Bioconjugate Chem. 2009, 20, 231–240
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Versatile Cyclic Templates for Assembly of Axially Oriented Ligands Neeraj Chopra,§ Wenxun Gan,‡ Hans Schreiber,§ Josh W. Kurutz,† and Stephen C. Meredith*,§,† Departments of Pathology, Chemistry, and Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois. Received July 21, 2008; Revised Manuscript Received November 12, 2008
In this paper, we describe two novel types of planar cyclic peptide templates for the facile addition of ligands that extend axially from the plane of the template ring. The first uses β-amino acids of alternating D- and L-chirality, since the insertion of the additional methylene group in the peptide backbone was predicted and subsequently shown by NMR and molecular modeling, to reorient ligands attached to amino acid side chain axially with respect to the template ring. A second contains alternating D- and L-amino acids with an achiral Gly residue interposed between each chiral amino acid. The inserted Gly residues also tend to reorient side chains axially rather than radially, as was demonstrated by NMR and molecular modeling. The axial orientation of attached ligands is intended to foster or allow interactions among attached ligands in situations in which this is desired. Two such situations that we consider are (1) development of immunological reagents with avidity effects and (2) modeling of oligomers in fibril-forming peptides. Toward the first of these goals, we demonstrated that these templates are suitable for attaching macromolecules, by incorporating two types of protein, neutravidin and trypsinogen. Toward the second goal, we demonstrate the attachment of two different fibril-forming peptides to the template. The templates described herein thus have many of the desirable traits of such molecules, i.e., (1) multivalency for the attachment of multiple ligands, (2) suitable chemical functions for facile attachment of ligands, (3) versatility as to the number and spacing of ligand attachment sites, (4) sufficient rigidity so that the attached ligands can be similarly oriented with respect to the template, and (5) sufficient flexibility to allow even large ligands, such as proteins, to attach and interact.
INTRODUCTION Cyclic peptides and their peptidomimetic homologues are versatile molecules with distinctive self-assembly properties. The concept of template-assisted peptide synthesis (TASP) was introduced and first developed by Mutter and colleagues (1-3) (see also refs 4-11 for review and more recent examples). Ghadiri and co-workers described planar, cyclic peptides containing alternating D- and L-amino acids that self-assemble into tubular structures with a β-sheet-like structure. The planarity of the rings derives from the cancellation of the innate twists of the β-strands by the alternation of D- and L-amino acids (12, 13). Some of these compounds have antibacterial activity because of their ability to stack into nanotubes that insert into bacterial membranes (14-16), while others self-assemble into artificial transmembrane ion channels (17, 18). In addition to these properties attributable to self-assembly, cyclic peptides and peptidomimetics can also serve as templates for attaching and assembling a wide variety of ligands (10, 11, 19-27). In the templates designed by Ghadiri et al. (12-17), the amino acid side chains are oriented radially from the ring, which, depending on the nature of the side chains, can either promote or prevent lateral association of the rings. In addition, the rigidity and β-sheet-like structure render these molecules prone to selfassociation into tubular aggregates, unless measures are taken to prevent this, such as N-methylation (28). As schematized in Figure 1A, the radial orientation of side chains would also orient any ligands attached to these side chains radially, which therefore tends to prevent interactions among these ligands. * To whom correspondence should be addressed: The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, U.S.A. Fax: (+1) 773-834-5251. E-mail:
[email protected]. Phone: 773-702-1267. § Department of Pathology. ‡ Department of Chemistry. † Department of Biochemistry and Molecular Biology.
Figure 1. Schematic depiction of planar cyclic peptide templates. Peptides made of alternating D- and L-R-amino acids, such as those described by Ghadiri and colleagues (12-17) have been observed to project their side chains radially, as depicted in (A). The use of alternating D- and L-β-amino acids (chiral R-carbon atom) or the addition of an odd number of atoms between alternating D- and L-Ramino acids, is predicted to project the side chains axially, as depicted in (B).
There are situations, however, in which it would be desirable to promote interaction among ligands attached to a template. Two such instances are (1) attachment of high-affinity singlechain antibodies fragments (scFv molecules) and high-affinity single-chain T-cell receptors (scTCR) “payloads”, in which avidity effects are desirable; and (2) controlled modeling of intermediates in the formation of amyloid fibrils. In both of these cases, it would be desirable to orient ligands attached to amino acid side chains oriented axially rather than radially, as demonstrated schematically in Figure 1B. The incorporation of amino acids with reactive side chains into the template could allow facile attachment of protein or other ligands to the ring. For these reasons, we have developed two strategies for synthesizing cyclic planar templates with axially oriented side chains. In addition to allowing interactions among attached ligands, we also reasoned that such templates would also have a reduced tendency toward aggregation into tubular β-sheetlike structures than previously described structures. The two strategies are the following:
10.1021/bc800312x CCC: $40.75 2009 American Chemical Society Published on Web 01/21/2009
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(1) We have synthesized cyclic peptides containing β-amino acids with alternating D- and L- chirality about the R-carbon atom, i.e., amino acids containing an “extra” methylene carbon atom in the backbone chain. Preliminary modeling studies suggested that the insertion of this additional odd-numbered atom into the backbone chain would have the effect of reorienting the side chains axially with respect to the template backbone, as opposed to radially. For brevity, these templates will be referred to as type 1 axial templates. (2) We have also synthesized cyclic peptides that retain the alternation of D- and L-R-amino acids, but insert the nonchiral amino acid, glycine, between each of the chiral amino acids. Like the scheme above using β-amino acids, preliminary modeling studies suggested that the addition of glycine would have the effect of reorienting side chains axially. In addition, the insertion of glycines in this manner would also impart additional flexibility to the ring, in those cases in which such flexibility is needed to allow interactions between ligands attached to the side chains. For brevity, these templates will be referred to as type 2 axial templates. In this paper, we describe the synthesis and structural characterization of these two types of templates and the facile attachment of protein ligands to reactive amino acid side chain groups. Ideally, such templates should have the following characteristics: (1) multivalency for the attachment of multiple ligands; (2) suitable chemical functions for facile attachment of ligands; (3) versatility as to the number and spacing of ligand attachment sites; (4) sufficient rigidity so that the attached ligands can be similarly oriented with respect to the template; and (5) sufficient flexibility to allow even large ligands, such as proteins, to attach and interact. Since these ligands could include scFv and scTCR molecules, the template should be flexible enough to allow avidity effects to occur and, in the case of fibril-forming peptide, to allow for conformational rearrangements to occur. We will show, below, that the templates significantly satisfy these criteria.
EXPERIMENTAL PROCEDURES Materials. Boc-Leu-OCH2-PAM resins, Boc-amino acids, S-trityl-β-mercaptopropionic acid and 2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) were from Peptides International. Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate) (BOP) and N-R-Fmoc-L-aspartic acid β-4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino} benzyl ester (R-Fmocβ-ODMAB-Asp) were from Novabiochem. 4-Dimethylaminopyridine (DMAP) was from Aldrich. N-R-Boc-N-β-Fmoc-D2,3-diaminopropionic acid (R-Boc-β-Fmoc-D-DAP) was from ChemImpex, Inc. N-R-Fmoc-L-aspartic acid R-t-butyl ester (Fmoc-Asp-R-OtBu) was from Bachem International. All other amino acids were from Novabiochem, Applied Biosystems, Midwest Biotech, or Anaspec. N,N-Diisopropylethylamine (DIEA) was obtained from Applied Biosystems. N,N-Dimethylformamide (DMF), dichloromethane (DCM), diethyl ether, HPLC-grade acetonitrile, and guanidine hydrochloride were from Fisher Chemicals. Trifluoroacetic acid (TFA) was obtained from Halocarbon Products. HF was from Matheson. Peptide Synthesis and Purification. Most peptides and peptide-R-thioesters were made manually by solid-phase peptide synthesis using tBoc chemistry with “in situ neutralization”, essentially as described previously (29, 30). In general, after chain assembly was complete, peptides were simultaneously deprotected and cleaved from the resin by treatment with anhydrous HF containing p-cresol (90:10, v/v) for 1 h at 0 °C. After evaporation of the HF under reduced pressure, crude products were precipitated and triturated with chilled diethyl ether, and the peptide products were dissolved in 50% aqueous acetonitrile and purified, or lyophilized for storage.
Chopra et al.
For synthesis of peptides with β-peptide bonds, i.e., Peptides 1, C1, and C1-Mal and its derivatives, we used standard FMOC chemistry to form β-peptide bonds using the reagents described above. PAM resin was activated with 1 mmol of R-FMOC-βODmab-Asp and 10% (w/v) 4-(dimethylamino)pyridine (DMAP), essentially as described (31-33), so that this amino acid was immobilized to the resin through its R-carboxyl group. For the cyclization reaction, the DMAB group was removed by three 20 min incubations with 2% hydrazine (v/v), followed by three washes with 5% DIEA (v/v). The cyclization reaction was carried out using BOP at 22 °C for 36 h, followed by HOBt/ DIC/DIEA for 48 h. Maleimide hexanoic acid (Sigma Chemicals) was added to the N-terminus or to DAP side chains of selected peptides (described below) by the usual coupling method, prior to HF cleavage. Crude peptides were purified by RP-HPLC on a preparative C18 (Zorbax) column using gradients of water (0.1% TFA, v/v) and acetonitrile (0.1% TFA, v/v). In some cases, isocratic mixtures of water and acetonitrile (0.1% TFA, v/v) were needed instead of gradients for peptides that were difficult to purify. Peptide purity was typically greater than 97% by analytical RPHPLC (Supporting Information, Figure S1). The molecular masses of the peptides were verified by ESI and MALDI-TOF mass spectrometry. Electron Microscopy. Peptide solutions were adsorbed onto a glow-discharged, 400-mesh, carbon-coated support film and stained with 1% uranyl acetate. Micrographs were recorded using a FEI Tecnai F30st-STEM Microscope at magnifications of 15 000×, 39 000×, and 98 000×. Circular Dichroism. Circular dichroic (CD) spectra were collected using an Aviv model 202 CD spectrophotometer (Lakewood, NJ) equipped with a temperature-controlled holder. Peptides were dissolved in 10 mM sodium phosphate, pH 7.40. A 1 or 10 mm path length was used for the measurements, which were collected at 0.2-0.5 nm intervals from 260 to 190 nm, with a 1 s averaging time and a 1 nm bandwidth. Five scans were collected, and the data were averaged and baselinecorrected. NMR. NMR was performed at 14 °C using a 600 MHz Varian Unity Inova spectrometer equipped with a cryogenic probe. Peptide concentration was ∼3.1 mM in 100 mM sodium phosphate, pH 4.52. A 1.0 mM solution of sodium 2,2-dimethyl2-silapentane-5-sulfonate (DSS, Cambridge Isotope Laboratories) was used for external chemical shift referencing. Twodimensional experiments included TOCSY and NOESY for spin assignments, CT-COSY for measurement of 3JNHR. Molecular Dynamics Simulations. The initial coordinates of cyclic peptides were modeled using the Chem3D program. Hydrogen atoms were built to the initial structure using the HBUILD module of the CHARMM molecular simulation program (34). The all-atom structure was then solvated in a 38.7 × 38.7 × 38.7 Å3 cubic solvent box with TIP3P (35) water so that water extended at least 10 Å away from the surface of the peptide. Four Na+ ions and seven Cl- ions were added to the system to neutralize the charge of the peptide. Periodical boundary conditions were applied with the images generated by the CRYSTAL module in CHARMM. Covalent bonds involving hydrogen atoms were constrained with SHAKE (36), and the particle mesh Ewald (PME (37)) was used to treat longrange electrostatic interactions. After an initial minimization of the system, MD simulations were performed for 1 ns at a constant temperature of 300 K using the CHARMM version 33a2 with the CHARMM27 force field (38).
RESULTS Design and Synthesis of Cyclic Template Peptides. A list of the peptides synthesized for these studies is given in Table
Cyclic Templates for Assembly of Ligands Table 1. Peptides Synthesized for These Studies name
sequence
(R-Boc-DDAP-R-tBu-Asp)2-R-Boc-DDAPAsp(R-PAM Resin) Peptide C1 Cyclo-(DDAP-Asp)3 Peptide C1-Mal Cyclo-(DDAP(β-hexanoylmaleimide)-Asp)3 Peptide 2 NH2-(DCysGlyArgGly)3-SCH2CH2C(O)-Leu-OH Peptide C2 Cyclo-(DCysGlyArgGly-)3 Peptide 3 NH2-(DCysGlyCysGly)3-SCH2CH2C(O)-Leu-OH Peptide C3 Cyclo-(DCysGlyCysGly)3 Peptide 4 Mal-PEG-GNNQQNY-OH Peptide 5 Mal-SDDYYYGFGSNKFGRPRDP-OH Peptide 6 Mal-GEGEGEGK-OH Peptide 6(Biotin) Mal-GEGEGEGK(Biotin)-OH Peptide 7 Cyclo-(DCys(S-Mal-GEGEGEGK-OH)-Gly-Arg-Gly)3 Peptide 7(Biotin) Cyclo-(DCys(S-Mal-GEGEGEGK(Biotin)-OH)-GlyArg-Gly)3 Peptide 1
1. Peptide 1, the linear precursor for Peptide C1 and Peptide C1-Mal, was synthesized as shown in Figure 2. In Peptides 1 and C1, all residues are linked by β-peptide bonds. Synthesis started by activating PAM resin (see Experimental Procedures) with 1 mmol of R-FMOC-β-ODmab-Asp and 10% (w/v) DMAP, so that this amino acid was immobilized to the resin through its R-carboxyl group. After removing the FMOC, the free R-amino group was coupled to R-Boc-β-FMOC-DDAP by standard FMOC chemistry. Standard FMOC chemistry was then employed to extend the peptide, alternately with Fmoc-Asp-ROtBu and R-Boc-β-FMOC-DDAP, until the hexapeptide, Peptide 1, had been synthesized. Peptide C1 is derived from Peptide 1 (Figure 2) by coupling the free β-COOH group of the C-terminal Asp to the free β-NH2 group of DDAP on the resin after the ODmab groups had been removed by three 20 min incubations with 2% (v/v) hydrazine, as described elsewhere (39-41). For this reaction, the free amino and carboxyl termini of the peptide were coupled by reaction for 36 h with BOP; followed by 48 h with HOBt/DIC/DIEA, at the end of which a ninhydrin test was negative. To produce Peptide C1-Mal, the R-tBOC protecting groups on the DAP residues were removed by TFA, after which maleimide hexanoic acid was coupled to the free R-amino groups. Finally, the derivatized, cyclized Peptide C1 or Peptide C1-Mal was removed from the resin using anhydrous HF. Type 2 axial template peptides were synthesized from thioester-containing linear precursors, such as Peptides 2 and 3. The scheme for synthesizing Peptides C2 (or C3) is shown in Figure 3. Peptides 2 and 3 contain both of the groups needed for native chemical ligation, i.e., an N-terminal Cys and a C-terminal thioester (44, 43). Briefly, the linear peptide was synthesized on a PAM resin loaded with Leu to which mercaptopropionic acid had been attached, producing a thioester upon extension of the peptide chain to produce Peptide 2 or 3 (29). Peptides were synthesized using optimized Boc solid phase peptide synthesis (30). The linear peptides were cyclized by native chemical ligation, as described elsewhere (44). Peptide 2C (Figure 3) is derived from Peptide 2 by native chemical ligation of the deprotected peptide. Peptides at ∼2 mg/mL were dissolved in 100 mM sodium phosphate, pH 7.20, containing 6 M guanidine HCl and 10 mM tris(2-carboxyethyl)phosphine (TCEP). To monitor the course of the reaction, small aliquots of the reaction mixture were examined by analytical RP-HPLC. Cyclization of the deprotected linear peptides was remarkably clean and rapid. Examples of reactions using this chemistry are shown in Supporting Information, Figures S2 and S3, for Peptide C1-Mal (m/z ) 1184.2, expected ) 1183.1); MALDI-TOF mass spectrometry of Peptide C2 showed m/z ) 1120 as compared to its linear precursor, for which m/z ) 1339, in both cases matching the expected masses. For Peptide C1-Mal, the reactions were performed on resin, while for Peptide C2, the reaction was carried out with the peptide in solution.
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Electron Microscopy. The inclusion of charged amino acids, Arg, Asp, and R,β-diamino propionic acid (DAP), into the sequence, was sufficient to yield highly soluble products and prevent the formation of insoluble nanotube structures. For Peptides 1, C1, 2, and C2, no precipitate was visible after centrifugation (10 min, 16 000 × g), and electron microscopy revealed only a small amount of amorphous material that appeared to be protein dried on the grid (Figure 4B shows Peptide C2, which is typical of these peptides). In contrast to Peptides 1, C1, 2, and C2, Peptide C3, cyclo-(DCysGlyCysGly)3, made well-ordered, insoluble nanotubes in buffer (Figure 4A). Circular Dichroic Spectroscopy. As anticipated for peptides containing residues of alternating chirality, Peptides 1, C1-Mal (Figure 5A), 2, and C2 (Figure 5B) showed little ellipticity. The spectrum could not be measured for Peptides 3 and C3 because of their poor solubility. The linear peptides 1 and 2 showed a small amount of negative ellipticity, increasingly so at wavelengths of
