H2O/MeCN gradient. B. Partially Protected Cyclic Disulfide Version of Site A Epitope Peptide. Several synthetic approaches (routes A-D, see Experimental Section) were explored to access the cyclic disulfide version of 21-residue FMDV site A peptide (O1BFS serotype) (C136SRNAVPNLRGDLQVLAQKCA156) in protected form;



Figure 6. HPLC analysis of epitope peptide prepared according to route D, after (A) 20 min and (B) 180 min cleavage and oxidation with piperidine/DMF (1:1). Peaks 1-3 are, respectively, a non-peptide byproduct, the dithiol form of the peptide, and the expected cyclic disulfide, which after preparative HPLC was obtained in highly homogeneous form (C). HPLC was performed on a C4 column eluted with a linear gradient of 5-95% B in 35 min (A and B) or 35-65% B in 30 min (C), flow rate 1.0 mL/min.

Figure 7. MALDI-TOF mass spectrum of the lipidated lysine core conjugated with the cyclic disulfide epitope peptide. The peak at 7558 Da is consistent with a conjugate consisting of three copies of the epitope bound to the Lys2-Lys-Ada2-NH2 core (theoretical mass 7570 Da).

three approaches were based on Fmoc chemistry (Scheme 1, routes A-C) and one on Boc chemistry (route D). Route A. In our first attempt to synthesize the site A cyclic disulfide in protected form, we opted for a maximal protection scheme relying on the 2′-chlorotrytyl chloride resin and trityl-type protection groups, which allowed (i) selective (iodine-mediated) Cys(Trt) deprotection, (ii) disulfide formation, and (iii) peptide-resin cleavage in a single operation (27). An initial synthesis was planned for a 20- (not 21-) residue target, namely, the above sequence lacking the Ala156 residue. In this case, the first amino acid coupled to the 2′-chlorotrityl chloride resin was Fmoc-Cys(Trt). A very low incorporation of this amino acid occurred (detectable by the weak absorbance of the Fm-piperidine adduct solution obtained upon deprotection), probably due to steric conflict between the Trt groups of both solid support and Cys side chain. Once chain assembly was complete, treatment with DCM/TFE/ HOAc (7:2:1) containing iodine (10 equivalent) was expected to achieve peptide-resin cleavage, Cys deprotection, and disulfide formation in a single step. However, MALDI-TOF or ES MS analysis of the resulting product showed no peaks assignable to the target sequence or related species. Since low solubility might be expected for the protected peptide and eventually complicate its detection, a small amount of peptide resin was cleaved and fully deprotected by means of reagent R (TFA/ thioanisole/EDT/anisole; 90:5:3:2) (37). Again, no peaks consistent with the expected structure could be detected in the cleavage mixture. A new synthetic attempt was done with a one-residue longer peptide as target, i.e., the sterically nonconflicting, native Ala156 anchored (0.38 mmol/g) to 2′-chlorotrityl

chloride resin instead of Cys155. Again, analysis of the fully deprotected (reagent R) crude by HPLC and MALDITOF (Figure 3) showed insatisfactory results: only traces of the expected peptide, plus a number of peaks attributable to single deletions (Arg, Leu, or Asn) were detected. Thus, despite its well-documented usefulness for the preparation of protected peptides, 2′-chlorotrityl chloride resin was in our hands inadequate to synthesize this particular peptide. Route B. Our next attempt to synthesize the 21-mer used PEG-PS as solid support, functionalized with the HMPB (28) handle. Since this handle is less acid-labile than 2′-chlorotrityl chloride resin, we opted for the Acm group as Cys side chain protection. In view of past difficulties, double couplings were done for Arg138,145, Asn139,143, and Leu144. To evaluate the quality of the synthetic product, a small aliquot was fully deprotected and cleaved, showing a rather homogeneous HPLC profile with the expected molecular weight by MALDITOF MS (expected 2255.2; found 2256.2). Once the presence of the correct sequence on the resin was assured, we performed selective solid-phase deprotection of the thiol group with 0.1 M Hg(OAc)2 in DMF. Mercury salts were removed from the polymer by extensive treatment with β-mercaptoethanol/DMF (1:9), and then the peptide-resin bond was cleaved with TFA/DCM (1:99) to give the partially protected peptide in reasonably pure dithiol form (Figure 4A), ready for oxidation in solution. Unfortunately, this protected precursor was totally insoluble in aqueous solution, even at the high dilution (49 µM) required to avoid aggregation and intermolecular disulfide bond formation. To improve its solubility we resorted to DMSO, a denaturing cosolvent known for its


added advantage of promoting thiol-disulfide oxidation (38, 39). In our case, the protected peptide could be dissolved in an oxidation buffer containing ca. 40% DMSO, but HPLC monitoring of the reaction showed a complex oxidation mixture (Figure 4B). In addition, and as described by other authors (40), we experienced severe difficulties in removing DMSO, either by reverse-phase HPLC or by selective adsorption of the peptide (carrying several aromatic side chain protections) on an aromaticbinding Diaion column (41). Since our efforts to devise efficient, preparative scale purification conditions were unsuccessful, this synthetic approach was finally abandoned. Route C. Oxidation of free bis-thiol precursors in the solid phase, i.e., while the partially protected peptide remains anchored to the polymeric support, is a wellknown strategy for disulfide formation (42-47). Use of a low-substitution resin creates pseudo-dilution conditions that favor intramolecular cyclization (48). Other obvious additional advantages are the facile removal of oxidizing agents by washing and filtration, and the fact that the solubility behavior of the peptide is not an issue. On the down side, the efficiency of solid phase cyclizations tends to depend strongly on the distance between the thiol groups. Though no systematic studies have been performed, not many cycles larger than 18 amino acids have been described (49). A number of Cys protecting groups, oxidizing agents, and conditions have been described for this type of strategy, both for Fmoc [e.g., Trt/iodine (50), Acm/iodine (51), Acm/thallium trifluoroacetate (52), etc.] and for Boc-based [Fm/piperidine (33)] synthetic chemistries. Our attempt of on-resin oxidation/cyclization used the same anchoring and protection scheme as those in route B. The Cys(Acm) residues were also deprotected with Hg(OAc)2, and after removal of Hg(II) salts, the resin-bound bis-thiol was treated with a mild oxidation mixture [NMP/DMSO (8:2)] (to our knowledge not previously reported) or, for comparison, with a well-known solidphase oxidation reagent, 20 mM CCl4/TEA in NMP (53). The reactions were monitored by qualitative Ellman test (31), which after 24 h gave only a very faint yellow coloration (vs strong orange color for the non-oxidized peptide resin). As shown in Figure 5, analytical HPLC profiles of small aliquots of both peptide-resins after full deprotection and cleavage (reagent R) show much cleaner materials than those obtained in solution (approach B). Difficulties, however, appeared when cleavage of the peptide in protected form from the solid support was attempted. Standard cleavage conditions (TFA/DCM (2: 98, v/v) were ineffectual. Higher percentages of TFA (5: 95 and 10:90, v/v) improved only slightly the cleavage yield but were associated to complex HPLC profiles indicative of undesired partial deprotection of side chains. Due to these difficulties, this initially promising scheme had also to be abandoned. Route D. In view of the shortcomings of the previous Fmoc-based synthetic schemes, a rather different approach was explored, on the basis of (i) Boc chemistry for chain elongation, (ii) minimalist protection scheme (Asn, Arg, and Gln side chains unblocked) to favor solubility of the partially protected cyclic disulfide epitope during coupling to the lipo-MAP core, and (iii) base-labile, fluorenyl-based chemistries for both Cys protection (Fm) and peptide anchoring to the solid support (HMFS handle, 54), which allow deprotection/oxidation and peptide-resin cleavage in a simultaneous step, under conditions orthogonal with other (acid-labile) protecting groups. A potential further advantage of Boc chemistry, vis-a`-

de Oliveira et al.

vis the difficulties experienced in building up the epitope sequence using Fmoc chemistry in routes A-C, would be a reduced risk of aggregation (and thus of ensuing incomplete couplings) by the use of TFA, an excellent disaggregating agent, at the repetitive NR-deprotection steps. In practice, this last approach turned out to be quite satisfactory. Thus, a single treatment of the peptideresin with piperidine readily produced the peptide epitope in (partially protected) cyclic disulfide form. An unusually large solvent-to-resin ratio was used at this step, to achieve high dilution conditions favoring the formation of the internal disulfide over other undesirable Cys pairings. Neutralization of the large amount of piperidine in the cleavage/oxidation mixture slightly complicated the work up: after filtering off the initially formed piperidine acetate, recurrent precipitation of this salt upon concentrating the solution in the rotary evaporator made several additional filtration steps necessary. This inconvenience aside, the procedure furnished in adequate purity and yield a correctly oxidized, minimally protected cyclic disulfide peptide epitope, which was further purified to satisfactory specifications by preparative HPLC (Figure 6). C. Conjugation of the Cyclic Disulfide Epitope to the Lipidated MAP Core. The purified, partially protected cyclic disulfide version of antigenic site A of FMDV prepared by route D above was incorporated to the tetravalent lipo-MAP by peptide bond formation between the C-terminal carboxyl of the epitope peptide and the amino groups of the lipidated lysine core. A 3-fold excess of peptide over each amino group (12 equiv in total) was used, with activation by carbodiimide (watersoluble) and HOOBt in DMF over 48 h. When HPLC monitoring of this conjugation reaction showed no significant progress (48 h at 25 °C), the reaction mixture was evaporated and submitted to acidolysis with anhydrous HF to remove the protecting groups remaining on the Ser, Asp, and Lys side chains of the disulfide epitope (see Figure 6). The resulting lipopeptide was shown by MALDI-TOF MS (Figure 7) to be predominantly the trivalent version of the conjugate (theoretical, 7565.1 Da; found, 7570.4 Da). The fact that no peak corresponding to the tetravalent conjugate could be detected in the mass spectra, together with the aforementioned lack of progress of the conjugation reaction after 48 h, led us to the temporary conclusion that the tetravalent version of the conjugate was not readily accessible, perhaps due to the predictably substantial steric crowding caused by the cyclic disulfide. This point is currently under investigation. Despite the less-than-optimal result in this particular case, the synthetic approach based on route D is in our opinion the most straightforward and reliable procedure for preparing dendrimeric conjugates of cyclic disulfide epitopes. The dendrimeric, lipidated, cyclic disulfide version of antigenic site A of FMDV which has been the object of the present study has been preliminarly evaluated as a vaccine candidate with very promising results (De Oliveira et al., manuscript in preparation). Thus, guinea pigs immunized with 500 µg of the trivalent conjugate and boosted with another 500 µg after 27 days elicited antibodies with neutralization titers comparable to control animals immunized with conventional (guinea pigadapted) FMDV vaccine. When challenged (experimentally infected) with live virus, two out of three animals turned out to be protected (vs three out of three for the conventional vaccine). We look forward to using this type


of conjugate, in combination with other replicas of FMDV antigenic sites, in future synthetic vaccine trials. ACKNOWLEDGMENT

Support from Generalitat de Catalunya (CERBA) and the Ministry of Education and Science (DGICYT Grants PB97-0873 and BIO99-0484), Spain, and from the European Union (Grant FAIR5-CT97-3577) is gratefully acknowledged. J.V. is a predoctoral fellow from DURSI (Generalitat de Catalunya, Spain). LITERATURE CITED (1) Arnon, R., and Horwitz, R. J. (1992) Synthetic peptides as vaccines. Curr. Opin. Immunol. 4, 449-453. (2) Ben Yedidia, T., and Arnon, R. (1997) Design of peptide and polypeptide vaccines. Curr. Opin. Biotechnol. 8, 442-448. (3) Patarroyo, M. E., Amador, R., Clavijo, P., Moreno, A., Guzmań, F., Romero, P., Tascoń, R, Franco, A., Murillo, L. A., Pontoń, G., and Trujillo, G. (1988) A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodium falciparum malaria. Nature 332, 158-161. (4) Meloen, R. H., Casal, J. I., Dalsgaard, K., and Langeveld, J. P. M. (1995) Synthetic peptide vaccines: success at last. Vaccine 13, 885-886, and references therein. (5) Van Regenmortel, M. H. V., and Muller, S. (1999) Immunization with peptides. In Synthetic Peptides as Antigens (Pillai, S., and Van der Vliet, Eds.) pp 136-167, Elsevier, Amsterdam. (6) Mu¨ller, G. M., Shapiro, M., and Arnon, R. (1982) Antiinfluenza response achieved by immunization with a synthetic conjugate. Proc. Natl. Acad. Sci. U.S.A. 79, 569-573. (7) Schutze, M. P., Deriaud, E., Przewlocki, G., and Leclerc, C. (1989) Carrier-induced epitopic suppression is initiated through clonal dominance. J. Immunol. 142, 2635-2640. (8) Pinto´, R. M., Gonza´lez-Dankaart, J. F., Sańchez, G., Guix, S., Go´mara, M. J., Garcıá, M., Haro, I., and Bosch, A. (1998) Enhancement of the immunogenicity of a synthetic peptide bearing a VP3 epitope of hepatitis A virus. FEBS Lett. 438, 106-110. (9) Mezo¨, G., Mezo¨, I., Pimm, M. V., Kajta´r, J., Sepro¨di, A., Teplań, I., Kovaćs, M., Vincze, B., Pa´lyi, I., Idei, M., Szekerke, M., and Hudecz, F. (1996) Synthesis, conformation, biodistribution, and hormone-related in-vitro antitumor activity of a gonadotropin-releasing hormone antagonist-branched polypeptide conjugate. Bioconjugate Chem. 7, 642-650. (10) 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. (11) Defoort, J.-P., Nardelli, B., Huang, W., Ho, D. D., and Tam, J. P. (1992) Macromolecular assemblage in the design of a synthetic AIDS vaccine. Proc. Natl. Acad. Sci. U.S.A. 89, 3879-3883. (12) Beekman, N. C. J. M., Schaaper, W. M. M., Turkstra, J. A., and Meloen, R. H. (1999) Highly immunogenic and fully synthetic peptide-carrier constructs targetting GNRH. Vaccine 17, 2043-2050. (13) Go´mara, M. J., Riedemann, S., Vega, I., Ibarra, H., Ercilla, G., and Haro, I. (2000) Use of linear and multiple antigenic peptides in the immunodiagnosis of acute hepatitis A virus infection. J. Immunol. Methods 234, 23-34. (14) See, however, Keah, H. H., Kecorius, E., and Hearn, M. T. W. (1988) J. Pept. Res. 51, 2-8, for a possible partial solution. (15) Schnolzer, M., and Kent, S. B. H. (1992) Constructing proteins by dovetailing unprotected synthetic peptides. Backbone-engineered HIV protease. Science 256, 221-225. (16) Lu, Y.-A., Clavijo, P., Galantino, M., Shen, Z.-Y., Liu, W., and Tam, J. P. (1991). Chemically unambiguous peptide immunogen preparation. Orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system. Mol. Immunol. 28, 623-630. (17) Zeng, W., Ghosh, S., Macris, M., Pagnon, J., and Jackson, D. C. (2001) Assembly of synthetic peptide vaccines by chemoselective ligation of epitopes: influence of different chemical linkages and epitope orientations on biological

Bioconjugate Chem., Vol. 14, No. 1, 2003 151 activity. Vaccine 19, 3843-3852. Cavallaro, V., Thompson, P. and Hearn, M. T. W. (2001) Solid-phase synthesis of a dendritic peptide related to a retinoblastoma protein fragment utilizing a combined Boc- and Fmoc-chemistry approach. J. Pept. Sci. 7, 262-269. (18) Cavallaro, V., Thompson, P. and Hearn, M. T. W. (2001) Solid-phase synthesis of a dendritic peptide related to a retinoblastoma protein fragment utilizing a combined Bocand Fmoc-chemistry approach. J. Pept. Sci. 7, 262-269. (19) Pereira, H. G. (1981) Foot-and-mouth disease. In Virus Diseases of Food Animals, Vol. 2, (Gibbs, E. P. J., Ed.) pp 333-336, Academic Press, New York. (20) Sobrino, F., and Domingo, E. (2001) Foot-and-mouth disease in Europe. EMBO Rep. 2, 459-461. (21) Brown, F. (1992) New approach to vaccination against footand-mouth disease. Vaccine 10, 1022-1026. (22) Bittle, J. L., Houghten, R. A., Alexander, H., Sutcliffe, J. G., Lerner, R. A., Rowlands, D. J., and Brown, F. (1982) Protection against foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence. Nature 298, 30-33. (23) McCullogh, K. C., De Simone, F., Brocchi, E., Capucci, L., Crowther, J. R., and Kihm, O. (1992) Protective immune response against foot-and-mouth disease. J. Virol. 66, 18351840. (24) Valero, M. L., Camarero, J. A., Haack, T., Mateu, M. G., Domingo, E., Giralt, E., and Andreu, D. (2000) Nativelike cyclic peptide models of viral antigenic site: finding a balance between rigidity and flexibility. J. Mol. Recognit. 13, 5-13. (25) Stewart, J. M., and Young, J. D. (1984) Solid-Phase Peptide Synthesis, pp 18-47, Pierce Chemical Co., Rockford, Illinois. (26) Fields, G. B., Tian, Z. and Barany, G. (1982) In Synthetic Peptides: A User’s Guide (Grant, G. A., Ed.) pp 77-183, Freeman, New York. (27) Barlos, K., Gatos, D., Kutsogianni, S., Papaphotiuo, G., Poulos, C., and Tsegenidis, T. (1991) Solid phase synthesis of partially protected and free peptides containing disulfide bonds by simultaneous cysteine oxidation-release from 2-chlorotrityl resin. Int. J. Pept. Protein Res. 38, 562-568. (28) Flo¨rsheimer, A., and Riniker, B. (1991) Solid-phase synthesis of peptides with the highly acid-sensitive HMPB linker. Peptides 1990. In Proceedings of the 21st European Peptide Symposium, Platja d’Aro, Spain, 1990 (Giralt, E., and Andreu, D., Eds.), pp 131-133, Escom, Leiden. (29) Veber, D. F., Milkowski, J. D., Varga, S. L., Denkewalter, R. G., and Hirschman, R. (1972) Acetamidomethyl, a novel thiol protecting group for cysteine. J. Am. Chem. Soc. 94, 5456-5461. (30) Liu, W.; Shiue, G. H., and Tam, J. P. (1989) A novel strategy for the desprotection of S-acetamidomethyl containing peptides: an approach to the efficient synthesis of endothelin. Peptides: Chemistry, structure and biology. Proceedings of the 11th American Peptide Symposium, San Diego, 1989 (Rivier, J. E., and Marshall, G. R., Eds) pp 271-271, Escom, Leiden. (31) Ellman, G. L. (1958) A colorimetric method for determining low concentrations of mercaptans. Arc. Biochem. Biophys. 74, 443-450. (32) Rabanal, F., Giralt, E., and Albericio, F. (1995) Synthesis and applications of a new base-labile fluorene derived linker for solid-phase peptide synthesis. Tetrahedron 51, 1449-1458. (33) Albericio, F., Hammer, R. P., Garcıá-Echeverrıá, C., Molins, M. A., Chang, J. L., Munson, M. L., Pons, M., Giralt, E., and Barany, G. (1991) Cyclization of disulfide-cointaining peptides in solid-phase synthesis. Int. J. Pept. Protein Res. 37, 402413. (34) Dubois, P., and Baleux, F. (1992) Novel version of multiple antigenic peptide allowing incorporation on a cysteine fuctionalized lysine tree. Int. J. Pept. Protein Res. 40, 7-12. (35) Liu, C. F., and Tam, J. P. (1994) Chemical ligation approach to form a peptide bond between unprotected peptide segments. Concept and model study. J. Am. Chem. Soc. 116, 4149-4153. (36) Gibbons, W. A. (1990) Synthesis, resolution and structural elucidation of lipidic amino acids and their homo- and heterooligomers. Liebigs Ann. Chem. 1175-1183.

152 Bioconjugate Chem., Vol. 14, No. 1, 2003 (37) Albericio, F., Kneib-Cordonier, N., Biancalana, S., Gera, L., Masada, R. I., Hudson, D., and Barany, G. (1990) Preparation and application of the 5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxyphenoxy)-valeric acid (PAL) handle for the solid-phase synthesis of C-terminal peptide amides under mild conditions. J. Org. Chem. 55, 3730-3743 (38) Wallace, T. J., and Mahon, J. J. (1965) Reactions of thiols with sulfoxides. III. Catalysis by acids and bases. J. Org. Chem. 30, 1502-1506. (39) Tam, J. P., Wu, C. R., Liu, W., and Zhang, J. W. (1991) Disulfide bond formation in peptides by dimethyl sulfoxide. Scope and applications. J. Am. Chem. Soc. 113, 6657-6662. (40) Munson, M. C., and Barany, G. (1993) Synthesis of a´-conotoxin SI, a bicyclic tridecapeptide amide with two disulfide bridges: Illustration of novel protection schemes and oxidation strategies. J. Am. Chem. Soc. 115, 10203-10210. (41) Vijayakumar, E. K. S., Roy, K., Chatterjee, S., Deshmukh, S. K., and Ganguli, B. N. (1996). Arthrichitin. A new cell wall active metabolite from Arthrinium phaeospermum. J. Org. Chem. 61, 19, 6591-93. (42) Eritja, R., Ziehler-Martin, J. P.,. Walker, P. A., Lee, T. D., Legesse, K., Albericio, F., and. Kaplan, B. E. (1987) On the use of S-tert-butylsulphenyl group for protection of cysteine in solid-phase peptide synthesis using Fmoc-amino acids. Tetrahedron 43, 2675-2680. Ponsati, B., Giralt, E., and Andreu, D. (1990) Solid-phase approaches to regiospecific double disulfide formation. Application to a fragment of bovine pituitary peptide. Tetrahedron 46, 8255-8266 (f). See also refs 32, 38, and 42. (43) Garcıá-Echeverrıá, C., Albericio, F., and Pons, M. (1989) Convenient synthesis of a cyclic peptide disulfide: A type II β-turn structural model. Tetrahedron Lett. 30, 2441-2444. (44) Ponsati, B., Giralt, E., and Andreu, D. (1990) Solid-phase approaches to regiospecific double disulfide formation. Application to a fragment of bovine pituitary peptide. Tetrahedron 46, 8255-8266 (45) Shih, H. (1993)New approaches to the synthesis of cystine peptides using N-iodosuccinimide in the construction of disulfide bridges. J. Org. Chem. 58, 3003-3008

de Oliveira et al. (46) Nishino, N., Mihara, H., Izumi, N., Fujimoto, T, Ando, S., and Ohba, M. (1993) Disulfide cyclization of protected peptide assembled on oxime resin. Tetrahedron Lett. 34, 1295-1298. (47) See also refs 33, 40, and 49. (48) Andreu, D., Albericio, F., Sole´, N. A., Munson, M. C., Ferrer, M., and Barany, G. (1994). Formation of disulfide bonds in synthetic peptides and proteins. In Methods in Molecular Biology 35: Peptide Synthesis Protocols. (Pennington, M. W., and Dunn, B. M., Eds.) pp 105-109. Humana Press, Totowa, NJ. (49) Camarero, J. A., Giralt, E., and Andreu, D. (1995) Cyclization of a large disulfide peptide in the solid phase. Tetrahedron Lett 36, 1137-1140. (50) Kamber, B., Hartmann, A.; Eiler, K., Riniker, B.; Rink, H., Sieber, P., and Rittel, W. (1980) The synthesis of cystine peptides by iodine oxidation of S-trityl-cysteine and Sacetamidomethyl cysteine peptides. Helv. Chim. Acta 63, 899-915. (51) Ruiz-Gayo, M., Albericio, F., Royo, M., Garcıá-Echevarrıá, C., Pedroso, E., Pons, M., and Giralt, E. (1989) A convenient procedure for synthesis of cystine peptides via iodine oxidation of S-acetamidomethyl-cysteine peptides. An. Quim. 85C, 116-118. (52) Fujii, N., Otaka, A., Funakoshi, S., Bessho, K., Watanabe, T., Akaji, K., and Yajima, H. (1987) Studies on peptides. CLI. Syntheses of cystine-peptides by oxidation of S-protected cysteine-peptides with thallium (III) trifluoacetate. Chem. Pharm. Bull. 35, 2339-2347. (53) Wenschuh, E., Heydenreich, M., Runge, R., and Fisher, S. (1989) CCl4 as mildes Oxidans in der Schwefelchemie: Oxidation von Thiolen zu Disulfiden. Sulfur Lett. 8, 251-260. (54) Nishiuchi, Y., Nishio, H., Inui, T., Bodi, J., and Kimura, T. (2000) Combined solid-phase and solution approach for the synthesis of large peptides or proteins. J. Pept. Sci. 6, 84-93.

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