Bioconjugate Chem. 1996, 7, 552−556
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Natural Peptides as Building Blocks for the Synthesis of Large Protein-like Molecules with Hydrazone and Oxime Linkages Keith Rose,*,† Weiguang Zeng,†,‡ Pierre-Olivier Regamey,† Igor V. Chernushevich,§ Kenneth G. Standing,§ and Hubert F. Gaertner† Department of Medical Biochemistry, University Medical Center, CH 1211 Geneva 4, Switzerland, Gryphon Sciences, Suite 90, 250 East Grand Avenue, South San Francisco, California 94080-3606, and Department of Physics, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Received April 8, 1996X
Methods are known for the production of synthetic protein-like molecules of nonlinear architecture with molecular masses in the 10-20 kDa range. To synthesize such compounds of higher molecular mass and complexity, chemoselective ligation of natural (as opposed to synthetic) peptide building blocks was studied. In preliminary experiments with model peptides, conditions for the formation of peptide oximes were investigated, and their stability at alkaline pH was examined, to resolve a literature controversy. It was found that low pH (down to 2.1) was suitable for polyoxime formation and that the oxime bond was stable for up to 65 h at pH 8 and for more than 2 h at pH 9. Then, using natural peptides, it was found to be possible to synthesize, and characterize by mass spectrometry, nine-component species with molecular masses >48 kDa. This is about twice the size of homogeneous artificial proteins previously described. Such complex molecules of defined structure are beginning to find applications as vaccine candidates, as radioimmunodiagnostic agents, and as nonviral gene therapy delivery vehicles.
INTRODUCTION
Unprotected peptides, in contrast to fully protected peptides, are generally soluble in aqueous media and easily purified and may be used as building blocks for the synthesis of fairly complex protein-like molecules of high molecular mass (1, 2). Thioether, disulfide, thioester, hydrazone, oxime, thiazolidine, and amide linkages have all been employed for such syntheses, which generally involve the spontaneous ligation of fragments made chemically and lead to well-defined products of branched structure. Linear products involving single or multiple condensations are accessible through chemical (3-5) or enzymic approaches (6). Until recently, the largest synthetic protein-like molecules made and fully characterized by mass spectrometry have been of branched architecture and have molecular masses in the region of 20-25 kDa (7-9). Rationally synthesized protein-like molecules, as opposed to well-known cross-linked hapten-carrier protein preparations, are beginning to show promise as vaccine candidates against viruses (10, 11) and parasites (malaria, E. Nardin, private communication) and as nonviral gene therapy delivery vehicles (D. Thatcher, private communication). This led us to try to incorporate not just relatively short synthetic peptides but also native proteins with a function, e.g., a tissue-targeting or immunostimulatory role. Our first hybrid consisted of cholera toxin B subunit, which is a mucosal-targeting protein of molecular mass of 11 600 Da, and a synthetic carrier molecule (12) to which five copies of a short synthetic peptide were attached, giving a product with a molecular mass of 20 550 Da (9). To discover how to make and work with larger and even more complex molecules, we first examined oxime formation as a function of pH. There is some controversy in this area in the literature. Early work with hydra* Author to whom correspondence should be addressed. † University Medical Center. ‡ Gryphon Sciences. § University of Manitoba. X Abstract published in Advance ACS Abstracts, July 1, 1996.
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zones in aqueous solution (13) had established that the rate of hydrazone formation increased with decreasing pH in the range 3-5 and that an equilibrium was reached which depended particularly on pH (lower yield at lower pH) and on concentration (lower yield at lower concentration). More recent work (1) threw doubt on the earlier study and reported that the reaction rate in aqueous solution increased with increasing pH in the range 4.2-5.7 for the formation of both peptide hydrazones and peptide oximes. Since yields must be optimized when working in dilute solution with native proteins, we first studied this aspect with synthetic peptides. A second important concern is product stability. It has been reported (1) that peptide oximes, while stable in acidic and neutral medium (1, 7), are unstable at pH 9. We therefore exposed peptide oximes to aqueous conditions in the range pH 7-10. Once these model studies had been completed, we went on to synthesize polyhydrazones and polyoximes of higher molecular mass using native proteins or protein fragments as building blocks. An insulin tetrahydrazone, octahydrazone, tetraoxime, and octaoxime (molecular masses of 24, 25, 48, and 48 kDa, respectively) and an F(ab′)3 trioxime of molecular mass 152 450 Da were made and characterized by mass spectrometry. The interesting biological properties of the F(ab′)3 have been reported elsewhere (14). This ability to extend the size range of accessible synthetic protein-like molecules augurs well for the fields of vaccination (10) and radioimmunotherapy (14) and also nonviral gene therapy, where it is necessary to bring together several peptides each with a separate function, such as cell targeting, DNA binding, and nuclear location (15). MATERIALS AND METHODS
Synthesis of Linear Peptides and Templates. Templates carrying four and eight aldehyde groups (Figure 1) were made manually according to standard techniques of peptide chemistry starting with Fmoc1 -Tyr(tBu)-Sasrin resin (Bachem). After removal of the Fmoc group with 50% piperidine in DMF for 15 min, the resinbound amino groups were acylated for 1 h with Fmoc© 1996 American Chemical Society
Polyoxime Artificial Proteins
Figure 1. Reaction scheme representing oxime formation between the octaaldehyde template (left) and aminooxyacetylinsulin to give the octaoxime. The branching Lys residues of the template are acylated on both their alpha and epsilon amino groups and are shown (together with the Tyr residue) unconventionally with their carboxyl groups to the right, for clarity. A tetraaldehyde template, which had one fewer rounds of Lys branching, was also made and used.
Lys(Fmoc) activated with TBTU/HOBT/DIEA, Fmocdeprotected then reacylated with Fmoc-Lys(Fmoc) on both alpha and epsilon amino groups, to give a resin with four protected amino groups. Acylation of these four amino groups (after Fmoc deprotection) with Boc-Ser(tBu) in similar fashion yielded the tetravalent template, whereas another round of acylation with Fmoc-Lys(Fmoc) prior to coupling of Boc-Ser(tBu) yielded the octavalent template in protected form. The templates were deprotected and cleaved from the resin by vortexing with a mixture consisting of TFA (85%) and thioanisole (15%) for 1 h at room temperature and then precipitating and washing with cold ether. The precipitate was taken up in water, filtered to remove resin, lyophilized, taken up again in water, and purified by HPLC. Pure tetravalent and octavalent templates were characterized by electrospray ionization mass spectrometry (ESI-MS) on the Geneva quadrupole instrument as described earlier (7): found 914.1 and 1776.3, calcd 914.0 and 1775.0, respectively. HPLC was performed using techniques and equipment that has previously been described (7). Linear peptides were synthesized according to standard automated techniques as previously described (7, 12). The R-melanocyte stimulating hormone (MSH) analog NH2-OCH2CO-Nle-Asp-His-(D-Phe)-Arg-Trp-LysNH2 has been described previously (16). The tripeptide Ser-Leu-Leu was oxidized with periodate under standard conditions (7) to give glyoxylyl-Leu-Leu, which was isolated by HPLC. All synthetic peptides and templates 1 Abbreviations: DMF, dimethylformamide; Fmoc, fluorenylmethyloxycarbonyl; TBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; HOBT, 1-hydroxybenzotriazole; DIEA, N,N-diisopropylethylamine; Boc, tert-butyloxycarbonyl; TFA, trifluoroacetic acid; HPLC, reversed phase highpressure liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; OSu, N-hydroxysuccinimide ester; MSH, R-melanocyte stimulating hormone; Msc, methylsulfonylethyloxycarbonyl.
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were single components on HPLC and had the expected mass spectrum when analyzed by ESI-MS. Des-AlaB30-insulinylcarbohydrazide, Des-AlaB30insulinyl-Phe-NHCH2CH2NH-COCH2ONH2, and AminooxyacetylB1-insulin. Porcine insulin, monocomponent grade, was purchased from Novo-Nordisk, Bagsvaerd, Denmark. Des-AlaB30-insulinylcarbohydrazide was prepared as previously described (12). Des-AlaB30-insulinylPhe-NHCH2CH2NH-COCH2ONH2 was prepared similarly by reverse proteolysis in 80% butane-1,4-diol using Phe-NHCH2CH2NH-COCH2ONH2 as nucleophile [itself prepared by acylation of Boc-NHOCH2CO-NH-CH2CH2NH2 (14) with Boc-Phe-OSu followed by deprotection with TFA]. Product was isolated by HPLC (yield 60%) and characterized by ESI-MS: found mass 5968.9 Da, calcd 5968.8 Da. AminooxyacetylB1-insulin was prepared according to standard techniques (17). Briefly, A1,B29-diMsc-insulin was prepared and acylated quantitatively with Boc-aminooxyacetyl-OSu in DMF. After isolation by HPLC, the Msc groups were quantitatively removed (17) and the product was recovered in Boc-protected form again by HPLC and characterized by ESI-MS on the Geneva quadrupole instrument (7): found mass 5948.1 Da, calcd 5950.6 Da. Just prior to use, the required amount of powder was quantitatively Boc-deprotected by dissolving in neat TFA (10 mg/mL) and incubating for 20 min at room temperature followed by removal of TFA under a stream of nitrogen. The deprotected protein was purified by HPLC, lyophilized to a fluffy white powder, and characterized by ESI-MS in Geneva: found mass 5852.7 Da; calcd 5850.6 Da. Creation of Aldehyde Groups on the Templates. The templates in their Ser form, prepared as above, were oxidized with periodate (7, 12). For this, template (200 µL, 15 mM in water for tetravalent template, 7.5 mM for octavalent, i.e. 12 µmol terminal serine) was diluted with 7.8 mL of imidazole buffer (50 mM, pH 6.95, chloride counterion) and then sodium metaperiodate (240 µL, 100 mM in water, i.e. 24 µmol) was added with mixing. After 5 min, the oxidation was quenched by adding 480 µL of ethylene glycol solution (100 mM in water, i.e. 48 µmol). The oxidized templates were isolated by semipreparative HPLC and characterized as the expected tetravalent and octavalent aldehydes (see Figure 1) through the products formed in oximation reactions: no signals were obtainable upon ESI-MS analysis of the polyaldehydes, probably because no positively charged group was present. Oxime Formation as a Function of pH. Solutions were prepared of the MSH analog NH2-OCH2CO-NleAsp-His-(D-Phe)-Arg-Trp-Lys-NH2 (5 mM in water) and glyoxylyl-Leu-Leu (10 mM in water), and oxime formation was started by mixing 40 and 60 µL, respectively, of these solutions with 100 µL of buffer: 0.2 M formate (Na), pH 3.0; 0.2 M acetate (Na), pH 4.6; 0.2 M acetate (Na), pH 5.3. The aldehydic peptide was thus initially in a 3-fold excess over the aminooxyacetyl-MSH derivative. At various times (2 min to 24 h), aliquots (10 µL) were analyzed by HPLC using a linear gradient of 1% B/min from 20 to 60%, and yields were calculated from a standard curve obtained with a solution of previously purified oxime of which the concentration had been determined by weight. The oxime was characterized by ESI-MS (found mass 1357.5 Da, calcd 1356.3 Da) and used for oxime stability tests as described just below. Oxime Stability. The small model peptide oxime obtained by coupling glyoxylyl-Leu-Leu to the MSH analog NH2-OCH2CO-Nle-Asp-His-(D-Phe)-Arg-Trp-LysNH2 was used to study the stability of the oxime bond under neutral to mildly alkaline conditions. A 5 mM solution of the oxime in water was diluted 10 times in different buffers [0.1 M phosphate (Na), pH 7.0 and 8.0;
554 Bioconjugate Chem., Vol. 7, No. 5, 1996
Figure 2. Extent of oxime formation, as a function of time and pH, between glyoxylyl-Leu-Leu (initial concentration 3 mM) and the MSH analog NH2-OCH2CO-Nle-Asp-His-(D-Phe)-Arg-TrpLys-NH2 (initial concentration of 1 mM). The pH values of the reaction mixtures were 3.0 ([), 4.6 (9), and 5.3 (b).
0.1 M NaHCO3/Na2CO3, pH 9.0 and 10), and the solutions were analyzed by HPLC after 2, 20, and 65 h at room temperature (approximately 22 °C). Polyoxime Formation as a Function of pH. Solutions were prepared of the hexaaldehyde template OdCHCO-Gly3-[Lys(COCHO)]5-Gly-OH (10 mM in water; see ref 7 for preparation) and the aminooxyacetyl peptide NH2-OCH2CO-KLEEQRPERVKG-OH (10 mM separately in each of the following: 0.1 M sodium acetate buffer, pH 4.6, 0.1% acetic acid, 0.1% TFA, and 0.1 M sodium phosphate buffer, pH 7.0; see ref 7 for peptide preparation). Oxime formation was initiated by mixing 25 µL (0.25 µmol) of template with 400 µL (4.0 µmol) of peptide derivative. The peptide derivative was thus present in a 2.7-fold excess over each aldehyde group on the template. After incubation at room temperature for various times, the reaction mixture was analyzed by HPLC. The polyoxime product was then isolated by shallow gradient semipreparative HPLC and characterized by mass spectrometry. Insulin Tetra- and Octahydrazone. Oxidized templates were dissolved (10 mM in acetonitrile/0.1% acetic acid, 4:1 v/v) and then diluted to 1 mM (i.e. 4 or 8 mM in aldehyde groups for the tetra- and octaaldehyde templates, respectively) with 0.1% acetic acid. Sufficient desAlaB30-insulinylcarbohydrazide (1 mM in 0.1% acetic acid) was then added to give a molar excess of 1.3 over aldehyde groups on the template. After incubation at room temperature for various times, the reaction mixture was analyzed by HPLC and the polyhydrazone product isolated by shallow gradient semipreparative HPLC and characterized by ESI-MS. Insulin Octaoxime. Oximation, isolation, and characterization followed the procedure used for insulin hydrazone formation except that, in place of des-AlaB30insulinylcarbohydrazide, des-AlaB30-insulinyl-Phe-NHCH2CH2NH-COCH2ONH2 and aminooxyacetylB1-insulin were used with the tetra- and octaaldehyde templates, respectively. After isolation, the insulin octaoxime gave no signal under our standard conditions (7), presumably because its m/z values lay beyond the range of our quadrupole instrument (m/z
