Toward New Designed Proteins Derived from ... - ACS Publications

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Bioconjugate Chem. 2001, 12, 726−741

Toward New Designed Proteins Derived from Bovine Pancreatic Trypsin Inhibitor (BPTI): Covalent Cross-Linking of Two ‘Core Modules’ by Oxime-Forming Ligation Nata`lia Carulla,† Clare Woodward,‡ and George Barany*,† Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, and Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota 55108. Received June 7, 2001; Revised Manuscript Received July 20, 2001

A 25-residue disulfide-cross-linked peptide, termed ‘oxidized core module’ (OxCM), that includes essentially all of the secondary structural elements of bovine pancreatic trypsin inhibitor (BPTI) most refractory to hydrogen exchange, was shown previously to favor nativelike β-sheet structure [Carulla, N., Woodward, C., and Barany, G. (2000) Synthesis and Characterization of a β-Hairpin Peptide That Represents a ‘Core Module’ of Bovine Pancreatic Trypsin Inhibitor (BPTI). Biochemistry 39, 79277937]. The present work prepares to explore the hypothesis that the energies of nativelike conformations, relative to other possible conformations, could be decreased further by covalent linkage of two OxCMs. Optimized syntheses of six ∼50-residue OxCM dimers are reported herein, featuring appropriate monomer modifications followed by oxime-forming ligation chemistry to create covalent cross-links at various positions and with differing lengths. Several side reactions were recognized through this work, and modified procedures to lessen or mitigate their occurrence were developed. Particularly noteworthy, guanidine or urea denaturants that were included as peptide-solubilizing components for some reaction mixtures were proven to form adducts with glyoxylyl moieties, thus affecting rates and outcomes. All six synthetic OxCM dimers were characterized by 1D 1H NMR; three of them showed considerable chemical shift dispersion suggestive of self-association and mutual stabilization between the monomer units.

INTRODUCTION

An ‘oxidized core module’ (OxCM),1 which is a 25residue disulfide-cross-linked peptide that includes essentially all of the secondary structural elements that in the full protein bovine pancreatic trysin inhibitor (BPTI: 58 residues, 3 disulfides, Figure 1a) are most refractory to hydrogen exchange, has recently been designed, synthesized, and characterized (1). NMR analysis showed that OxCM favors nativelike β-sheet structure in rapid equilibrium with populations of non-native β-sheets (1). Based on these encouraging observations, ways were sought to further stabilize nativelike conformations in OxCM. The working hypothesis is that covalent linkage of two OxCMs may enhance self-association and mutual stabilization, resulting in a more compact, kinetically accessible, and thermodynamically favored structural ensemble resembling a protein native state. This design strategy was reinforced by findings about the full-length protein, i.e., a dimeric form of BPTI exists in solution and undergoes rapid associationdissociation (2, 3). Further, molecular modeling studies on dimeric BPTI indicate that the residues at the hydrophobic monomer-monomer interface are primarily in OxCM (4) (Figure 1b). The chemistry chosen to link two OxCMs is oximeforming ligation (5). In its most general form, this method * To whom correspondence should be addressed at the Department of Chemistry, University of Minnesota, 207 Pleasant St. S.E., Minneapolis, MN 55455. Phone: 612-625-1028. Fax: 612-626-7541. E-mail: [email protected]. † University of Minnesota, Minneapolis. ‡ University of Minnesota, St. Paul.

allows the joining of two unprotected peptide segments in an unambiguous manner by the chemoselective reaction of two unique and mutually reactive functionalities: (a) an (aminooxy)acetyl moiety and (b) any of a variety of carbonyl-containing moieties including aldehydes, methyl ketones, and R-oxo aldehydes (glyoxylyl 1 Abbreviations used for amino acids and the designations of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature (34). The following additional abbreviations are used: Abu or X, R-amino-n-butyric acid; Alloc, allyloxycarbonyl; Aoa or U, (aminooxy)acetyl; Boc, tert-butyloxycarbonyl; BPTI, bovine pancreatic trypsin inhibitor; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; Dpr or Z, R,β-diaminopropionic acid; DSS, sodium 2,2-dimethyl-2silapentane-5-sulfonate; ESMS, electrospray mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; FPLC, fast protein liquid chromatography; GdnHCl, guanidine hydrochloride; Gxy or J, glyoxylyl; HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate Noxide; HOAt, 1-hydroxy-7-azabenzotriazole (3-hydroxy-3H-1,2,3triazolo-[4,5-b]pyridine); HOBt, 1-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; MALDI-TOF, matrixassisted laser desorption/ionization time-of-flight (mass spectrometry); Mpa or B, β-mercaptopropionic acid; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; OxCM, oxidized core module; PAL, ‘peptide amide linker’ [5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid]; PEG-PS, poly(ethylene glycol)-polystyrene (resin support); Pfp, pentafluorophenyl; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; PyAOP, 7-azabenzotriazol-1-yl-N-oxytris(pyrrolidino)phosphonium hexafluorophosphate; SEC, size-exclusion chromatography; tBu, tert-butyl; TFA, trifluoroacetic acid; Tmob, 2,4,6-trimethoxybenzyl; TOCSY, total correlation spectroscopy; TP, model tetrapeptide amide Tyr-Dpr(Gxy)-Ala-Lys-NH2; Trt, triphenylmethyl or trityl.

10.1021/bc015518m CCC: $20.00 © 2001 American Chemical Society Published on Web 08/31/2001

Oxime Ligation of BPTI Core Modules

Bioconjugate Chem., Vol. 12, No. 5, 2001 727

optimize conditions for oxime-forming ligations of BPTI core modules. The chosen dimerization chemistry accommodates changes in the position of the cross-link (Figure 2a) and variations in the length of the cross-link (Figure 2b). Six examples of OxCM dimers were synthesized. Onedimensional proton (1D 1H) NMR was used as a qualitative criterion to gauge the extent of conformational families present in each OxCM dimer. Three dimers showed considerable chemical shift dispersion, and one of them is likely to be amenable for further structural characterization by multidimensional homo- and heteronuclear NMR methods. EXPERIMENTAL PROCEDURES

Figure 1. Ribbon representations of (a) crystallographic structure of BPTI, showing positions of the three native disulfides and highlighting in dark blue those residues that correspond to the oxidized core module (OxCM), and (b) a model for the BPTI dimer, reorienting two BPTI structures from (a) in the way proposed by Zielenkiewicz et al. (4), but without repeating those authors’ energy minimization calculations. Residues Arg17 in monomer I (left) and Leu29 (nearer to Arg17) and Asn24 (further from Arg17) in monomer II (right) are shown in red. Both diagrams in (a) and (b) were produced using the program MOLSCRIPT (33). (c) Representation of a simpler model for OxCM dimer that, unlike what is shown in (b), ignores the nativelike twist of the β-sheet strands and suggests that Arg17 in monomer I is spatially closer to Asn24 in monomer II. Block arrows are β-sheet strands (oriented N at tail to C at arrowhead), solid boldface ribbons are β-turns, and solid lines are the flexible loops including the disulfide bond that cyclizes each OxCM.

function). Oxime-forming ligations have been used primarily in the production of peptide dendrimers (5-7), ‘template assisted synthetic proteins’ (TASPs) (8, 9), and functional proteins such as the heterodimeric transcription factor cMyc-Max (10). When carried out intramolecularly, oxime-forming ligation provides macrocyclic peptides (11, 12). For the present application (Scheme 1), it was observed that the presence of guanidine hydrochloride (GdnHCl) or urea as denaturants in the buffers used to dissolve peptide materials altered the rates and outcomes of the ligations. The underlying chemistries were elucidated in a model tetrapeptide system, and the resultant insights were applied to

General. Materials, instrumentation, and procedures for synthesis, spectroscopy, chromatography, and analysis are detailed in the Supporting Information; some of these follow precedents in earlier publications from our laboratories (13-15), and others are specific for this work. Refer to the Supporting Information for conditions A through I used in reversed-phase analytical or semipreparative HPLC and conditions J and K for sizeexclusion chromatography (SEC). The matrix used for sample analysis by matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) was R-cyano-4hydroxycinnamic acid (4-HCCA; Hewlett-Packard, obtained as solution in methanol), diluted 1:1 with 0.1% TFA in CH3CN/H2O (1:1), whereas samples for electrospray mass spectrometry (ESMS) were infused in 0.2% acetic acid in CH3CN/H2O (1:1). The final isolated yields were calculated by comparison of amino acid analyses of the purified peptide to the initial loading of the resin. In describing core module monomers, capped monomers, and dimers, the notation presented in Figures 2, 3, and 8, and the accompanying text, is used. When closely related procedures were followed, this section provides representative descriptions while further characterizations are reported in the Supporting Information. NMR Spectroscopy. NMR samples for the tetrapeptide model studies were 11 mM dissolved in 2H2O at pH 4.6 (measured at electrode and uncorrected for isotope effects). 2H2O was from Aldrich, and urea-15N2 was from Cambridge Isotope Laboratories, Inc. (Andover, MA). GdnDCl-d6 and urea-15N2,d4 were prepared respectively by dissolving GdnHCl or urea-15N2 in an excess of 2H2O, lyophilization, and repeating the solution/lyophilization cycle 3 more times. Experiments were performed at 25 °C on Varian 300, 500, 600, or 800 MHz Inova instruments, and the pulse sequences used were obtained from the Varian library (macros: gCOSY, gHMQC, and gHMBC). 1H/1H-gCOSY were acquired using 2K × 333 data points and a spectral width of 12 ppm in both dimensions. 1H/13C-gHMQC and 1H/13C-gHMBC were acquired using 2K × 512 data points and a spectral width of 12 ppm in the 1H dimension and 240 ppm in the 13C dimension. 15N/ 1 H-gHMQC and 15N/1H-gHMBC were acquired using 2K × 128 data points and a spectral width of 12 ppm in the 1H dimension and 200 ppm in the 15N dimension. A shifted sine bell or a Gaussian function was applied to both dimensions during data processing. NMR samples for the capped monomers were 0.3-0.5 mM, pH 4.5, and for the OxCM dimers were 0.2-0.4 mM, pH 2. The lyophilized pure peptides or proteins were dissolved in water at the desired pH and adjusted when required with aliquots of 0.1 N 2HCl/2H2O or 0.1 N NaO2H/2H2O. 1H NMR spectra were obtained in 90:10 H2O/2H2O on a Varian 600 MHz Inova instrument.

728 Bioconjugate Chem., Vol. 12, No. 5, 2001

Carulla et al.

Scheme 1. Synthesis of IR17K(U)-IIL29K(J) Dimer by Oxime-Forming Ligation To Connect Corresponding OxCM Monomersa

a

See later in this paper (Figures 2 and 3 and accompanying text) for explanations of schematic and nomenclature conventions.

Proton chemical shifts of capped monomers were measured from an internal DSS standard defined as 0 ppm. NMR data acquisition and processing followed methods described previously (1). The hydrogen isotope exchange rate for the most downfield amide proton resonance of IR17K(U)-IIL29K(J) was obtained by measuring peak integrals vs time in a series of 1D spectra at pH 2 and 5 °C (Supporting Information Figure 2). The pseudo-first-order rate constant was determined from nonlinear least-squares fit of an exponential rate equation to experimental data. Peptide Synthesis. Starting with Fmoc-PAL-PEGPS resin (13, 16) (1 g, 0.24 mmol/g, 0.24 mmol), the core module monomer linear sequences were assembled by automated Fmoc solid-phase synthesis using an Applied Biosystems Pioneer automated peptide synthesizer. Sidechain protecting groups were provided by appropriate tert-butyl (tBu) ethers and esters for Thr, Tyr, Asp, and Glu; tert-butyloxycarbonyl (Boc) for Lys (when not part of cross-link); 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) for Arg; 2,4,6-trimethoxybenzyl (Tmob) for Asn and Gln; and trityl (Trt) for Cys. Washes between chemical steps were carried out with DMF. Fmoc removal was achieved with piperidine/DMF (1:4, 5 min). Couplings of all residues other than Cys were 1 h, in DMF, as mediated by HBTU/HOBt/DIEA (4:4:8 equiv with respect to peptide-resin; 3.5 min preactivation) using 4 equiv of Fmoc-amino acids. Fmoc-Cys(Trt)-OPfp was used for 1 h, in DMF, to introduce Cys with minimal racemization (17). As expected, completed peptide-resins had a lower substitution (i.e., 0.12 mmol/g), due to weight gain from the peptide portion. Peptide Synthesis of Core Module Monomers: IR17K(U), IR17K(UG), and IR17Z(U) (Supporting Information Scheme 1), and IIL29K(J), IIL29K(JG), IIL29Z(J), and IIN24K(J) (Supporting Information Scheme 2 and Figure 4). IR17K(U). In the linear core module sequence, Arg17 was replaced by Fmoc-Lys(Alloc)-OH. A portion of the completed peptide-resin (1 g, 0.12 mmol/g, 0.12 mmol) was washed with CH2Cl2 (10 × 0.5 min), and the N-Alloc protecting group of Lys17 was cleaved under an argon atmosphere at 25 °C by adding Me2NH‚BH3 (84.8 mg, 1.44 mmol, 12 equiv) in CH2Cl2 (4.7 mL), followed 1 min later by Pd(PPh3)4 (27.7 mg, 0.024 mmol, 0.2 equiv) in CH2Cl2 (0.8 mL). After 10 min treatment, the peptideresin was drained and washed with CH2Cl2 (3 × 0.5 min),

and the deprotection procedure was repeated twice. The peptide-resin was then washed successively with CH2Cl2 (8 × 0.5 min), 0.2% TFA in CH2Cl2 (2 × 1 min), DIEA/ CH2Cl2 (1:19) (3 × 1 min), and CH2Cl2 (5 × 0.5 min). An aliquot of the peptide-resin at this stage was cleaved, and analyzed by HPLC (tR 16 min, condition D). The identity of the peptide in the HPLC peak was confirmed by MALDI-TOF analysis; calcd monoisotopic mass: 2826.4, found: 2827.6 [M+H]+. The peptide-resin (1 g) with the Alloc group removed was placed into a column of the Applied Biosystems Pioneer synthesizer. Next, Boc-Aoa-OH (8 equiv) was incorporated onto the N-amino side chain of Lys17 using PyAOP/HOAt/DIEA (8:8:16 equiv with respect to peptideresin). Boc-Aoa-OH, PyAOP, and HOAt were weighed as solids into the same vial, and were dissolved by addition of equal volumes of 1 M DIEA-DMF and neat DMF; after a 3.5 min preactivation period, this mixture was added to the peptide-resin to initiate coupling. Two more such couplings were required to complete the incorporation of Boc-Aoa-OH. Upon completion of on-resin steps, the dried protected peptide-resin was cleaved portion-wise with freshly prepared Reagent R [TFA/thioanisole/1,2-ethanedithiol/anisole (90:5:3:2)]. Peptide-resin (450 mg, 58.4 µmol of peptide) was treated with cleavage cocktail (5 mL) for 1 h at 25 °C. The filtrate was collected and set aside at 25 °C, while the remaining peptide-resin was cleaved twice more, 1 h each, with fresh cocktail (5 mL). The separate filtrates were concentrated under a stream of N2, and Et2O (∼25 mL, freshly distilled from Na) was added at 0 °C with thorough agitation. The precipitated peptide was collected by low-speed centrifugation, washed with cold freshly distilled Et2O (3 × 25 mL), and dissolved in ∼100 mL of H2O/CH3CN (9:1) for lyophilization. The crude peptide (126 mg, 74% cleavage yield) was characterized by analytical HPLC (tR 26.3 min, >90% purity; condition A), and the identity of the main HPLC peak was confirmed by MALDI-TOF analysis; calcd monoisotopic mass: 2899.45, found: 2900.32 [M+H]+. When the precipitated peptide was washed with reagent grade Et2O from a can (not distilled), a byproduct was observed by HPLC (tR 21 min; condition D) and the identity of this HPLC peak was confirmed by MALDI-TOF; found: 2940.1 [M+H]+ (40 amu above the expected mass).


To form the disulfide bridge, the crude reduced peptide (60 mg, 20.7 µmol) was dissolved in 6 mL of 0.01 N aqueous HCl, and then ∼300 mL of a mixture of dimethyl sulfoxide/6 M GdnHCl in 0.1 M sodium phosphate buffer, pH 7 (1:18), was added. The reaction was monitored by analytical HPLC (tR 26.3 min and tR 21.8 min for reduced and oxidized, respectively; condition A). After oxidation had gone to completion in 12 h at 25 °C, the mixture was concentrated over a 1 day period using an Amicon ultrafiltration cell (YM1 membrane, MWCO ∼1000) (final volume ∼25 mL). Crude oxidized peptide was loaded in five batches onto semipreparative HPLC (condition E). Purified peptide (10.2 mg, 6%) was characterized by analytical HPLC (tR 21.8 min; condition A), amino acid analysis (Asp, 2.02; Thr, 0.93; Glu, 1.02; Gly, 3.02; Ala, 1.99; Val, 0.97; Ile, 1.35; Leu, 1.02; Tyr, 2.86; Phe 1.97; Lys, 2.92; Arg, 1.03), and MALDI-TOF; calcd monoisotopic mass: 2897.43, found: 2898.29 [M+H]+. For synthesis and characterization of IR17K(UG) and IR17Z(U), see Supporting Information. IIL29K(J). In the linear core module sequence, Leu29 was replaced by Fmoc-Lys(Alloc)-OH. Alloc deprotection was carried out in the same way as already described with the procedure for IR17K(U). Coupling of Fmoc-Ser(tBu)-OH toward IIL29K(J) was difficult, but accomplished by the same procedures used to add Boc-Aoa-OH toward IR17K(U). The NR-Fmoc group of Ser was removed immediately before cleavage. The dried protected peptide-resin was cleaved essentially as described for IR17K(U), except that Reagent K [TFA/phenol/thioanisole/H2O/1,2-ethanedithiol (82.5:5:5:5:2.5)] was used instead of Reagent R. The crude peptide (130 mg, 75% cleavage yield) was characterized by analytical HPLC (tR 21 min, >90% purity; condition A), and the identity of the main HPLC peak was confirmed by ESMS analysis; calcd average mass: 2958.46, found: 2957.7 ( 1.3. When Reagent R was used to cleave the peptide-resin, a byproduct (∼25% of major) was observed. This was analyzed and isolated by HPLC (tR 22.8 min; condition A) and identified by ESMS; found: 3054.2 ( 0.6 (96 amu above the expected mass). There follows a description of experimental details for three different scenarios matching Figure 4 and the accompanying text discussion. Figure 4a. Crude reduced peptide was loaded in six batches, each of 20 mg/5 mL of 0.01 N aqueous HCl, onto semipreparative HPLC (condition F) (25 mg, 19% purification yield). To form the disulfide bridge, the pure reduced peptide (15 mg, 5 µmol) was dissolved in 4 mL of 0.01 N aqueous HCl, ∼200 mL of a mixture of dimethyl sulfoxide/2 M GdnHCl in 0.1 M sodium phosphate buffer, pH 7 (1:18), was added, and the reaction was stirred for 24 h at 25 °C. The reaction was monitored by analytical HPLC (tR 22.8 min and tR 18.5 min for reduced and oxidized, respectively; condition A). The identity of the oxidized peptide in the HPLC peak was confirmed by MALDI-TOF analysis; calcd monoisotopic mass: 2954.46, found: 2955.3 [M+H]+. Directly following disulfide bond formation and retaining the same milieu, the Ser residue branched off the Lys side chain was oxidized by adding aqueous sodium periodate (2 mL, 5 mM, 2 equiv). After 10 min reaction in the dark at 25 °C, the reaction was quenched with aqueous ethylene glycol (2 mL, 10 mM, 4 equiv). The resultant glyoxylyl peptide was characterized by analytical HPLC [tR 18.6 min (P1) and 19 min (P2); ratio 2:9; condition A]. The reaction mixture (200 mL) was placed in an Amicon ultrafiltration cell (YM1 membrane, MWCO ∼1000) that was part of an Amicon diafiltration setup. During diafiltration, H2O was introduced into the cell while the buffer components (GdnHCl,


sodium phosphate) and undesired species (DMSO, periodate, ethylene glycol, and formaldehyde generated by the serine oxidation) were removed from the reaction mixture. After 600 mL of H2O (3 times the volume of the reaction mixture) had passed through the cell, the mixture was concentrated (final volume ∼50 mL) and finally lyophilized. Lyophilized peptide (17 mg, 10%) was characterized by analytical HPLC [tR 19 min (P2); condition A], amino acid analysis (Asp, 1.99; Thr, 0.94; Glu, 1.01; Gly, 3.09; Ala, 1.99; Val, 0.86; Ile, 0.98; Tyr, 2.58; Phe, 1.92; Lys, 2.93; Arg, 2.03), ESMS; calcd average mass: 2925.39, found: 2942.6 ( 0.5 [18 amu (H2O) above expected with glyoxylyl end group], 2924.7 ( 0.7 [expected], 2906.7 ( 0.2 [18 amu (H2O) below expected with glyoxylyl end group], and by MALDI-TOF; calcd monoisotopic mass: 2923.43, found: 2924.1 [M+H]+. Figure 4b. To form the disulfide bridge, the crude reduced peptide (60 mg, 20.2 µmol) was dissolved in 6 mL of 0.01 N aqueous HCl, ∼300 mL of a mixture of dimethyl sulfoxide/6 M GdnHCl in 0.1 M sodium phosphate buffer, pH 7 (1:18), was added, and the reaction was stirred for 24 h at 25 °C. The crude oxidized peptide was characterized by analytical HPLC (tR 18.5 min, >90% purity; condition A), and the identity of the main peak was confirmed by MALDI-TOF analysis; calc monoisotopic mass: 2954.46, found: 2955.3 [M+H]+. Directly following disulfide bond formation and retaining the same milieu, the Ser residue branched off the Lys side chain was oxidized by adding aqueous sodium periodate (3 mL, 13.5 mM, 2 equiv). After 10 min reaction in the dark at 25 °C, the reaction was quenched with aqueous ethylene glycol (3 mL, 27 mM, 4 equiv). The glyoxylyl peptide was characterized by analytical HPLC [tR 18.6 min (P1) and 19 min (P2), ratio 1:1; condition A]. After periodate oxidation, the mixture was concentrated over a 1 day period using an Amicon ultrafiltration cell (YM1 membrane, MWCO ∼1000) (final volume ∼25 mL). Crude glyoxylyl peptide was loaded in five batches onto semipreparative HPLC (condition G) to provide separately analytical HPLC pure P1 and P2 which were each characterized by ESMS; calcd average mass: 2925.39, found: for P1, 2943.6 ( 1.2 [18 amu (H2O) above], 2924.9 ( 0.5 [expected], 2906.4 ( 0.2 [18 amu (H2O) below]; and for P2, 2942.6 ( 0.5 [18 amu (H2O) above], 2924.7 ( 0.7 [expected], 2906.7 ( 0.2 [18 amu (H2O) below]. Purified P1 and P2 were indistinguishable by MALDI-TOF; calcd monoisotopic mass: 2923.43, found: 2924.2 [M+H]+, and by amino acid analysis (Asp, 2.00; Thr, 0.80; Glu, 0.98; Gly, 3.08; Ala, 1.96; Val, 0.96; Ile, 1.62; Tyr, 2.78; Phe 1.98; Lys, 2.88; Arg, 2.08). Including fractions that had both components, the final overall isolated yield of P1 plus P2 was 34 mg (20%). When either pure P1 or pure P2 was dissolved in various solvent milieus, reequilibration occurred as described in the text. Figure 4c. The same procedures as in the previous paragraph were followed, except that 6 M GdnHCl was replaced by 8 M urea. Purified peptide (34 mg, 20%) was characterized by analytical HPLC [tR 19 min (P3); condition A], amino acid analysis (Asp, 2.20; Thr, 0.85; Glu, 1.15; Gly, 3.27; Ala, 2.32; Val, 0.81; Ile, 1.59; Tyr, 3.25; Phe 2.21; Lys, 3.31; Arg, 2.16), ESMS; calcd average mass: 2925.39, found: 2984.4 ( 1.4 [60 amu (urea) above], 2925.0 ( 0.9 [expected], 2906.7 ( 0.5 [18 amu (H2O) below], and MALDI-TOF; calcd monoisotopic mass: 2923.43, found: 2924.7 [M+H]+. For synthesis and characterization of IIL29K(JG), IIL29Z(J), and IIN24K(J), see Supporting Information. Tyr-Dpr(Ser)-Ala-Lys-NH2. Starting with Fmoc-PALPEG-PS resin (1 g, 0.24 mmol/g, 0.24 mmol), the title


precursor to model tetrapeptide TP was assembled by Fmoc solid-phase synthesis using an Applied Biosystems Pioneer automated peptide synthesizer. As also done for synthesis of IIL29Z(J), the dipeptide building block FmocDpr(Boc-Ser(tBu))-OH was incorporated at the third cycle. The dried protected peptide-resin (500 mg, 120 µmol) was cleaved using Reagent K. Crude reduced peptide was loaded onto semipreparative HPLC (condition H). Fractions with the desired peptide were pooled and lyophilized to provide a white powder (47 mg, 71%), pure by analytical HPLC (tR 7.3 min; condition C), with the expected amino acid analysis (Ser, 0.80; Ala, 1.00; Tyr, 0.98; Lys, 0.95) and ESMS; calcd average mass: 552.62, found: 554.2 [M+H]+. Tyr-Dpr(Gxy)-Ala-Lys-NH2. Purified Tyr-Dpr(Ser)Ala-Lys-NH2 (10 mg, 18.1 µmol) was dissolved in 10 mL of 0.1 M sodium phosphate buffer, pH 7, and then aqueous sodium periodate (0.1 mL, 0.36 M, 2 equiv) was added. After 10 min reaction in the dark, the reaction was quenched with aqueous ethylene glycol (0.1 mL, 0.72 M, 4 equiv). The crude glyoxylyl peptide, characterized by analytical HPLC [tR 7.30 min (T1); condition C], was loaded in two batches onto semipreparative HPLC (condition H). Fractions with the desired peptide were pooled and lyophilized to provide a white powder (29 mg, 43%), characterized further by amino acid analysis (Ala, 1.00; Tyr, 0.70; Lys, 0.97) and ESMS; calcd average mass: 521.57, found: 540.2 [M+H2O+H]+, 522.2 [M+H]+, 504.3 [M-H2O+H]+. Corresponding oxidation experiments were carried out in the presence of GdnHCl or urea. In 2, 6, and 8.5 M GdnHCl in 0.1 M sodium phosphate buffer, pH 7, the peptide products were recognized by analytical HPLC [tR 7.3 min (T1) and tR 7.6 min (T3); ratio 4:1, 3:2, and 1:1, respectively; condition C] and characterized by HPLC/ ESMS; calcd average mass: 521.57, found: for T1, 540.2 [M+H2O+H]+, 522.2 [M+H]+, 504.3 [M-H2O+H]+; and for T3, 539.8 [M+H2O+H]+, 522.3 [M+H]+, 504.1 [M-H2O+H]+. In 8 M urea in 0.1 M sodium phosphate buffer, pH 7, peptide product was recognized by analytical HPLC [tR 7.4 min (T2); condition C] and characterized by HPLC/ESMS; calcd average mass: 521.57, found: 582.2 [M+urea+H]+, 540.2 [M+H2O+H]+, 522.2 [M+H]+, 504.3 [M-H2O+H]+. Oxime-Forming Ligation Reactions. Capping of Monomers. (A) IR17K(U*). IR17K(U) (2.9 mg, 1 µmol) was dissolved in 1 mL of 0.01 N aqueous HCl, and acetone (10 µL, 136 µmol) was added. The reaction was complete at 25 °C within 30 s, as shown by analytical HPLC (tR 25.1 min; condition A). Crude capped monomer was purified by semipreparative HPLC (condition I) to give 2 mg of IR17K(U*) as a white solid after lyophilization (70%, based on IR17K(U)). Purified peptide was characterized by amino acid analysis (Asp, 2.00; Thr, 0.92; Glu, 1.07; Gly, 3.02; Ala, 2.00; Val, 0.96; Ile, 1.44; Leu, 1.01; Tyr, 2.71; Phe, 1.87; Lys, 2.91; Arg, 0.96) and MALDI-TOF; calcd monoisotopic mass: 2937.47, found: 2938.22 [M+H]+. For characterization of IR17K(U*G) and IR17Z(U*), see Supporting Information. (B) IIL29K(J*). IIL29K(J) (2.9 mg, 1 µmol) was dissolved in 0.87 mL of 6 M GdnHCl in 0.1 M NaOAc buffer, pH 4.6, and then, CH3ONH2‚HCl (25-30 wt % solution in water) (0.13 mL, 400 µmol) was added, and the reaction mixture was stirred for 24 h at 25 °C. Crude capped monomer was purified by semipreparative HPLC (condition G) to give 1.5 mg of IIL29K(J*) as a white solid after lyophilization (50%, based on IIL29K(J)). Pure peptide was characterized by analytical HPLC (tR 20.3 min; condition A), amino acid analysis (Asp, 2.04; Thr, 0.92; Glu, 1.01; Gly, 2.98; Ala,

Carulla et al.

1.99; Val, 0.97; Ile, 1.35; Tyr, 2.78; Phe, 1.97; Lys, 2.96; Arg, 2.03), and ESMS; calcd average mass: 2954.43, found: 2953.6 ( 1.4. For characterization of IIL29K(J*G), IIL29Z(J*), and IIN24K(J*), see Supporting Information. Dimer Formation. IR17K(U)-IIL29K(J). Reaction was initiated by combining purified IR17K(U) (2.9 mg, 1 µmol) and IIL29K(J) (P1 or P2) (2.9 mg, 1 µmol) in 1 mL of 6 M GdnHCl in 0.1 M NaOAc buffer, pH 4.6, and maintaining the reaction for 3 days at 25 °C while monitoring by analytical HPLC (Supporting Information Figure 1). Crude dimer was loaded in two batches for SEC (condition J) to give 3.3 mg of IR17K(U)-IIL29K(J) as a white solid after lyophilization (57%, based on IR17K(U)). Pure peptide was characterized by analytical HPLC (tR 29.2 min; condition B), amino acid analysis (Asp, 3.82; Thr, 1.67; Glu, 1.98; Gly, 5.94; Ala, 3.85; Val, 1.88; Ile, 3.11; Leu, 1.10; Tyr, 5.02; Phe, 3.65; Lys, 5.45; Arg, 2.86), and ESMS; calcd average mass: 5806.76, found: 5806.3 ( 1.6. Contrasting to the optimal conditions to carry out the oxime-forming ligation given in the previous paragraph, when the reaction was initiated by combining IR17K(U) (2.9 mg, 1 µmol) and IIL29K(J) (P3) (2.9 mg, 1 µmol) in 1 mL of 8 M urea in 0.1 M NaOAc buffer, pH 4.6, the reaction was extremely slow due to the low reactivity of P3 (see Figure 7, dotted line and accompanying text). Moreover, during that lengthy time period, IR17K(U) was mostly converted to the corresponding carbamylated byproduct, recognized by analytical HPLC (tR 27.1 min; condition B) and identified by MALDI-TOF; found: 2941.09 [M+H]+ (42.6 amu above the expected mass for IR17K(U)). For characterization of IR17Z(U)-IIL29K(J), IR17Z(U)-IIL29Z(J), IR17K(U)-IIL29K(JG), IR17K(UG)-IIL29K(JG), and IR17K(U)-IIN24K(J), see Supporting Information. RESULTS AND DISCUSSION

Dimer Design Considerations: Positioning and Length of Cross-Link between Monomers. The general rationale for creating and studying dimers of OxCM has already been stated. Presented herein is an iterative design strategy that is integrated with the available chemical options dictated by the central choice of oxime formation in solution as the means to link two monomers (Scheme 1). Since an entirely rigorous nomenclature would be cumbersome, several intuitive pictorial and written shorthands are used to describe the target molecules (Figures 2 and 3). The disulfide-bridged OxCM monomer comprises ∼25 residues, and these are numbered to comply with the corresponding residues in native BPTI; e.g., the Nterminal Mpa and C-terminal Cys of OxCM are respectively 14 and 38 (Figure 3). Solid-phase methods to assemble such molecules, to cleave them from the support, and to produce the spanning disulfide bridge have already been described (1). As 2 monomers are linked to form a dimer of ∼50 residues, symmetry is lost: the monomer which will be modified to include an (aminooxy)acetyl (Aoa or U) residue is referred to as I, and the monomer which will eventually provide a glyoxylyl (Gxy or J) functionality is referred to as II. The choice of where to position the I-II cross-link is suggested by modeling studies of Zielenkiewicz et al. (4), which propose a large BPTI dimer interface involving numerous intermolecular hydrophobic contacts but only three intermolecular hydrogen bonds. The hydrophobic residues of BPTI involved in this antiparallel self-complementary association are primarily the ones also found in the OxCM. Inspection



Figure 2. Schematic representation of, and nomenclature system for, the six OxCM covalent dimers synthesized in this work, emphasizing (a) different positions of the cross-link and (b) different length cross-links between the monomers. See accompanying text for details. Each solid boldface ribbon depicts an OxCM, and the spanning disulfide bond between Mpa14 and Cys38 is indicated by the solid line connecting the N and C termini. Arrows indicate residue numbers that are connected; the type I monomer is drawn directly beneath the type II monomer in all representations. All atoms in the cross-links, starting with the β-carbons, are shown. The dimer IR17K(U)-IIL29K(J) is drawn twice so that it can be compared to other dimers addressing points (a) and (b).

of the BPTI dimer model reveals that Arg17 in one monomer is near Leu29 in the other monomer (Figure 1b). A simpler model that ignores the nativelike twist of the β-sheet strands suggests that Arg17 in one monomer is near Asn24 in the other monomer (Figure 1c). Each of these residues (i.e., Arg17, Asn24, Leu29) can be replaced in the linear sequence by an orthogonally protected NR,ωdiamino CR-monoacid, i.e., Lys or Dpr (Figure 3c-e). At the appropriate stage of the overall solid-phase synthesis process (see Supporting Information Schemes 1 and 2 and later text for details), the Nω-amino group can be exposed selectively, and extended further to accomplish one or more of the following: (i) vary the linker length, i.e., by coupling a protected Gly residue (this is in addition to control of length by the earlier choice of Dpr vs Lys); (ii) for monomer I, ultimately introduce the Aoa functionality by coupling the corresponding protected building block; and (iii) for monomer II, couple a protected Ser derivative which serves as a precursor to the Gxy moiety that is established later by a periodate oxidation step carried out in solution.

Based on these principles and strategies, six OxCM dimers were assembled (Figure 2). All dimers replaced Arg17 in monomer I that would contribute the Aoa component of the oxime linkage. In one set of studies (Figure 2a), the ‘matching’ residue that was replaced in monomer II was either Leu29 or Asn24. In brief (details later), NMR studies show that the dimer with the oxime linkage between residue 17 in monomer I and residue 24 in monomer II, i.e., IR17K(U)-IIN24K(J), has less chemical shift dispersion than the dimer with the oxime linkage between residue 17 in monomer I and residue 29 in monomer II, i.e., IR17K(U)-IIL29K(J). Therefore, a further set of studies (Figure 2b) kept constant the preferred crosslinkage site between residue 17 in monomer I and residue 29 in monomer II, but varied from 10 to 22 atoms the cross-linking length (counting atoms between, but not including, the R-carbon of position 17 in I and the R-carbon of position 29 in II). Synthesis of Core Module Monomers Suitable for Oxime-Forming Ligation. (A) Assembly, Cleavage, and Disulfide Formation in Unprotected OxCM Monomers


Figure 3. Amino acid sequences for (a) residues 14-38 in native BPTI, (b) OxCM [ref (1); underlined residues are different from the native sequence], (c) monomers of type I, replacing Arg17, (d) monomers of type II, replacing Leu29, and (e) monomer of type II, replacing Asn24. More detailed chemical structures drawn at the bottom include B (β-mercaptopropionic acid), X (R-amino-n-butyric acid), and the cross-linking substituents Γ and ϑ, for (c), (d), and (e). The glyoxylyl endgroup of ϑ is drawn in its aldehyde form to explain its reactivity in oxime-forming ligation; NMR experiments reported later prove that this endgroup exists in the hydrated dihydroxyacetyl form (see Figure 5a for accurate structure in a model peptide).

Containing the (Aminooxy)acetyl Functionality. Linear protected sequences were assembled by automated stepwise Fmoc solid-phase peptide chemistry starting with Fmoc-PAL-PEG-PS supports (Supporting Information Scheme 1). Leading to the eventual elaboration of the cross-link, the synthetic strategy next required on-resin

Carulla et al.

removal of an N-Alloc protecting group from Lys17. Application of Pd(Ph3)4 (0.1 equiv) and Me2NH‚BH3 (6 equiv) in CH2Cl2 (18) worked smoothly, and set the stage for addition of Boc-Aoa-OH to the liberated amino group. Couplings were sluggish and required repeat treatments; the best conditions used Boc-Aoa-OH (8 equiv) mediated by PyAOP (8 equiv) and DIEA (16 equiv) in DMF for 3 × 1 h to achieve a negative ninhydrin test result. The aforementioned series of steps gave monomer IR17K(U) (see previous section for explanation of nomenclature); interpolation of one coupling/deprotection cycle of Fmoc-GlyOH gave monomer IR17K(UG). Preparation of a third monomer of interest, i.e., IR17Z(U), was expedited because Fmoc-Dpr(Boc-Aoa)-OH, a commercially available building block, could be incorporated directly during the linear assembly. Cleavages and manipulations of these peptides presented new challenges, due to the ready reactivity of the Aoa functionality with aldehydes and ketones to provide stable oxime derivatives. During routine work, peptides are precipitated from acidolytic cleavage cocktails by addition of ether; they may also become exposed to acetone either from glassware last washed with this solvent or from the dry ice/acetone baths used to shellfreeze aqueous samples before lyophilization (10). Often, a major byproduct was observed, present at a level comparable to or even higher than the desired Aoacontaining product. These byproducts all show a mass (ESMS) 40 amu above the expected, corresponding to addition of acetone (or propionaldehyde) and elimination of H2O. Fortunately, such byproducts were eliminated entirely when the ether used to precipitate the peptide was freshly distilled from sodium, and when precautions were taken to avoid exposure to incidental or residual acetone. Cleavages were carried out conveniently using TFA/thioanisole/1,2-ethanedithiol/anisole (90:5:3:2) (Reagent R) (13), and provided the peptides with all free side chains including two thiols from the termini (Mpa14 and Cys38-NH2). For OxCM, disulfide bond formation was carried out by dissolving the peptide in DMSO/2 M GdnHCl in 0.1 M phosphate buffer, pH 7 (1:18), for 24 h at 25 °C (1). However, the substitution of Arg17 by Γ (Figure 3) changes the solubility characteristics of the peptide, and a precipitate is observed as the oxidation proceeds under the previously optimized conditions. GdnHCl sometimes aids in solubilizing peptides as well as creating a denaturing environment that favors disulfide formation (19). Several GdnHCl concentrations (1-6 M) were tested, and the best results were obtained with DMSO/6 M GdnHCl in 0.1 M phosphate buffer, pH 7 (1:18), for 12 h at 25 °C. Other conditions, which varied pH (6 or 7) and/or used different percentages of DMSO without GdnHCl, were less satisfactory because disulfide product precipitated. Procedures concluded by concentration of reaction mixtures and peptide purification by reversedphase HPLC. The overall isolated yield was typically 6%, based on the starting PAL-PEG-PS resin. (B) Assembly, Cleavage, Disulfide Formation, and Periodate Oxidation of Unprotected OxCM Monomers Containing the Glyoxylyl Functionality. Again, linear protected sequences were assembled by automated stepwise Fmoc solid-phase peptide chemistry starting with Fmoc-PAL-PEG-PS supports (Supporting Information Scheme 2). Toward monomer IIL29K(J), the N-Alloc protecting group of Lys29 was removed as already described, and Fmoc-Ser(tBu)-OH was coupled as mediated by PyAOP/HOAt/DIEA. The NR-Fmoc group of Ser was removed from the resultant peptide-resin immediately



Figure 4. Chemistries and chromatographic analyses of three different scenarios that were followed to generate the disulfide bond and the glyoxylyl functionality in monomers of type II. Elucidation of accurate structures for P1, P2, and P3, based in part on studies with a model peptide TP, is covered extensively in the accompanying text.

before acidolytic cleavage. Toward monomer IIL29K(JG), an additional coupling/deprotection cycle incorporated FmocGly-OH onto the N-amine of Lys29, prior to addition of Fmoc-Ser(tBu)-OH. Finally, preparation of the IIL29Z(J) monomer featured direct incorporation of commercially available Fmoc-Dpr(Boc-Ser(tBu))-OH. The cleavage and further processing of these modified monomers revealed several unanticipated problems, which were systematically elucidated and circumvented. Recall

that a central part of the plan was to eventually oxidize the Ser residue with periodate in order to generate the glyoxylyl functionality. Acidolytic cleavage with Reagent R (composition given in the previous section) was invariably accompanied by formation of a byproduct, at a level typically 25% that of desired, which was shown by MALDI-TOF to have a mass 96 amu above what was expected. Moreover, the byproduct (as part of crude mixture) did not react further upon periodate treatment.


This information was consistent with a tentative conclusion that the byproduct is an O-trifluoroacetyl ester of the N-terminal serine residue, although an N-trifluoroacetyl species formed by O f N migration cannot be ruled out (20). Consistent with this hypothesis, and of obvious practical import, the trifluoroacetyl byproduct was not formed when cleavage was carried out with TFA/phenol/ thioanisole/H2O/1,2-ethanedithiol (82.5:5:5:5:2.5) (Reagent K) (21). This result is attributed to the presence in the latter cocktail of water, which either scavenges any activated trifluoroacetyl species or hydrolyzes any ester that is produced. Because of concerns about incompatibility of free thiols with the oxidation conditions planned for converting Ser to the glyoxylyl moiety (22), the disulfide-forming step was carried out next in the overall process. In turn, solubility considerations as already described made it necessary to add substantial levels of denaturants (GdnHCl or urea), along with the DMSO (5% v/v) used as the oxidizing reagent. Three scenarios were followed (Figure 4). In each case, peptide was first dissolved fully in 0.01 N aqueous HCl, next the mixture of pH 7 phosphate buffer, denaturant, and DMSO was added, and then disulfide formation was carried to completion (24 h, single major peak by HPLC). Subsequently, in the same milieu, NaIO4 (2 equiv) was added, and 10 min later, oxidation was quenched with ethylene glycol (4 equiv) (23). HPLC analysis showed three products (P1, P2, and P3 in Figure 4), of which only P2 was later proven to correspond to the expected structure. The highest relative level of P2, i.e., ∼80%, occurred when the crude peptide had been purified first, and then dissolved in 2 M GdnHCl (Figure 4a). When the crude peptide was used directly, the concentration of GdnHCl required for solubilization was higher, i.e., 6 M, and consequently the relative level of P2 dropped to ∼50% (determined after the periodate step; Figure 4b). The byproduct in the experiments with GdnHCl was P1, for which the following properties were deduced: (i) the most intense ESMS peak corresponds to the aldehyde, and two other peaks, half the intensity of the major, correspond to the hydrated and dehydrated forms of the aldehyde; (ii) the unique MALDI-TOF signal corresponds to the aldehyde form; (iii) the byproduct was quite unreactive for the oxime-forming ligation step (see ‘Synthesis of OxCM Dimers’); and (iv) the byproduct was converted to the desired P2, which has the anticipated good reactivity, by extensive diafiltration with H2O (Figure 4a), albeit with reduction in the overall yield due to solubility problems. In contrast, ESMS data on the desired P2 showed that the most intense peak corresponds to the hydrated form of the aldehyde, while the intensities of the free aldehyde and the dehydrated form were about 1/3 and 1/6 respectively; MALDI-TOF analysis of P2 again showed only the aldehyde form. Finally, in experiments carried out in the presence of 8 M urea, neither P1 nor P2 formed, but the exclusive unwanted product was P3. Purified P3 had the following characteristics: (i) the most intense ESMS peak corresponds to the peptide aldehyde having reacted with urea (60 amu above what was expected), with the aldehyde form and the dehydrated forms also present each at about 1/4 intensity; (ii) the unique MALDI-TOF signal corresponds to the aldehyde form; and (iii) P3 was exceptionally unreactive for the oxime-forming ligation step (see ‘Synthesis of OxCM Dimers’). Model Studies To Determine Structures of Byproducts Obtained after Periodate Oxidation in the Presence of Denaturants. To understand the formation of unexpected byproducts P1 and P3 after oxidation

Carulla et al. Table 1. Effects of Denaturants on the Distribution of Products from Periodate Oxidation at pH 7 of Model Peptide H-Tyr-Dpr(Ser)-Ala-Lys-NH2a milieu (aqueous buffer, denaturant)

T1

T2

T3

0.1 M phosphate, pH 7 2 M GdnHCl in 0.1 M phosphate, pH 7 6 M GdnHCl in 0.1 M phosphate, pH 7 8.5 M GdnHCl in 0.1 M phosphate, pH 7 8 M urea in 0.1 M phosphate, pH 7

100 82 63 49 0

0 0 0 0 100

0 18 37 51 0

a Products are designated in order of HPLC elution, and relative amounts are expressed in percent so as to add up to 100%. As noted in the text, TP products T1, T2, and T3 correlate to type II monomer-derived products P2, P3, and P1, respectively.

of a Ser residue in the presence of GdnHCl or urea, the model peptide H-Tyr-Dpr(Ser)-Ala-Lys-NH2 (corresponding to BPTI residues 23-26, with replacement of Asn24) was synthesized and evaluated. Mirroring the results with the full 25-residue OxCM type II monomers, treatment of the model with periodate produced two products (T1, T3) with varying HPLC-reported ratios depending on the GdnHCl concentration (2-8.5 M) that was used, and a unique product (T2) when urea (8 M) was used (Table 1). In the absence of denaturant, only T1 formed, suggesting that this corresponds to the desired H-TyrDpr(Gxy)-Ala-Lys-NH2. Further supporting the correlations P1/T3, P2/T1, and P3/T2, HPLC/ESMS analysis was inconclusive about the true structures of T1 and T3, but supportive of T2 being a urea adduct. The Gxy-containing T1 was readily purified, and then redissolved in each of three different milieus for NMR analysis: (a) 2H2O at pH 4.6, (b) 6 M GdnDCl-d6 in 2H2O at pH 4.6, and (c) 8 M urea-15N2,d4 in 2H2O at pH 4.6. Milieu (a) allowed structural conclusions about pure T1, whereas milieu (b) promoted a rapid equilibration to a 3:2 ratio of T1 to T3 (confirmed by HPLC), and T1 was converted quantitatively to T2 within 12 h in milieu (c). From the NMR data and detailed interpretations that follow, it is concluded (for the tetrapeptide structure) that in T1, the terminal Gxy residue exists as the corresponding hydrate, i.e., a dihydroxyacetyl residue; that an amino group of guanidine reacts with the terminal aldehyde of Gxy to form a covalent hemiaminal adduct (two diastereomers) as exemplified in T3; and that the amino group of urea reacts in an analogous fashion as exemplified in T2 (Figure 5 shows all of the proposed structures, including numbering of key atoms in the positions of interest). It is highly likely that these structural inferences generalize to systems (P1, P2, and P3 in Figure 4) for which the observations were originally made. It is instructive to compare the NMR-established structural conclusions with the more ambiguous information from mass spectrometry; more specifically, MALDI-TOF suggested only the expected aldehyde structures (without the proper hydration), whereas the more “gentle” ESMS technique was clear only for the urea adduct, hinted at the aldehyde hydrate (major peak, but unhydrated and dehydrated species also noted), and entirely missed the guanidine adduct (unhydrated as major, and hydrated as well as dehydrated aldehyde forms as minor, all present). Since the environments of the rapidly equilibrating species in these samples change as they are prepared for and then analyzed by mass spectrometry, these findings are not especially surprising. Supporting the structural conclusions, consider first the 1D 1H NMR spectra (Figure 6), backed up by 1H/1HgCOSY experiments which allowed assignments of all nonexchangeable hydrogen resonances (Supporting Information Table 1). The most significant differences are



Figure 5. Structures of (a) T1, the hydrated Gxy-containing model tetrapeptide, (b) T3, the two hemiaminal diastereomers obtained by reversible reaction of guanidine with the terminal aldehyde of the Gxy residue in T1, and (c) T2, the two hemiaminal diastereomers obtained by reversible reaction of urea with the terminal aldehyde of the Gxy residue in T1. Arrows identify key atoms in the structural identifications (see also Table 2).

between 5.4 and 5.8 ppm. In the absence of denaturants (Figure 6a), a unique singlet at 5.52 ppm is assigned to the H-C(2) proton of the dihydroxyacetyl residue [the former H-C(R) of Ser]. In an 1H/13C-HMQC experiment, the 1H signal at 5.52 ppm correlates to a 13C signal at 89.6 ppm, again consistent with a hydrated aldehyde (24) (Figure 5a). When spectra are recorded in the presence of GdnDCl-d6 (Figure 6b), the singlet at 5.52 ppm was supplemented by two more signals, of equal height, at 5.71 and 5.72 ppm. The latter two signals had consistent chemical shifts regardless of what field strength (300, 500, 600, or 800 MHz) was used for spectral acquisition; hence, they are derived from two unique chemical environments rather than from splitting of a single resonance. Further information obtained from 1H/13CHMQC and 1H/13C-HMBC spectra (Table 2) reprise the observations about T1 already noted, and provide a way to characterize structures of the two new species corresponding to T3. Thus, the 1H signals at 5.71 and 5.72 ppm both correlated in 1H/13C-HMQC to the same 13C signal at 76.8 ppm, consistent with a hemiaminal, and in 1H/13C-HMBC to two 13C signals at 172.5 and 159.6 ppm. While the 172.5 ppm signal is assigned to the C(1) carbon (originally CdO of Ser), the 159.6 ppm peak is

particularly conclusive because it is derived from guanidine. The 13C chemical shift is close to, but different from, 157.2 ppm, which is the shift of the carbon from GdnHCl itself (24). These data establish the covalent connectivity of the adduct (Figure 5b). The two close, but separate, 1H peaks in the 1D NMR spectra are now readily explained by the chirality of C(2) after nucleophilic attack by a guanidine nitrogen on the Gxy aldehyde. Spectroscopic examination of the Gxy-containing peptide in the presence of urea (Figure 6c) reveals similarities as well as differences from the same peptide in the presence of GdnHCl. The absence of a signal at 5.5 ppm is taken to mean that the aldehyde function of the model tetrapeptide had reacted quantitatively with urea. As before, 1H signals at 5.57 and 5.61 ppm were assigned to the H-C(2) proton, correlated by 1H/13C-HMQC to the same 13C signal at 76 ppm and by 1H/13C-HMBC to two 13C signals at 175.3 ppm [assigned to the C(1) carbon] and 162.7 ppm [similar to the shift of urea itself at 163.5 ppm (24)]. Also as before, the two 1H shifts are independent of field strength and explained by the chirality of C(2). The availability of [15N]urea allowed correlation in 1 H/15N-HMBC of the 1H signals at 5.57 and 5.61 ppm, respectively, to 15N signals at 100.8 and 101.0 ppm


Carulla et al.

Figure 6. 1H NMR spectra, recorded at 500 MHz, of the purified Gxy-containing model tetrapeptide redissolved in (a) 2H2O at pH 4.6, (b) 6 M GdnDCl-d6 in 2H2O at pH 4.6, and (c) 8 M urea-15N2,d4 in 2H2O at pH 4.6. The proton resonance(s) of H-C(2) in each milieu is/are indicated in the spectra. Full 1H assignments are compiled in Supporting Information Table 1. The residual H2O signal was suppressed by use of presaturation techniques. The signal around 6.9 ppm in (b) corresponds to residual GdnHCl. In (c), the signal of residual protons from urea is not observed because urea exchanges rapidly with water, and the urea signal is saturated concomitant with H2O suppression. Table 2. Diagnostic 1H, 13C, and 15N Chemical Shifts for H-Tyr-Dpr(Gxy)-Ala-Lys-NH2, Recorded at 500 MHz in 2H2O at pH 4.6 in the Absence and Presence of Denaturantsa residue Gxy-derived denaturant-derived

position H-C(2) C(1)c C(2)e N(1)f C(3)c

2H

2O

5.52 d 89.6

8 M urea-15N2,d4

6 M GdnDCl-d6 5.52b d 89.7b

5.71 172.5 76.8

5.72 172.5 76.8

159.6

159.6

5.57 175.3 76.0 101.0 162.7

5.61 175.3 76.0 100.8 162.7

a

Refer to Figure 5 to define the atoms in the Ser to Gxy conversion, and the atoms derived from denaturants. Sample preparations are defined in the legend to Figure 6, and the 1H chemical shifts in the first row of this table overlap with that figure. b These signals, which overlap those observed in 2H2O in the absence of denaturant, are attributed to the original species present in 2H2O, and not to the Gdn adduct. c Chemical shifts of C(1) and C(3) were obtained from 1H/13C-HMBC experiments. d No correlation was observed between the H-C(2) proton and C(1) in the 1H/13C-HMBC experiment. Two bond coupling constants are small, and their observation depends on electronic properties of the bond. e Chemical shifts of C(2) were obtained from 1H/13C-HMQC experiments. f Chemical shifts of N(1) were obtained from 1H/15N-HMBC experiments.

(compared to 15N of urea itself at 76 ppm), further supporting the urea adduct structure (Figure 5c). It is also plausible that additional stabilization could be provided by formation of a hydrogen bond involving the oxygen of OdC(3) and the hydrogen of OH-C(2); note this gives a six-membered ring. The observations described in this and the preceding section are of considerable practical importance, given the subsequent goal to apply glyoxylyl-containing peptides for oxime-forming ligation reactions. In aqueous media, the glyoxylyl moiety itself exists in the dihydroxyacetyl (hydrated) form; this follows classical expectations about aldehyde chemistry and will not affect its ultimate reactivity with strong nucleophiles. Formation under mild aqueous conditions of the described intermolecular adducts of glyxoxylyl functions with guanidine or urea is unprecedented and surprising due to the low nucleophilicities of guanidino or amide nitrogens. The high concentrations of denaturants do drive the equilibrium toward adduct, although in the case of guanidine, enough adduct reverts to the glyoxylyl species so that reactions with Aoa-containing products can proceed (albeit much slower; see next section). A review of organic literature

databases showed numerous examples of related transformations (i.e., amides or carbamates react with glyoxylyl or simpler aldehyde derivatives), although invariably under nonaqueous conditions with heating and/or long reaction times. The recent report by Rose et al. (25) of a rapid intramolecular cyclization (six-membered ring forms, specifically facilitated by structural constraint of the Pro residue), involving irreversible nucleophilic attack in aqueous media by a peptide amide nitrogen on an N-terminal glyoxylyl function, provides a somewhat analogous precedent to what was found in the present work. Synthesis of OxCM Dimers: Oxime-Forming Ligation. At this stage of the work, methodologies were on hand to obtain both families of functionalized monomers that needed to be connected by an oxime-forming ligation, but the identification of covalent adducts between glyoxylyl moieties and either GdnHCl or urea denaturants was a source of concern. To create the desired OxCM dimers, it was necessary to react the functionalized monomers under acidic aqueous conditions at pH 4.6, based on literature precedents (5-7, 10). Several strategies were explored to combine IR17K(U) with


Figure 7. Oxime-forming reactions connecting IR17K(U) and IIL29K(J) (1 mM each) as a function of time and denaturant. The denaturant used to solubilize components in the reaction mixture was 6 M GdnHCl (solid line), 8.5 M GdnHCl (dashed line), and 8 M urea (dotted line). The ordinate, expressed as a percentage, was calculated (based on HPLC areas) as the amount of IR17K(U)-IIL29K(J) formed divided by the total amount of all species (product, starting materials, adducts of II with either GdnHCl or urea, and adduct of I with cyanate).

IIL29K(J), in the context that the use of denaturants could not be avoided due to the need to keep peptide materials in solution. Later, the best procedures were extended to prepare the other dimers. In one experiment, the Ser-containing precursor of IIL29K(J) was oxidized with periodate in 6 M GdnHCl in 0.1 M phosphate buffer, pH 7, generating a 1:1 ratio of P1 and P2 as already indicated (Figure 4). These two products were separated by semipreparative reversedphase HPLC and separately dissolved in 6 M GdnHCl in 0.1 M NaOAc buffer, pH 4.6, for reaction with IR17K(U). There was an early burst of initial reactivity corresponding to P2 (free glyoxylyl) vs P1 (guanidine hemiaminal), i.e., 10% vs 2% of IR17K(U)-IIL29K(J) formed after 3 min. However, within 1 h both reaction mixtures had equilibrated, so that regardless of whether pure P1 or pure P2 had been present at the outset, these materials were now in a 1:1 ratio. From this point on, the kinetics of the ligations were essentially the same in both reactions (Figure 7, solid line). The experiment was repeated at higher GdnHCl concentration, i.e., 8.5 M. In this case, equilibration within 1 h gave P1 and P2 in a ratio of 1.4: 1, and the overall kinetics were slightly slower (Figure 7, dashed line). Experiments in the other direction, i.e., with lower GdnHCl concentration, were not feasible due to solubility problems. These data are consistent with the idea that the higher the concentration of GdnHCl, the more guanidine hemiaminal adduct (P1) is formed, and the slower the reaction. In another experiment, the peak obtained from periodate oxidation in the presence of 8 M urea, i.e., P3 (Figure 4c), was reacted with IR17K(U) in 8 M urea in 0.1 M NaOAc buffer, pH 4.6. This reaction was the slowest of the experiments carried out (Figure 7, dotted line), and was accompanied by slow formation of a IR17K(U)-derived byproduct which by MALDI-TOF was 42.6 amu higher than the starting monomer. It is known that in aqueous solution, urea is in equilibrium with ammonium cyanate (26, 27), and that cyanate reacts with amino groups to yield carbamyl derivatives (26). It is reasonable to propose that, over the long time course of reaction in the presence of urea [where the urea adduct


of IIL29K(J) is particularly unreactive], the Aoa moiety of IR17K(U) eventually reacts with the slowly accumulating cyanate. From these experiments, optimized conditions were established for the oxime-forming ligation. Equivalent amounts of purified IR17K(U) and IIL29K(J) (the latter obtained from periodate oxidation in 6 M GdnHCl in 0.1 M phosphate buffer, pH 7) were combined in 6 M GdnHCl in 0.1 M NaOAc buffer, pH 4.6. The course of the reaction was followed by analytical reversed-phase HPLC (Supporting Information Figure 1); after 3 days, no appreciable further product was formed, and product was isolated in 57% yield after SEC purification. Five further dimers (Figure 2) were created by the identical protocols with comparable yields. Evaluation of Conformational Integrity of OxCM Monomers Modified for Incorporation into Dimers. Having developed chemical means to combine two OxCM monomers and form the corresponding dimers, it was also necessary to check that the antiparallel β-sheet conformations sampled by OxCM are not significantly perturbed by the structural modifications that later facilitate cross-linking. Accordingly, each monomer was appropriately ‘capped’, and characterized by a spectroscopic criterion the reliability of which had been established in earlier work (1). Monomers of type I, with an (aminooxy)acetyl functionality, were treated with acetone, whereas monomers of type II, with a glyoxylyl functionality, were treated with CH3ONH2‚HCl (Figure 8; note the ‘*’ convention to indicate oxime capping). For each capped monomer, the CRH chemical shifts were determined as a function of residue number, and the deviation from literature random coil values (28, 29) was calculated. The baseline experiment (Figure 8, red traces) was the previously reported analysis of OxCM (1), in which residues 25-28 (a turn in native BPTI) show a negative deviation (upfield shift), and residues 18-24 and 29-35 (strands in native BPTI) show positive deviations (downfield shift). IR17K(U*), IR17K(U*G), IIL29K(J*), IIL29K(J*G), and IIL29Z(J*) have CRH chemical shift deviations similar to OxCM both in the strands and in the turn (Figure 8), consistent with the conclusion that the structures of these monomers do not differ significantly from that observed in OxCM. However, IR17Z(U*) has CRH chemical shift deviations similar to OxCM for turn residues but smaller CRH chemical shift deviations for strand residues. The latter result indicates that replacement of Arg17 in OxCM by the Dpr(Aoa) unit is a sufficiently drastic change to impact strand conformation. A more dramatic example for a poorly tolerated substitution was revealed with IIN24K(J*), where surrounding the modified site at residue 24, residue 23 which was in a strand for OxCM now appears to be in a turn, and residue 25 which was in the OxCM turn now is indistinguishable from random. Structural Characterization of the OxCM Dimers. Each synthesized OxCM dimer was evaluated by 1H NMR and size-exclusion chromatography. Synthetic dimers were dissolved to a concentration of 0.3 mM in CH3CN/10 mM aqueous NaCl (1:19) at pH 2, and applied at 3 °C to a size-exclusion chromatography column made of cross-linked agarose and dextran. The salt and CH3CN were included to suppress, respectively, ionic and hydrophobic interactions between the packing material and the proteins, whereas the pH was selected for consistency with what pilot NMR studies had shown to be most promising. Under these conditions, all synthesized proteins elute as a single peak in the order expected by their molecular weights, without any evidence for noncovalent association (i.e., they were physically monomeric, with


Carulla et al.

Figure 8. Along the right side are shown schematic representations, all drawn according to the same conventions used earlier (Figure 2), of capped monomers of (a) type I, replacing Arg17, and with acetone used to cap (aminooxy)acetyl functions; (b) type II, replacing Leu29, and with CH3ONH2‚HCl to cap glyoxylyl moieties; and (c) type II, replacing Asn24, again capped with CH3ONH2‚ HCl. Each panel compares the CRH chemical shift deviations, obtained at pH 4.5 and 3 °C, of OxCM (red trace) to a capped monomer (black trace) defined and named underneath, with an asterisk (*) used to indicate the presence of the appropriate oxime cap.

the preceding term not to be confused with the “monomer/ dimer” terminology used to describe the chemical constructs of this work) (Supporting Information Table 2). The potential for folding of each protein variant was qualitatively assessed by 1D 1H NMR spectra, recorded at 5 °C and pH 2 (Figure 9). Well-folded proteins have relatively narrow line widths, while conformational heterogeneity and increased internal mobility on the millisecond to microsecond time scale, which characterize molten globule and aggregated states, result in broad and/or heterogeneous line widths. Spectra of well-folded proteins are also characterized by pronounced chemical shift dispersion, which arises from the variety of unique, local magnetic environments present in a compact protein. To evaluate the best position for the cross-link, IR17K(U)IIN24K(J) was compared to IR17K(U)-IIL29K(J). The former is characterized by broad proton line widths and negligible chemical shift dispersion (Figure 9a), whereas the latter shows narrower line widths and considerable chemical shift dispersion (Figure 9b). The problems with IR17K(U)-

IIN24K(J) may have been anticipated by the NMR studies on IIN24K(J*) (see Figure 8c and accompanying discussion in previous section) which showed that replacement of Asn24 changes the structure of the monomer itself. In addition, the present working model for the antiparallel BPTI dimerization interface shows Arg17 to be closer to Leu29 (Figure 1b). Asn24, which on the basis of a simpler two-dimensional representation (Figure 1c) was predicted to be close to Arg17, is now believed to be on the opposite side due to the nativelike ‘twist’ of the β-sheets. With the I17-II29 optimal cross-link position established, variations in the length of the cross-link were considered next. Although intuition suggested that selfassociation between covalently joined monomers might be improved if they were closer, experiments showed better results with longer cross-links. 1D 1H NMR of IR17Z(U)-IIL29Z(J) (Figure 9c) and IR17Z(U)-IIL29K(J) (Figure 9d) show broad line widths and reduced chemical shift dispersion. These results can be explained in part by the NMR studies on IR17Z(U*) (Figure 8a, left), which showed a decrease in structure for the strand residues with


Figure 9. Aromatic and amide regions of 1D 1H NMR spectra at 600 MHz, obtained at pH 2 and 5 °C, are shown for OxCM dimers (a) IR17K(U)-IIN24K(J), (b) IR17K(U)-IIL29K(J), (c) IR17Z(U)IIL29Z(J), (d) IR17Z(U)-IIL29K(J), (e) IR17K(UG)-IIL29K(JG), and (f) IR17K(U)IIL29K(JG).


respect to OxCM. In addition, unwanted steric interactions between monomers may be a factor. 1D 1H NMR spectra of IR17K(UG)-IIL29K(JG) (Figure 9e) and IR17K(U)IIL29K(JG) (Figure 9f) show narrower line widths and similar chemical shift dispersion, by comparison to IR17K(U)-IIL29K(J), demonstrating the advantages of a longer cross-link. Preliminary Characterization of OxCM Dimers IR17K(U)-IIL29K(J) and IR17K(UG)-IIL29K(JG). The first dimer that gave evidence of folded structure beyond what is inherent in the OxCM monomer was IR17K(U)-IIL29K(J). Strong evidence in further support of this conclusion comes from 1D 1H/2H isotope exchange studies at pH 2 and 5 °C, monitored in the region 7.5-10.5 ppm where the amide protons resonate (Supporting Information Figure 2). While a subset (∼36%) of the protons exchange within a day, the majority of the amides (particularly the more downfield ones) are still observable after 6 days. The most downfield NH at pH 2 and 5 °C (labeled ‘a’ in Supporting Information Figure 2) was sufficiently resolved from the amide envelope to allow determination of the hydrogen isotope exchange rate (kobs ∼ 10-4 min-1, t1/2 ∼ 110 h). By comparison, exchange of strand residues in OxCM (t1/2 ∼ 1 h, pH 4.1 and 6 °C) is 2 orders of magnitude faster, and exchange in the most protected regions of a partially folded protein such as [14-38]Abu (30) (t1/2 ∼ 10 h, pH 4.6 and 1 °C) is 1 order of magnitude faster. Of additional promise, the 1D 1H NMR spectrum of IR17K(U)-IIL29K(J) in 2H2O at pH 2 at 5 °C shows a characteristic singlet at the unusually upfield position of 0.35 ppm. This signal is likely due to a methyl group from an aliphatic residue side chain that is in a structured environment. As the temperature was increased to 60 °C, the upfield signal decreased in a cooperative transition with an apparent Tm ∼39 °C. By comparison, the transition of [14-38]Abu at pH 6 occurs at ∼19 °C (31). Efforts to characterize the structure of the IR17K(U)IIL29K(J) dimer further were stymied by the fact that relatively few cross-peaks were observed in the TOCSY spectra. These findings, while disappointing, reflect a situation precedented in the literature for partially folded proteins (32). One possible explanation recognizes multiple conformations in chemical exchange, leading to relatively short T2 relaxation times which in turn broaden peaks to the extent that they are not recognized. The iterative development of dimers to date culminates with IR17K(UG)-IIL29K(JG), the 1D 1H NMR spectrum (Figure 9e) of which shows narrow line widths and good dispersion. TOCSY spectra show considerably more crosspeaks, allowing for optimism on the prospects for further structural analysis. Conclusions. Optimized syntheses of six OxCM dimers via oxime-forming ligation have been worked out. Issues that have been investigated include practical aspects of the preparation and manipulation of (aminooxy)acetylfunctionalized peptides, as well as the effects of using denaturants (GdnHCl or urea) in the buffers to dissolve glyoxylyl-functionalized peptides, or their precursor peptides, for further steps. In the latter regard, it is recommended to avoid the use of urea entirely, and to use GdnHCl only at the lowest possible concentration consistent with peptide solubility. Three of the dimers, IR17K(U)-IIL29K(J), IR17K(U)-IIL29K(JG), and IR17K(UG)-IIL29K(JG), were judged by 1D 1H NMR spectroscopy to show evidence of compact structure. Among them, IR17K(UG)-IIL29K(JG) seems to be particularly suited for further structural characterization. Such stud-


ies may ultimately validate the proposed strategy to create new proteins by combining core modules. ACKNOWLEDGMENT

We thank Drs. Fernando Albericio, Jordi Alsina, Dominique Lelie`vre, and Scott Yokum for helpful discussions on the synthetic work and Drs. David Live and Letitia Yao for help with NMR. NMR experiments were performed at the University of Minnesota High Field NMR Laboratory, and mass spectrometry data were collected at the University of Minnesota Mass Spectrometry Consortium for the Life Sciences. N.C. holds a Doctoral Dissertation Fellowship from the Graduate School of the University of Minnesota. Experimental work was supported by National Institutes of Health Grant GM 51628 (G.B. and C.W.). Supporting Information Available: Detailed general materials, instrumentation, and procedures for synthesis, spectroscopy, chromatography, and analysis. Additional information about syntheses and characterizations of peptides not covered in the representative text descriptions. Figures documenting HPLC analysis to monitor oxime-forming ligation, and 1H/2H isotope exchange of the OxCM dimer IR17K(U)-IIL29K(J). Detailed schemes outlining syntheses and cleavages of OxCM monomers of type I and type II. Tables showing complete 1H NMR assignments for the model peptide TP, and presenting elution behavior of the OxCM dimers on a SEC column (12 pages). This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Carulla, N., Woodward, C., and Barany, G. (2000) Synthesis and Characterization of a β-Hairpin Peptide That Represents a ‘Core Module’ of Bovine Pancreatic Trypsin Inhibitor (BPTI). Biochemistry 39, 7927-7937. (2) Gallagher, W. H., and Woodward, C. K. (1989) The Concentration Dependence of the Diffusion Coefficient for Bovine Pancreatic Trypsin Inhibitor: A Dynamic Light Scattering Study of a Small Protein. Biopolymers 28, 2001-2024. (3) Ilyina, E., Roongta, V., Pan, H., Woodward, C., and Mayo, K. H. (1997) A Pulsed-Field Gradient NMR Study of Bovine Pancreatic Trysin Inhibitor Self-Association. Biochemistry 36, 3383-3388. (4) Zielenkiewicz, P., Georgalis, Y., and Saenger, W. (1991) SelfAssociation of Bovine Pancreatic Trypsin Inhibitor: Specific or Nonspecific? Biopolymers 31, 1347-1349. (5) Rose, K. (1994) Facile Synthesis of Homogeneous Artificial Proteins. J. Am. Chem. Soc. 116, 30-33. (6) Shao, J., and Tam, J. P. (1995) Unprotected Peptides as Building Blocks for the Synthesis of Peptide Dendrimers with Oxime, Hydrazone, and Thiazolidine Linkages. J. Am. Chem. Soc. 117, 3893-3899. (7) Rose, K., Zeng, W., Regamey, P. O., Chernushevich, I. V., Standing, K. G., and Gaertner, H. F. (1996) Natural Peptides as Building Blocks for the Synthesis of Large Protein-like Molecules with Hydrazone and Oxime Linkages. Bioconjugate Chem. 7, 552-556. (8) Tuchscherer, G., Lehmann, C., and Mathieu, M. (1998) New Protein Mimetics: The Zinc Finger Motif as a Locked-In Tertiary Fold. Angew. Chem., Int. Ed. Engl. 37, 2990-2993. (9) Scheibler, L., Dumy, P., Boncheva, M., Leufgen, K., Mathieu, H.-J., Mutter, M., and Vogel, H. (1999) Functional Molecular Thin Films: Topological Templates for the Chemoselective Ligation of Antigenic Peptides to Self-Assembled Monolayers. Angew. Chem., Int. Ed. Engl. 38, 696-699. (10) Canne, L. E., Ferre´-D’Amare´, A. R., Burley, S. K., and Kent, S. B. H. (1995) Total Chemical Synthesis of a Unique Transcription Factor-Related Protein: cMyc-Max. J. Am. Chem. Soc. 117, 2998-3007.

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