Solid-phase Peptide Synthesis Chemistry Research Articles

OaAEP1 Ligase-Assisted Chemoenzymatic Synthesis of Full Cysteine-Rich Metal-Binding Cyanobacterial Metallothionein SmtA

Among all approaches used for the semisynthesis of natural or chemically modified products, enzyme-assisted ligation is among the most promising and dynamically developing approaches. Applying an efficient C247A mutant of Oldenlandia affinis plant ligase OaAEP1 and solid-phase peptide synthesis chemistry, we present the chemoenzymatic synthesis of a complete sequence of the cysteine-rich and metal-binding cyanobacterial metallothionein Synechococcus metallothionein A (SmtA). Zn(II) and Cd(II) binding to the newly synthesized SmtA showed identical properties to the protein expressed in Escherichia coli. The presented approach is the first example of the use of OaAEP1 mutant for total protein synthesis of metallothionein, which occurs in mild conditions preventing cysteine thiol oxidation. The recognition motif of the applied enzyme could naturally occur in the protein structure or be synthetically or genetically incorporated in some loops or secondary structure elements. Therefore, we envision that this strategy can be used for efficiently obtaining SmtA and for a wide range of proteins and their derivatives.

68Ga-Labeled Cystine Knot Peptide Targeting Integrin αvβ6 for Lung Cancer PET Imaging.

Integrin αvβ6 has been considered as a promising biomarker for lung cancer, and its expression is often related to poor prognosis. An αvβ6-binding cystine knot peptide R01-MG was previously engineered and validated. Here, we developed a positron emission tomography (PET) probe of R01-MG for imaging αvβ6-positive lung cancer. Cystine knot peptide R01-MG was synthesized through solid-phase peptide synthesis chemistry and radiolabeled with 68Ga after being conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA). The stability of 68Ga-DOTA-R01-MG was analyzed in phosphate-buffered saline (PBS) (pH 7.4) and fetal bovine serum (FBS). The cell uptake assay of the probe was evaluated using αvβ6-positive (A549 and H1975) and αvβ6-negative (H1299) lung cancer cell lines. In addition, small animal PET imaging and biodistribution studies of 68Ga-DOTA-R01-MG were performed in αvβ6-positive and αvβ6-negative lung cancer models. Our study showed that 68Ga-DOTA-R01-MG exhibited excellent stability in PBS and FBS. Small animal PET imaging and biodistribution data revealed that 68Ga-DOTA-R01-MG displayed rapid and good tumor uptake in animal models with αvβ6-positive lung cancer, and the probe was rapidly cleared from the normal tissues, resulting in good tumor-to-normal tissue contrasts. Meanwhile, no obvious tumor uptake of 68Ga-DOTA-R01-MG was observed in animal models with αvβ6-negative lung cancer, demonstrating specific binding of the probe to integrin αvβ6. In conclusion, 68Ga-DOTA-R01-MG has great potential to be a promising PET tracer for imaging αvβ6-positive lung cancer.

Computational-aided design: minimal peptide sequence to block dengue virus transmission into cells

Dengue virus (DV) infection is one of the main public health concerns, affecting approximately 390 million people worldwide, as reported by the World Health Organization. Yet, there is no antiviral treatment for DV infection. Therefore, the development of potent and nontoxic anti-DV, as a complement for the existing treatment strategies, is urgently needed. Herein, we investigate a series of small peptides inhibitors of DV antiviral activity targeting the entry process as the promising strategy to block DV infection. The peptides were designed based on our previously reported peptide sequence, DN58opt (TWWCFYFCRRHHPFWFFYRHN), to identify minimal effective inhibitory sequence through molecular docking and dynamics studies. The in silico designed peptides were synthesized using conventional Fmoc solid-phase peptide synthesis chemistry, purified by RP-HPLC and characterized using LCMS. Later, they were screened for their antiviral activity. One of the peptides, AC 001, was able to reduce about 40% of DV plaque formation. This observation correlates well with the molecular mechanics-Poisson–Boltzmann surface area (MM-PBSA) analysis – AC 001 showed the most favorable binding affinity through 60 ns simulations. Pairwise residue decomposition analysis has revealed four key residues that contributed to the binding of these peptides into the DV2 E protein pocket. This work identifies the minimal peptide sequence required to inhibit DV replication and explains the behavior observed on an atomic level using computational study. Communicated by Ramaswamy H. Sarma

Total synthesis of bovine pancreatic trypsin inhibitor and the protein diastereomer [Gly37D‐Ala]BPTI using Boc chemistry solid phase peptide synthesis

AbstractBovine pancreatic trypsin inhibitor (BPTI) is a well‐studied model for investigation of protein folding and stability. Here, we report the synthesis and characterization of wild‐type BPTI and a diastereomeric protein analogue [Gly37D‐Ala]BPTI. Each 58‐residue polypeptide chain was made by native chemical ligation of two peptide segments, BPTI[1‐29]‐αthioester and the appropriate version of the Cys30‐58 BPTI peptide segment. Boc chemistry in situ neutralization solid phase synthesis was used to prepare the peptide segment reactants. The resulting full‐length polypeptide chains were folded in a cysteine/cystine redox buffer to give synthetic protein molecules containing three disulfide bonds. The diastereomeric analogue [Gly37D‐Ala]BPTI folded as efficiently as the native protein. Synthetic proteins were characterized by analytical LCMS and by natural‐abundance 1H‐15N HSQC NMR fingerprinting. These results illustrate the power of Boc chemistry peptide synthesis and its utility for the total chemical synthesis of protein molecules.

Synthesis and Application of a Long-Circulating Radiolabeled Peptide for Targeting of Osteosarcoma.

The small peptide TMTP1 (NVVRQ) has been proved to target a series of highly metastatic tumor cells. The aim of this study was to develop a new agent based on TMTP1 conjugated with Evans blue (EB), to increase tumor uptake and modify the pharmacokinetic characteristics of the resulting radiolabeled agent. DOTA-EB-TMTP1 was prepared through conventional solid-phase peptide synthesis chemistry. Then, it was successfully labeled with Cu-64 to obtain [64Cu]DOTA-EB-TMTP1. The tumor targeting properties were evaluated in vivo using 143B xenografts. DOTA-EB-TMTP1 was successfully labeled with Cu-64 in a yield of 87.3 ± 5.2%. In a small animal positron emission tomography/X-ray computed tomography (PET/CT) study in osteosarcoma 143B xenograft mice, [64Cu]DOTA-EB-TMTP1 was found to rapidly accumulate in the tumor tissue. The tumor uptake increased over time and reached a plateau of 6.50 ± 0.88% ID/g 8h after tail vein injection. The radioactivity remained in the tumor tissue 48h postinjection with a negligible decrease. Overall, the introduction of the EB motif to TMTP1 significantly changed its pharmacokinetics in vivo, and this strategy fulfills the purpose of prolonging the blood circulation and enhancing the tumor uptake. [64Cu]DOTA-EB-TMTP1 is a promising agent for osteosarcoma targeting. Moreover, our study highlights that DOTA-EB-TMTP1 is a good candidate for labeling with different radionuclides for potential theranostic applications.

Benzimidazolinone-Free Peptide o-Aminoanilides for Chemical Protein Synthesis.

The thioester surrogate 3,4-diaminobenzoic acid (Dbz) facilitates the efficient synthesis of peptide thioesters by Fmoc chemistry solid phase peptide synthesis and the optional attachment of a solubility tag at the C-terminus. The protection of the partially deactivated ortho-amine of Dbz is necessary to obtain contamination-free peptide synthesis. The reported carbamate protecting groups promote a serious side reaction, benzimidazolinone formation. Herein we introduce the Boc-protected Dbz that prevents the benzimidazolinone formation, leading to clean peptide o-aminoanilides suitable for the total chemical synthesis of proteins.

Detecting Vulnerable Atherosclerotic Plaques by 68Ga-Labeled Divalent Cystine Knot Peptide

Integrin αvβ3 has been considered as a promising biomarker for vulnerable atherosclerotic plaques, and it is highly expressed by those instability-associated factors, such as macrophages, vessel endothelial cells, and smooth muscle cells. Our previous study successfully showed that the 64Cu-labeled divalent (containing two RGD motifs) cystine knot peptide, 64Cu-NOTA-3-4A, had high binding affinity and specificity in targeting vulnerable carotid atherosclerotic plaques with increased αvβ3 levels. Therefore, considering that 68Ga has excellent nuclear physical properties for positron emission tomography (PET), this study aimed to investigate the feasibility of using 68Ga-NOTA-3-4A for PET study of vulnerable atherosclerotic plaques. The vulnerable carotid atherosclerotic plaques were induced and maintained in ApoE-/- mice through carotid artery ligation and a high-fat diet. Divalent knottin peptide 3-4A was synthesized through solid-phase peptide synthesis chemistry and radiolabeled with 68Ga after being conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The probe stability was analyzed in phosphate buffered saline (PBS) buffer and mouse serum. ApoE-/- mice with atherosclerotic plaques ( n = 4) were imaged by PET/CT at 1 and 2 h after the tail vein injection of 68Ga-NOTA-3-4A. The targeting specificity was determined by coinjection of 68Ga-NOTA-3-4A and nonradioactive c(RGDyK) peptide. The carotid artery tissues were removed, and immunofluorescent staining was performed to evaluate αvβ3 integrin expression. It was found that 68Ga-NOTA-3-4A displayed high stability in both PBS buffer and mouse serum. Small animal PET/CT images and quantification analysis indicated the quick and high plaque uptake of 68Ga-NOTA-3-4A (6.67 ± 1.44 and 2.97 ± 0.46%ID/g at 1 and 2 h, respectively). The plaque-to-normal artery ratio was 15.88 and 9.90 at 1 and 2 h, respectively. Furthermore, the plaque accumulation of 68Ga-NOTA-3-4A was significantly inhibited via coinjection of c(RGDyK). Finally, immunostaining identified integrin αvβ3 expressed by macrophages, vessel endothelial cells, and smooth muscle cells. In summary, 68Ga-NOTA-3-4A has high potential to be a promising PET tracer for imaging vulnerable atherosclerotic plaques.

Scope and Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin.

We have systematically explored three approaches based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry solid phase peptide synthesis (SPPS) for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the "hybrid method", in which maximally protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[O-β-(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High-resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies could yield an efficient total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS.

Obviation of hydrogen fluoride in Boc chemistry solid phase peptide synthesis of peptide-αthioesters.

Under suitable conditions, trifluoromethanesulfonic acid performs comparably to hydrogen fluoride for the on-resin global deprotection of peptides prepared by Boc chemistry solid phase peptide synthesis (SPPS). Obviation of hydrogen fluoride in Boc chemistry SPPS enables the straightforward synthesis of peptide-αthioesters for use in native chemical ligation.

64Cu-Labeled Divalent Cystine Knot Peptide for Imaging Carotid Atherosclerotic Plaques

The rupture of vulnerable atherosclerotic plaques that lead to stroke and myocardial infarction may be induced by macrophage infiltration and augmented by the expression of integrin αvβ3. Indeed, atherosclerotic angiogenesis may be a promising marker of inflammation. In this study, an engineered integrin αvβ3-targeting PET probe, (64)Cu-NOTA-3-4A, derived from a divalent knottin miniprotein was evaluated in a mouse model for carotid atherosclerotic plaques. Atherosclerotic plaques in BALB/C mice, maintained on a high-fat diet, were induced with streptozotocin injection and carotid artery ligation and verified by MR imaging. Knottin 3-4A was synthesized by solid-phase peptide synthesis chemistry and coupled to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) before radiolabeling with (64)Cu. PET probe stability in mouse serum was evaluated. Mice with carotid atherosclerotic plaques were injected via the tail vein with (64)Cu-NOTA-3-4A or (18)F-FDG, followed by small-animal PET/CT imaging at different time points. Receptor targeting specificity of the probe was verified by coinjection of c(RGDyK) administered in molar excess. Subsequently, carotid artery dissection and immunofluorescence staining were performed to evaluate target expression. (64)Cu-NOTA-3-4A was synthesized in high radiochemical purity and yield and demonstrated molecular stability in both phosphate-buffered saline and mouse serum at 4 h. Small-animal PET/CT showed that (64)Cu-NOTA-3-4A accumulated at significantly higher levels in the neovasculature of carotid atherosclerotic plaques (7.41 ± 1.44 vs. 0.67 ± 0.23 percentage injected dose/gram, P < 0.05) than healthy or normal vessels at 1 h after injection. (18)F-FDG also accumulated in atherosclerotic lesions at 0.5 and 1 h after injection but at lower plaque-to-normal tissue ratios than (64)Cu-NOTA-3-4A. For example, plaque-to-normal carotid artery ratios for (18)F-FDG and (64)Cu-NOTA-3-4A at 1 h after injection were 3.75 and 14.71 (P < 0.05), respectively. Furthermore, uptake of (64)Cu-NOTA-3-4A in atherosclerotic plaques was effectively blocked (∼90% at 1 h after injection) by coinjection of c(RGDyK). Immunostaining confirmed integrin αvβ3 expression in both the infiltrating macrophages and the neovasculature of atherosclerotic plaques. (64)Cu-NOTA-3-4A demonstrates specific accumulation in carotid atherosclerotic plaques in which macrophage infiltration and angiogenesis are responsible for elevated integrin αvβ3 levels. Therefore, (64)Cu-NOTA-3-4A may demonstrate clinical utility as a PET probe for atherosclerosis imaging or for the evaluation of therapies used to treat atherosclerosis.

Total chemical synthesis of fully functional Photoactive Yellow Protein

The Photoactive Yellow Protein (PYP) is a structural prototype for the PAS superfamily of proteins, which includes hundreds of receptor and regulatory proteins from all three kingdoms of life. PYP itself is a small globular protein that undergoes a photocycle involving a series of conformational changes in response to light excitation of its p-coumaric acid chromophore, making it an excellent model system to study the molecular basis of signaling in the PAS super family. To enable novel chemical approaches to elucidating the structural changes that accompany signaling in PYP, we have chemically synthesized the 125 amino acid residue protein molecule using a combination of Boc chemistry solid phase peptide synthesis and native chemical ligation. Synthetic PYP exhibits the wildtype photocycle, as determined in photobleaching studies. Planned future studies include incorporation of site-specific isotopic labels into specific secondary structural elements to determine which structural elements are involved in signaling state formation using difference FTIR spectroscopy.

Radiofluorinated Rhenium Cyclized α-MSH Analogues for PET Imaging of Melanocortin Receptor 1

In order to accomplish in vivo molecular imaging of melanoma biomarker melanocortin 1 receptor (MC1R), several α-melanocyte-stimulating hormone (α-MSH) analogues have been labeled with N-succinimidyl-4-¹⁸F-fluorobenzoate (¹⁸)F-SFB) and studied as positron emission tomography (PET) probes in our recent studies. To further pursue a radiofluorinated α-MSH peptide with high clinical translation potential, we utilized 4-nitrophenyl 2-¹⁸F-fluoropropionate (¹⁸F-NFP) to radiofluorinate the transition metal rhenium cyclized α-MSH metallopeptides for PET imaging of MC1R positive malignant melanoma. Metallopeptides Ac-d,Lys-ReCCMSH(Arg¹¹) (two isomers, namely RMSH-1 and RMSH-2) were synthesized using conventional solid phase peptide synthesis chemistry and rhenium cyclization reaction. The two isomers were then conjugated with ¹⁹F-NFP or ¹⁸F-NFP. The resulting cold or radiofluorinated metallopeptides, (¹⁸/¹⁹)F-FP-RMSH-1 and (¹⁸/¹⁹)F-FP-RMSH-2, were further evaluated for their in vitro receptor binding affinities, in vivo biodistribution, and small-animal PET imaging properties. The binding affinities of ¹⁹F-FP-RMSH-1 and ¹⁹F-FP-RMSH-2 were determined to be within low nanomolar range. In vivo studies revealed that both F-labeled metallopeptides possessed good tumor uptake in the B16F10 murine model with high MC1R expression, while possessing much lower uptake in A375M human melanoma xenografts. Moreover, ¹⁸F-FP-RMSH-1 displayed more favorable in vivo performance in terms of higher tumor uptake and much lower accumulation in the kidney and liver, when compared to that of ¹⁸F-FP-RMSH-2 at 2 h postinjection (p.i.). ¹⁸F-FP-RMSH-1 also displayed lower liver and lung uptake when compared with that of the same peptide labeled with ¹⁸F-SFB (named as ¹⁸F-FB-RMSH-1). Small animal PET imaging of ¹⁸F-FP-RMSH-1 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptake and poorer tumor/normal organ contrast were observed for A375M model compared to those of the B16F10 model. ¹⁸F-FP-RMSH-1 also exhibited higher tumor uptake and better tumor retention when compared with ¹⁸F-FB-RMSH-1. ¹⁸F-FP-RMSH-1 demonstrates significant advantages over ¹⁸F-FB-RMSH-1 and ¹⁸F-FP-RMSH-2. It is a promising PET probe for imaging MC1R positive melanoma and MC1R expression in vivo.

Jeffamine Derivatized TentaGel Beads and Poly(dimethylsiloxane) Microbead Cassettes for Ultrahigh-Throughput in Situ Releasable Solution-Phase Cell-Based Screening of One-Bead-One-Compound Combinatorial Small Molecule Libraries

A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated poly(dimethylsiloxane) (PDMS) cassette for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting trifunctional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry resulting in beads with increased loading capacity, hydrophilicity, and porosity at the outer layer. We have found that such bead configuration can facilitate ultrahigh-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 min) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel were then layered over the microbead cassette to immobilize the compound-beads. After 24 h of incubation at 37 °C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were resynthesized and found to be cytotoxic (IC(50) 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultrahigh-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays.

PET of Malignant Melanoma Using 18F-Labeled Metallopeptides

Melanocortin type 1 receptor (MC1R), also known as alpha-melanocyte-stimulating hormone (alpha-MSH) receptor, is an attractive molecular target for melanoma imaging and therapy. An (18)F-labeled linear alpha-MSH peptide ((18)F-FB-Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys-NH(2) [NAPamide]) shows promising melanoma imaging properties but with only moderate tumor uptake and retention. A transition metal rhenium-cyclized alpha-MSH peptide, ReO[Cys(3,4,10),d-Phe(7),Arg(11)]alpha-MSH(3-13) (ReCCMSH(Arg(11))), has shown high in vitro binding affinity to MC1R and excellent in vivo melanoma-targeting profiles when labeled with radiometals. Therefore, we hypothesized that ReCCMSH(Arg(11)) could be a good platform for the further development of an (18)F-labeled probe for PET of MC1R-positive malignant melanoma. In this study, the metallopeptide Ac-d-Lys-ReCCMSH(Arg(11)) was synthesized using conventional solid-phase peptide synthesis chemistry and a rhenium cyclization reaction. The resulting peptides were then labeled with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB). The (18)F-labeled metallopeptides were further tested for their in vitro receptor binding affinities, in vivo biodistribution, and PET imaging properties. Both isomers of Ac-d-Lys-ReCCMSH(Arg(11)), named as RMSH-1 and RMSH-2, were purified and identified by high-performance liquid chromatography. The binding affinities of RMSH-1 and RMSH-2 and their respective (19)F-SFB-conjugated peptides ((19)F-FB-RMSH-1 and (19)F-FB-RMSH-2) were all determined to be within nanomolar range. Both (18)F-labeled metallopeptides showed good tumor uptake in the B16F10 murine model, with high MC1R expression, but much lower uptake in the A375M human melanoma xenografted in mice, indicating low MC1R expression. (18)F-FB-RMSH-1, when compared with (18)F-FB-RMSH-2, displayed more favorable in vivo performance in terms of slightly higher tumor uptakes and much lower accumulations in the kidney and liver at 2 h after injection. Small-animal PET of (18)F-FB-RMSH-1 and -2 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptakes and poorer tumor-to-normal organ contrasts were observed for the A375M model than for the B16F10 model. (18)F-FB-RMSH-1 and -2 showed higher tumor uptake and better tumor retention than did (18)F-FB-NAPamide. Specific in vivo targeting of (18)F-FB-RMSH-1 to malignant melanoma was successfully achieved in preclinical models with high MC1R expression. Thus, the radiofluorinated metallopeptide (18)F-FB-RMSH-1 is a promising molecular probe for PET of MC1R-positive tumors.

Synthesis of Dideoxymycobactin Antigens Presented by CD1a Reveals T Cell Fine Specificity for Natural Lipopeptide Structures

Mycobacterium tuberculosis survival in cells requires mycobactin siderophores. Recently, the search for lipid antigens presented by the CD1a antigen-presenting protein led to the discovery of a mycobactin-like compound, dideoxymycobactin (DDM). Here we synthesize DDMs using solution phase and solid phase peptide synthesis chemistry. Comparison of synthetic standards to natural mycobacterial mycobactins by nuclear magnetic resonance and mass spectrometry allowed identification of an unexpected alpha-methyl serine unit in natural DDM. This finding further distinguishes these pre-siderophores as foreign compounds distinct from conventional peptides, and we provide evidence that this chemical variation influences the T cell response. One synthetic DDM recapitulated natural structures and potently stimulated T cells, making it suitable for patient studies of CD1a in infectious disease. DDM analogs differing in the stereochemistry of their butyrate or oxazoline moieties were not recognized by human T cells. Therefore, we conclude that T cells show precise specificity for both arms of the peptide, which are predicted to lie at the CD1a-T cell receptor interface.

Total chemical synthesis and biophysical characterization of the minimal isoform of the KChIP2 potassium channel regulatory subunit

The potassium channel accessory subunit KChIP2 associates with Kv4.2 channels in the cardiac myocyte and is involved in the regulation of the transient outward current (I(to)) during the early phase of repolarization of the action potential. As a first step to biophysically probe the mechanism of KChIP2, we have chemically synthesized its minimal isoform, KChIP2d, using Boc chemistry solid phase peptide synthesis in conjunction with native chemical ligation. The synthetic KChIP2d protein is primarily alpha-helical as predicted and becomes more structured upon binding calcium as assessed by (1)H-NMR and CD spectroscopy. Synthetic KChIP2d is in a monomer-dimer equilibrium in solution, and there is evidence for two monomer binding sites on an N-terminal peptide of Kv4.2. Planned future studies include the incorporation of fluorescent and spin labeled probes in KChIP2d to yield structural information in parallel with electrophysiologic studies to elucidate KChIP2d's mechanism of action.

Mass Spectrometry Identifiable Cross-Linking Strategy for Studying Protein−Protein Interactions

A new mass spectrometry identifiable cross-linking strategy has been developed to study protein-protein interactions. The new cross-linker was designed to have two low-energy MS/MS-cleavable bonds in the spacer chain to provide three primary benefits: First, a reporter tag can be released from cross-link due to cleavage of the two labile bonds in the spacer chain. Second, a relatively simple MS/MS spectrum can be generated owing to favorable cleavage of labile bonds. And finally, the cross-linked peptide chains are dissociated from each other, and each then can be fragmented separately to get sequence information. Therefore, this novel type of cross-linker was named protein interaction reporter (PIR). To this end, two RINK groups were utilized to make our first-generation cross-linker using solid-phase peptide synthesis chemistry. The RINK group contains a bond more labile than peptide bonds during low-energy activation. The new cross-linker was applied to cross-link ribonuclease S (RNase S), a noncovalent complex of S-peptide and S-protein. The results demonstrated that the new cross-linker effectively reacted with RNase S to generate various types of cross-linked products. More importantly, the cross-linked peptides successfully released reporter ions during selective MS/MS conditions, and the dissociated peptide chains remained intact during MS(2), thus enabling MS(3) to be performed subsequently. In addition, dead-end, intra-, and inter-cross-linked peptides can be distinguished by analyzing MS/MS spectra.

Convenient synthesis of human calcitonin and its methionine sulfoxide derivative

The human calcitonin peptide chain was assembled using Fmoc solid-phase peptide synthesis chemistry. The combinations of cleavage Reagent H with trans-[Pt(en) 2Cl 2] 2+ and Reagents B, K, and R with trans-[Pt(CN) 4Cl 2] 2− provide convenient methods for the synthesis of human calcitonin and its methionine sulfoxide derivative; the formation of intramolecular disulfide bonds by the above Pt(IV) oxidants is essentially quantitative.

Evaluation of the trifluoromethanosulfonic acid/trifluoroacetic acid/thioanisole cleavage procedure for application in solid-phase peptide synthesis.

As an extension of our investigation of peptidyl-resin linkage stability towards different cleavage procedures used in the solid-phase peptide synthesis (SPPS) technique, the present paper evaluated the trifluoromethanesulfonic acid (TFMSA)/trifluoroacetic acid (TFA)/thioanisole method, varying the type of resin (benzhydrylamine-resin, BHAR; methylbenzhydrylamine-resin, MBHAR and 4-(oxymethyl)-phenylacetamidomethyl-resin, PAMR) and peptide resin-bound residue (Gly and Phe). The vasoactive angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue linked to those resins used routinely in tert-butyloxycarbonyl (Boc)-SPPS chemistry were submitted comparatively to a time course study towards TFMSA/TFA cleavage. At 0 degrees C, [Gly8]-AII was completely removed from all resins in less than 6 h, but the hydrophobic Phe8 moiety-containing AII sequence was only partially cleaved (not more than 15%) from BHAR or MBHAR in this period. At 25 degrees C, [Gly8]-AII cleavage time decreased to less than 2 h irrespective of the solid support, and quantitative removal of AII from PAMR and MBHAR occurred in less than 3 h. However, about 10-15 h seemed to be necessary for cleavage of AII from BHAR, and in this extended cleavage reaction a significant increase in peptide degradation rate was observed. Regardless of the cleavage temperature used, the decreasing order of acid stability measured for resins was BHAR>MBHAR>PAMR. Collectively, these findings demonstrated the feasibility of applying TFMSA/TFA solution as a substitute for anhydrous HF at the cleavage step in Boc-SPPS methodology. Care should be taken however, as the cleavage efficacy depends on multiple factors including the resin, peptide sequence, the time and temperature of reaction.

Solid-phase precipitation and extraction, a new separation process applied to the isolation of synthetic peptides.

A new method for separation and purification is described. The process, referred to as solid-phase precipitation and extraction (SPPE), was developed and applied to postcleavage isolation of synthetic peptides. The technique uses normal approaches of chromatography and solid-phase extraction sorbents with a precipitation or drying procedure so that the sorbent becomes a support matrix for thin-film deposition of the compounds of interest. This procedure causes precipitated compounds of interest to be trapped on the large surface area or in the pores of the matrix so that by-products and impurities can be removed by strong wash solvents. In application to solid-phase peptide synthesis chemistry, by-products from the cleavage and deprotection are selectively extracted from the crude sample mixture under mild conditions. In comparison to the ether precipitation method used in peptide chemistry, the SPPE process provides isolated products that are 14-17% (w/w) higher purity.