Synthesis and construction of a novel multiple peptide conjugate system: strategy for a subunit vaccine design

Peptides 21 (2000) 9 –17

Synthesis and construction of a novel multiple peptide conjugate system: strategy for a subunit vaccine design Robert A. Boykinsa, Manju Joshia, Chaing Syinb, Subhash Dhawanc,*, Hira Nakhasia,1 a

Laboratory of Parasitic Biology and Biochemistry, Division of Allergenic Products and Parasitology, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20892, USA b Division of Blood Applications, Center For Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20892, USA c Immunopathogenesis Section, Laboratory of Molecular Virology, Division of Emerging Transfusion Transmitted Diseases, Center For Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20892, USA Received 8 September 1999; accepted 24 October 1999

Abstract We describe the design and synthesis of a novel well characterized multi-peptide conjugate (MPC) system containing antigens from human malaria parasite and the Tat protein of HIV type-1 (HIV-1-Tat). Construction of the MPC utilizes Fmoc solid-phase peptide synthesis coupled with solution chemistry. In the first phase, a core template that serves as primary anchor for the synthesis and attachment of multiple antigens is synthesized. Serine(trityl) and multiple lysine branches with epsilon groups blocked during chain assembly are incorporated forming a tetrameric core. Cysteine whose side chain thiol serves to couple haloacetyl or S-protected haloacetyl peptides is added to complete assembly of the core template. Modification to the coupling solvent, addition of key amino acid derivatives (N-[1-hydroxy-4methoxybenzyl]) in the peptide sequence allows the synthesis of base peptides on the core template with molecular mass greater than 7500 kDa. Base peptides are then reacted with high performance liquid chromatography purified haloacetyl peptides to generate multiple peptide conjugates with molecular masses of 10 to 13 kDa. MPC constructs thus formed are further characterized by matrix assisted laser desorption-time of flight mass spectroscopy (MALDI-MS), amino acid analysis, size exclusion chromatography, and SDS-polyacrylamide gel electrophoresis (PAGE). To our knowledge, this is the first report describing a chemically well defined multiple conjugate system with potential for development of synthetic subunit vaccines. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Linear peptide: Peptide sequence with N-terminal haloacetyl; Base peptide: Peptide sequence synthesized on core template

1. Introduction Most traditional vaccines have been prepared by either using an attenuated version of a pathogenic organism, by inactivation of specific organisms (pertussis, typhoid, polio), an entity of the organism such as cell wall components of organism or polysaccharide of Haemophilus, Meningococcus [10,31]. These approaches have produced many highly effective vaccines. Although these vaccines have not been produced without difficulties, namely the presence of possible adventitious agents, reversion to virulence in the * Corresponding author. Tel.: ⫹1-301-827-0796; fax: ⫹1-301-4807928. E-mail address: [email protected] (S. Dhawan) 1 Co-corresponding author. E-mail address: [email protected] (H. Nakhasi)

case of viruses, basic complexity in strain properties, achieving complete inactivation (i.e. inactivated vaccines), the possibility of cross-reactivity with host antigens. An alternative strategy to conventional vaccines has been to identify epitopes and immunogens that are responsible for a specific response and use the synthetic peptide of the immunogenic epitopes as a vaccine [21]. Such synthetic molecules are devoid of many of the unwanted risks as compared to those derived from human pathogens. In addition, synthesis of peptides with lipophilic or glycosidic functional groups can allow improve delivery and aid in targeting to antigen presenting cells. It is generally thought that the conformational B-cell epitopes involved in neutralization of some organisms would be difficult to mimic by the use of a simple synthetic linear molecule [1,32]. One very practical approach to address this issue has been described by Tam and others in

0196-9781/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 0 ) 0 0 1 7 2 - 2

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providing a high density of peptide epitope and a three dimensional conformation in the peptide structure called the multiple antigen peptide (MAP) [1] [8,23–25]. However, the classic solid phase synthesis methodology employed in the traditional MAP is not without difficulty. Amino acid sequence of the desired peptide, overall length of the molecule and yield of the desired product play a crucial role in obtaining enough well defined material for vaccine studies. Problems exist in microheterogenity [9,11] in the synthetic product making isolation and purification extremely laborious and in many cases somewhat impractical to obtain a reasonable amount of material for use. In the present report, we have successfully overcome some of the major obstacles in the synthesis and construction of highly complex and well defined multimeric peptide molecules consisting of antigens from malaria and HIV-Tat protein sequences that could be useful for vaccine studies.

2. Methods 2.1. Synthesis of core template The core template was synthesized on an ABI Model 430 peptide synthesizer by using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry [2,20] mediated by 2-[1-H-Benzotriazole-1-yl]-1.13.3-tetramethyluronium hexafluorophosphate/ 1-hydroxybenzotriazole (HBTU/HOBt) activation on the Rink Amide[4,2⬘,4⬘Dimethoxyphenyl-Fmoc-aminomethyl] phenoxyacetamido-norleucyl-MBHA resin (0.45 mmol/g resin substitution) (Novabiochem, La Jolla, CA, USA) at the 0.25 mmol scale for synthesis. Bulk amino acids derivatives were purchased from Novabiochem and 1.0 mmol of each desired residue was used initially in the coupling reaction. Double couple or recoupling required two or more millimole per residue. For the core template, amino acids were incorporated with the following side chain protection: Lys(Fmoc), Lys(Boc), Ser(Trt), and Cys(Trt). Side chain protection used in all other peptides were: Glu(Otbu), His(Trt), Asp(OtBu), Cys(Trt), Lys(Boc), Asn(Trt), Arg(Pmc), Ser(tBu), Cys(tButhio), Lys(Fmoc), Val(Fmoc Hmb), Leu(Fmoc Hmb), Gln(Trt), Trp(Boc). 2.2. Synthesis of base peptides The base peptides consisting of antigens from malaria and HIV-Tat protein were synthesized following characterization of the core template. These base peptides were subsequently used to prepare four multi-peptide conjugates (MPCs). N-[2-hydroxy-4-methoxybenzyl] (Hmb) derivatives were incorporated at specific residues (see Fig. 1) to reduce potential aggregation in the growing peptide chains [12,13,26,28,29]. The coupling time for a single coupling was 1 h. Coupling efficiencies were monitored using the Kaiser or trinitrobenzene sulfonic acid (TNBS) test after completion of each coupling step to ensure greater than 99%

Fig. 1. Amino acid sequence of various functional peptides from malaria and HIV-Tat protein. (a) Letters shown in bold and underlined represent Hmb derivatized amino acids for malaria peptides; (b) letters shown in bold show the position of the seven cysteine(tButhio) residues in the HIV-Tat peptide (HIV-1-Tat-1).

completion. Double couple/recouple cycles were introduced when necessary. Cycles that failed to achieve at least 99% after a double/recouple cycle were capped with 2 mmol benzoic anhydride to terminate any unreacted amines. An antigen from Plasmodium falciparum (LSA-1) [7] previously identified as T3 was used to construct the base peptide T3. The T3-CSP base peptide was synthesized using T3 base peptide as its template following removal of 1/2 of the T3 peptidyl resin from the synthesizer reaction flask after deprotection thus allowing the synthesis to be continued with an additional eight residues added per branch of the T3 construct. Both T3 and T3CSP base peptides were subjected to cleavage by acidolysis and purification. The HIV-Tat base peptide Tat-2-Tat-3 was obtained by direct solid phase synthesis under similar conditions as described at the 0.25 mmol scale followed by cleavage and purification. Mass spectral analysis was performed on both base peptides to confirm agreement with theoretical mass for each species. 2.3. Synthesis of linear haloacetyl peptide (malaria) The peptides were synthesized as described using standard ABI Fmoc cycles with a final cycle added for modification of the N-terminal amino group with a haloacetyl group [30]. Briefly, 1.0 mmol N-bromoacetyl-NHS ester, Cl2Ac2O or Br2Ac2O that is prepared by dissolving 1.0 mmol BrCH3COOH in dichloromethane (DCM) to which 0.5 mmol N,N[prime]-dicylohexylcarbodiimide (DCC) in dichloromethane (DCM) is added with mixing. The reaction was carried out for 30 min at room temperature and the

R.A. Boykins et al. / Peptides 21 (2000) 9 –17

solution is filtered to remove the dicyclohexyl urea (DCU) precipitate formed. The activated amino acid residue [active ester/anhydride] was then added to the reaction vessel containing the resin bound deprotected peptide and allowed to couple for 1 h at room temperature. Coupling of the anhydride was carried out in DCM. Following synthesis, peptides were cleaved from the resin according to a specific cleavage protocol described [5] followed by purification by reverse phase-high performance liquid chromatography (RP-HPLC). The peptides were lyophilized and stored at ⫺70°C until needed. 2.4. Synthesis of linear [Cys-tButhio] haloacetyl peptide (HIV-1-Tat) The HIV-Tat [4] peptides were synthesized essentially the same way as the malaria peptides, except that all cysteine sulfhydryl residues of Tat-1 peptide were protected with the tButhio blocking group. Chloroacetylation of the amino terminal end of the peptide was chosen specifically for the Tat peptide to minimize reactivity with other highly reactive side chains present in both the base and linear peptides during final assembly of the Tat MPC. Cleavage of this molecule produced a stable N-chloroacetyl peptide whose side chain thiol groups remained blocked throughout the purification. Subsequently, the haloacetyl linear peptide Tat-1 was coupled to the base peptide Tat-2-Tat-3. The sulfhydryl protection was released following coupling of the haloacetyl peptide to the previously deprotected cysteine residues on the base peptide. Deprotection was accomplished with an excess of tri-n-butylphosphine (Bu3P) reagent as used earlier in the assembly process. 2.5. Peptide cleavage Following deprotection of the N-terminal amino group, the base peptides were cleaved with a cocktail of trifluoroacetic acid (TFA), water, ethanedithiol (EDT), triisopropylsilane (TIS), phenol, thioanisole (88:4:2:1:1:4). Following cleavage, base peptides were precipitated and washed in cold methyl Tert-butyl Ether. Before further processing, peptides were dissolved in water or 0.1% TFA or acetic acid followed by the addition of 1.0 ml ␤-mercaptoethanol. For the haloacetyl peptides, the cleavage cocktail consisted of TFA, water, phenol (90:5:5). Thiol scavengers were not used because thiol compounds could react with the haloacetyl moiety as well as could result in the deprotection of the Cys (tButhio) in the Tat linear peptide. 2.6. Peptide purification Both linear and base peptides were purified by RP-HPLC using 6.8 ⫻ 25 cm Vydac C4, C8, or C18 columns (The Separation Group, Hesperia, CA, USA) with a gradient of 0.1% TFA/H20 and 0.1% TFA/acetonitrile. Following HPLC purification, the acetonitrile was removed by rotary

11

evaporator and the peptides were lyophilized and stored at ⫺20o or ⫺70°C (haloacetyl peptides) until needed. Peptide identities were established by matrix assisted laser desorption-time (MALDI-MS) mass spectrometric analysis. The homogeneity of each base peptide was assessed by Edman sequence and quantitative amino acid composition analyses to determine specific molar ratio for each residue. 2.7. Coupling of base construct and linear haloacetyl (malaria) peptide Into a 50 ml Teflon flask fitted with a nitrogen tube, the base peptide was dissolved in 0.5 M NaHCO3 or 0.1–1M Tris/guandine-HCl pH 8.0 buffer as needed. A solution of Bu3P in 1-propanol or triscarboxyethylphosphine (TCEP) was freshly prepared. The haloacetyl peptides were dissolved in 1 to 2 ml (0 – 6 M) guandinine-HCl and added to the reaction flask under nitrogen. For example, to 10 mg base peptide T3 (1.7 ␮m) 0.6 ␮m reducing agent was added for reduction of the base cysteinyl residues. The solution was placed on a stirrer for 1 h followed by the addition of 0.8 mg (0.29 ␮m) haloacetyl peptide (T1) to the reaction mixture. The coupling reaction was then carried out for 1.5 to 3 h at 25°C for a bromoacetyl peptide and up to 6 h when using the chloroacetyl derivative. The reaction mixture was monitored with MALDI-MS for the presence of conjugate material. ␤-mercaptoethanol (0.2 ml) was then added to the reaction flask. The conjugate mixture was then desalted by RP-HPLC and further purified by size exclusion chromatography to isolate the desired multiple peptide conjugate. 2.8. Coupling of base peptide and linear (sulfhydryl protected) haloacetyl peptide (HIV-1-Tat) HIV-Tat peptide (Tat-1) peptide contains seven protected cysteine residues. A similar strategy, as previously described, was used to couple the N-chloroacetyl modified HIV-Tat peptide to the base peptide Tat-2-Tat-3. The base peptide was dissolved in 1M Tris/6 M guanidine HCl buffer, pH 8.0. A fivefold excess (based on thiol content) of Bu3P in 1-propanol was added to effect reduction of the base cysteine residues. Essentially, to 10 mg (1.4 ␮m) of the base peptide Tat-2-Tat-3, 0.25 ␮m Bu3P reagent was added. Following reduction of the cysteinyl residues in the base molecule, 1.6 mg (0.5 ␮m) chloroacetyl peptide was dissolved in 6 M guanidine-HCl and added to the reaction mixture. The solution was adjusted to 20% (v/v) with 1-propanol to increase solubility of the Tat haloacetyl peptide, and allowed to proceed 2 to 6 h under nitrogen. Reaction mix was again monitored qualitatively by MALDI-MS by observing the presence of a signal consistent with the expected mass of the desired MPC. Following conjugation of the base and haloacetyl peptide, excess Bu3P (1.9 ␮mol) was added for final deprotection of the cysteine residues. The deprotection was carried out for 2 h at 25°C. The

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conjugate mixture was then desalted by RP-HPLC as described below. 2.9. Desalting of conjugate mix The peptide conjugate mixtures were desalted by RPHPLC using a C18 column (10 ␮, 19 mm ⫻ 150 mm) Waters, Milford, MA, USA, by using a one step gradient of 0.1% TFA/H2O and 10% acetonitrile/H2O to elute the low molecular weight salts followed by 70% acetonitrile to elute the MPC or any unconjugated peptides. The conjugated mixtures were then lyophilized and stored at ⫺20°C. 2.10. Size exclusion chromatography Size exclusion chromatography was performed using a Diol-S5 column (YMC, Wilmington, NC, USA) to isolate the desired MPC peptide from the reaction mixture. The elution buffer consisted of 0.1 M phosphate buffer, 0.2 M NaCl, pH 7.0, containing 0.02 M sodium azide. The column was standardized using a GPC standard molecular weight mixture obtained from Bio Rad (Hercules, CA, USA). 2.11. Mass spectrometry Mass spectrometry was used to identify and/or confirm the molecular mass of the various peptide species. Mass analysis was performed by using a Voyager DE-RP MALDI-TOF mass spectrometer (Perseptive Biosystems, San Jose, CA, USA). The ionization matrix for the analysis was carried out in 10 mg Sinapinic acid/␣-cyano-4-hydroxy cinnamic acid (␣-CHCA) dissolved in 50% acetonitrile/ 0.1% TFA. All mass analyses were performed in the positive ion mode. 2.12. Edman sequence analysis The amino acid sequence and the relative degree of purity of the base peptides were established by Edman sequence analysis on an Applied Biosystems Model 494A sequencer, Foster City, CA, USA. 2.13. SDS-PAGE The MPCs were subjected to SDS-PAGE using 10 to 20% Tricine gels (Novex, San Diego, CA, USA) under reducing conditions. After completion of electrophoresis, gels were stained with Commassie blue to visualize the position of the MPCs.

3. Results The present report describes a novel synthesis and construction of the MPC. Using solid phase synthesis coupled with conventional solution chemistry, we have successfully

Fig. 2. Flow chart for synthesis and construction of the MPC.

produced MPC molecules that are chemically well defined. These MPCs were generated using various functional peptides from malaria and the Tat protein of human immunodeficiency virus type-1 (Fig. 1). Our results indicate that this system can be universally adapted to produce a well characterized MPC molecule virtually from any known protein sequence. To our knowledge, this is the first report describing synthesis for a homogeneous multiple antigenic peptide system that has remained a challenge for many years. A step by step strategy used to prepare a multiple peptide conjugate is shown in Fig. 2. As shown, solid phase peptide synthesis followed by thiol coupling chemistry was utilized to synthesize and construct the MPC molecules. The key component of the MPC system involves the solid phase synthesis of a core template that serves as primary anchor for the synthesis of a base peptide and subsequent attachment of additional peptides through specific site directed coupling (Fig. 3). Initially, Serine (Trt) representing the C terminal of the MPC is attached to the solid support followed by the sequential addition of multiple branched lysine residues forming a tetrameric core whose epsilon amino groups remain blocked throughout the synthesis process (Fig. 3a). Cysteine whose side chain thiol group serves to form a stable thioether in the final assembly of the MPC is

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Fig. 4. Mass spectroscopy analysis by MALDI-MS of the base peptides comprising of T3 (a), T3-CSP (b), and Tat-2-Tat-3 (c), respectively.

Fig. 3. (a) Structure of the core template; (b) structure of the base peptide, shown with lysine branching and SPPS carried out at alternate amino positions of the multiple branched lysines; (c) MPC.

then coupled to the dual branched lysines forming a completed core template. The second step of assembly involves the synthesis of a base peptide consisting of one or more antigens (Fig. 3b). By using the strategies described, the base peptide constructs were synthesized from various peptides listed in Fig. 1. Peptides were then deprotected and cleaved from the resin. The crude peptides were purified by RP-HPLC. Fractions containing the desired peptide as determined by mass spectral analysis, were pooled and lyophilized. Identities of the base peptides were established by MALDI-MS. The mass analysis of base peptides T3, T3CSP, and Tat-2-Tat-3 is shown in Fig. 4. The observed molecular ion for each construct was found to be consistent with that obtained from the theoretical sequence (Table 1). The identities were further confirmed by performing quantitative amino acid analysis [17] and Edman sequence analysis to estimate homogeneity in the base constructs. The molar ratio of each amino acid was found to be consistent

with that of the theoretical values (Table 2). In the final phase of the assembly, with identities established by mass spectral and amino acid analyses, HPLC purified linear or S-protected haloacetyl peptides comprising one or more functional epitopes were then coupled to the cysteine thiol groups of the base peptide as described above to complete the assembly of the MPC molecule (see Fig. 3c). The MPC, thus formed was further characterized by mass spectroscopy, size exclusion chromatography, and SDS-gel electroTable 1 Mass spectral analysis of base peptides and multiple peptide conjugates MPC

T3 (base peptide) T3-CSP (base peptide) Tat-2-Tat-3 (base peptide) T1-T3 T1-CSP-T3 T3-MSP Tat-1-Tat-2-Tat-3

Mass [MH⫹] Theoretical

Observed

5930.56 7515.15 7303.65 11 139.24 12 723.73 10 615.03 11 997.35

5931 7513 7306 11 144 12 737 10 711 12 460

Theoretical mass reported for the tetrameric MPC molecules.

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Table 2 Amino acid composition of the base peptides Amino Acid

Asx Thr Ser Glx Pro Gly Ala Cysb Val Met Ile Leu Tyr Phe His Lys Trp Arg

a

Table 4 Relative degree of conjugation Experimental

Theoretical

Haloacetyl peptide

T3-CSP

Tat-2-Tat-3

T3-CSP

Tat-2-Tat-3

9 — 4 7 2 1 2 2 — — 2 3 — — — 5 — —

1 1 4 6 4 1 2 2 — — 1 — — — 4 3 1 4

8.7

0.8 0.7 3.5 5.7 3.6 1.2 2.0 1.8

3.6 6.8 2.0 0.8 2.0 1.8

1.5 2.7

4.7

0.8

3.7 2.8 0.8 3.8

Theoretical ⫽ peptide plus Core molecule. Cysteine determined as cysteic acid.

a

b

T1 MSP-1 Tat-1 T1

⫹

Base peptide

mole CMC/mole MPC

T3 T3 Tat-2-Tat-3 T3-CSP

1.5 1.8 1.4 1.5

Calculations based on determination of carboxymethylcysteine (CMC) after acid hydrolysis of conjugated peptide by amino acid analysis.

and T1-CSP-T3 were observed to be 10 kDa, 12 kDa, 13 kDa and that of the HIV-1-Tat MPC (Tat-1-Tat-2-Tat-3) to be 12 kDa, respectively (Fig. 6). Overall purity of the tetrameric MPC molecules were greater than 80% with the absolute percent dependent upon isolation and removal by size exclusion chromatography (SEC) of the base and linear peptides used as reactants. Analysis of the final MPC molecules by mass spectroscopy and SDS-gel electrophoresis indicates the presence of material corresponding to a trimeric species. Analysis of these data suggest the presence of a trimer may be the result of incomplete coupling to the thiol groups on the core molecule. In all the MPCs reported

phoresis. The yields of the various base constructs and the efficiency of conjugation are summarized in Table 3 and Table 4, respectively. As shown in Table 4, the carboxymethylcysteine (CMC) values derived from acid hydrolysis of the MPC were somewhat less than predicted. However, these results clearly indicate that the extent of conjugation was nearly complete. Under ideal conditions, 2 mole of CMC/mole of MPC should be released during hydrolysis of the conjugate molecule. Fig. 5 illustrates representative profiles of two MPC molecules analyzed by MALDI-MS. Mass analysis of the T1-T3 construct produces an ion signal centered at 11,144 m/z (Fig. 5a). Analysis of the T1-CSP-T3 produces a similar ion cluster centered at 12,737 m/z (Fig. 5b and Table 1). The level of purity for each MPC was determined by analytical reverse phase HPLC on a Vydac C18, 5 ␮ column. Fig. 5c illustrates a typical chromatogram obtained from such analysis. Molecular weight of the MPCs was determined by SDSPAGE under reducing conditions. The apparent molecular weights of malaria peptide conjugates T1-T3, T1-MSP-1, Table 3 Summary of yields for the base peptides Base peptide

T3 T3-CSP Tat-2-Tat-3

Yield (mg) Crude

HPLC purified

680 845 1750

258 302 490

All yields calculated from acid hydrolysis of protein content as determined by amino acid analysis.

Fig. 5. Representative profiles of two MPC molecules analyzed by MALDI-MS. (a) MALDI-MS analysis of T1-T3 MPC; (b) T1-CSP-T3 MPC; and (c) Analytical RP-HPLC of the T1-CSP-T3 MPC on C18 column using an acetonitrile/TFA gradient from 0 to 60%.

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Fig. 6. SDS-PAGE of the MPCs. Coomassie blue staining of 10 to 20% Novex Tricine polyacrylamide gel loaded with HPLC-purified MPC. The molecular weight markers are unstained Novex Mark 12 standards.

here, the trimer represents approximately 20% relative to the tetramer.

4. Discussion Synthesis and construction of the MPC can provide a useful system to be used as synthetic vaccine candidates that may require several functionally antigenic peptides [33]. For a number of years, several attempts by a number of laboratories have been made to produce a model system containing multiple functional epitopes of a protein. However, due to lack of purity and low yields of the final product, successful development of such molecules has been hindered [9,16]. Although tremendous advances have occurred in solid phase peptide chemistry since its inception more than 30 years ago, there still remain limitations to the successful synthesis in reasonable yields of well characterized complex peptide molecules encompassing multiple antigens. In the present study, we have attempted to overcome a majority of limitations associated with the synthesis and construction of such a well characterized, multi-epitope peptide. We have utilized both solid phase and solution chemistry to construct the MPC. By selective coupling to alternate lysine branches of a tetrameric core molecule we have synthesized and obtained highly purified base peptides up to 7500 kDa by the solid phase approach, which provides sufficient yields and quantity for the final solution coupling step with an HPLC purified haloacetyl peptide.

There are many questions that must be addressed, especially when synthesizing multiple peptide sequences with many reactive sites within the molecule by the solid phase peptide synthesis (SPPS) approach. Aggregation and steric hindrance of the growing peptide chain in SPPS is thought to be a major factor for microheterogeniety found in the traditional multiple antigen peptide by the direct synthesis approach [9,25]. Many of these problems are thought to be sequence related possibly caused by inter and intra-chain hydrogen bonding by the peptide backbone forming beta sheets or other secondary structures [14]. This may lead to massive steric hindrance, thus reducing the acylation or deprotection reactions or both [22]. It has been reported that protected peptide bonds (i.e. containing a tertiary nitrogen) prevent hydrogen bonding thought to be the primary cause of sequence related problems [12]. Other groups have shown that peptide aggregation may occur as early as the fifth residue being dependent upon peptide sequence and side chain blocking groups [3]. Therefore, for completion of a successful synthesis, a general rule has to be applied: “the peptides chains must always be fully extended and the solid support must always be completely solvated.” To minimize these possible deleterious effects during chain assembly, we added 10% of DMSO to the N-methylpyrrolidone (NMP) solvent and incorporated several Hmb amino acids throughout the peptide chain. Furthermore, incorporation of a serine residue as the C-terminal amino acid with its side chain protected by a trityl group allow ease of removal and further modification, if desired, before cleavage from the solid

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support. The side chain of this residue could be useful in the attachment of one or more antigens, adjuvant or other component. Assembly of a tetrameric core template and coupling to alternate lysine residues with the epilson position remaining blocked is believed to provide greater access to the resin bound amino groups during the acylation and/or deprotection reactions, thus ensuring a higher degree of coupling efficiency at each cycle especially when coupling residues with bulky side chains. In the synthesis of all base peptides we obtained greater than 99% coupling yield for each residue as determined by Kaiser or TNBS assay. We have therefore demonstrated that a carefully designed synthesis strategy for the base peptide antigen is crucial in obtaining characterizable species in high yield after conjugation to the linear molecule. In this report the peptide length is limited to 31 residues per branch on the base peptide. However, based on the results of the present study, and previously published studies [5,6] we believe that a peptide length of 50 or more residues per branch could be achieved with similar levels of purity and yield. These studies are currently in progress in our laboratory. Reactions involving nucleophilic substitution of alkyl halides have been well documented by others [18,19]. This chemistry has been used extensively in the conjugation of proteins and peptides that result in the formation of stable thioether bonds [15]. In the final malaria MPCs, we observe by SDS gel electrophoresis, a faint lower band consistent with the molecular weight of a trimeric species (i.e. the base peptide and one linear peptide coupled) present in the mixture with the desired tetramer molecule. Mass analysis by MALDI-MS and electrospray ionization mass spectroscopy (ESI-MS) also confirms this finding. In the case of Tat MPC, similar observations were made. However, the average yield of the MPC constructs was greater than 70% tetramer. We believe the presence of additional species could be the result of limited solubility of the reactants or steric effects thereby limiting accessibility to the thiol group and therefore reducing conjugation. In this study, we have utilized both Tris and bicarbonate buffers in addition to guanidine hydrochloride to carry out the thiol coupling step. We conclude that the choice of buffer for solubility of the base and linear peptides is crucial in the optimization of the conditions for the thiol reaction. Measurement of the CMC ratio was performed to determine the degree of conjugation [15]. Our results indicate the presence of slightly less than 2 moles CMC/mole of each MPC construct. Under ideal conditions 2 moles of CMC/ mole of conjugate should be liberated upon acid hydrolysis. Among the many factors that may inhibit production of the tetramer molecule are solubility of the peptides and steric hindrance. Some of these factors could contribute to variable yields that, in addition, could be dependent upon the physical parameters of the individual species. To facilitate the solubility of the chloroacetyl Tat peptide [Tat-1], we added increasing amounts of 1-propanol. We also found that in the case of the more hydrophobic peptides, 1 M Tris/6 M

guanidine-HCl was required to minimize the formation of the trimeric MPC. Specifically, for the MPC containing the Tat-3 peptide, where the overall yield was somewhat lower. The lower yield of the Tat MPC could likely be attributed to the release of a small number of thiol blocking groups [tButhio] in the peptide sequence, following addition of the haloacetyl peptide into the reaction mixture containing the base peptide Tat-2-Tat-3. The release could occur as a result of the left over Bu3P used in the initial reduction step. Our data suggest that poor solubility of the Tat MPC is a factor in the SEC analysis to isolate only the MPC and remove the unreacted starting peptides (base and linear peptide). Therefore, the Tat MPC has higher levels of these peptides that may be a factor in suppression of the ionization of the MPC when analyzed by MALDI-MS. However, these low intensity signals observed seem to be consistent with the expected molecular mass indicating the presence of both tetramer and trimer species (data not shown). In summary, our strategy for synthesis of an MPC has several advantages over other published methodologies. These include: 1) synthesis of a well defined core template molecule that minimizes steric hindrance by coupling to only two branches per synthetic cycle instead of the traditional four, or eight, as described by others [8,23,24,27]; 2) core molecule spacing is similar to that used in the synthesis of a tetramer. However, in the case of the MPC, only two residues are assembled per cycle at alternate ␣ amino groups on the tetrameric core with the opposite ⑀ positions remaining protected throughout the synthesis of the base molecule; 3) incorporation of a serine residue with a trityl group used as side chain protection that can be modified on the solid phase for greater functionality without cleavage of the peptide resin link. The addition of this residue at the C terminal with an acid labile side chain could be useful in the attachment of antigens or an adjuvant molecule; 4) synthesis of a base molecule with specific thiol sites for attachment of additional peptides; 5) site directed coupling for the linear peptide; 6) modification to coupling solvent N-methylpyrollidone by adding 10% DMSO to improve solubility of peptide chains thereby enhancing the acylation and/or deprotection reaction to improve the coupling efficiency; 7) addition of the Hmb protected amino acids at specific points in the peptide chain to further minimize aggregation of the growing peptide chains thereby minimizing the risk of lower yields in the desired peptide; 8) use of the tButhio side chain blocking group on all cysteinyl residues in peptide sequence, the N-terminal amino group of which is modified with a haloacetyl group; 9) selective deprotection of all internal cysteinyl residues only after formation of the final multiple peptide conjugate; 10) analytical characterization of the core template, base and final MPC molecules; 11) immunologic recognition of antigens used in the MPC; and 12) additional side chain modification to the lysine and cysteine residues on the core template leading to an alternate use of the core template functionality in the replace-

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ment of the Boc group at the branched ⑀ lysine position by a quasi-orthogonally protected lysine derivatives such as Fmoc-Lys(Dde) or Fmoc-Lys(ivDde)-OH. The use of such derivatives allow selective deprotection with dilute hydrazine or other similar amines without cleavage from the solid support. The use of Lys(Mtt) or Lys(Tfa) allows selective deprotection with dilute acid solutions. Similarly, the use of Cys(Mmt)-OH as a replacement for the current Cys(Trt) allows the selective deprotection of the thiol with dilute acid thereby enabling the sulfhydryl to be readily exploited to produce a thioether linkage or other reactions involving the thiol. In essence, the basic core molecule described in this paper can provide increased versatility in the use of multiple strategies for the covalent attachment of additional peptide antigens, adjuvant component, or other desirable functionalities. Thus, the methodology described in this report may provide strategies for producing well characterized multiple peptide conjugates useful in the development of a subunit vaccine in significant yields for experimental and potential commercial applications. Acknowledgments M. B. Joshi is supported by a postdoctoral fellowship from the Oak Ridge Institute for Science and Education. References [1] Arnon R, Horwitz RJ. Synthetic peptides as vaccines. Curr Opinion Immunol 1992;4:449 –53. [2] Atherton E, Sheppard RC. Solid-phase peptide synthesis. Oxford: IRL Press, 1989. [3] Bedford J, Hyde C, Johnson T, Jun W, Owen D, Quibell M, Shepard RC. Amino acid structure and difficult sequences in solid-phase peptide synthesis. Int J Peptide Protein Res 1992;40:300 –7. [4] Boykins RA, Mahieux R, Shankavaram UT, et al. A short peptide domain from HIV-1-Tat protein mediates pathogenesis. J Immunol 1999;163:15–20. [5] Boykins R, Oravecz T, Unsworth E, Syin C. Chemical synthesis and characterization of chemokine RANTES and its analogues. Cytokine 1999;11:8 –15. [6] Clark–Lewis I, Moser B, Walz A, Baggiolini M, Scott GJ, Aebersold R. Chemical synthesis, purification and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8 (IL-8)) and neutrophil activation peptide 2. Biochem 1991;30:3128 – 35. [7] Connelly M, King CL, Bucci K, et al. T-cell immunity to peptide epitopes of liver stage antigen-1 in an area of Papua New Guinea in which malaria is holoendemic. Infection and Immunity 1997;65: 5082–7. [8] Drijfhout JW, Bloemhoff W, Poolman JT, Hoogerhout P. Solid-phase and applications of N-(S-acetylmercaptoacetyl) peptides. 1990;187: 349 –54. [9] Drijfhout JW, Bloemhoff W. A new synthetic functionalized antigen carrier. Int J Peptide Protein Res 1991;37:27–32. [10] Frasch CE. Vaccines for the prevention of meningococcal disease. Clin Microbiol Rev 1989;2(suppl.)134 – 8. [11] Grant GA, Crankshaw MW, Gorka J. Edman sequencing as tool for characterization of synthetic peptides. Methods in Enzymology 1997; 289:395– 419.

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