Gregg B. Fields

Human matrix metalloproteinase specificity studies using collagen sequence‐based synthetic peptides

The matrix metalloproteinase (MMP)/matrixin family has been implicated in both normal tissue remodeling and a variety of diseases associated with abnormal turnover of extracellular matrix components. To better understand MMP behaviors and to aid in the design of MMP inhibitors, a variety of sequence specificity studies have been performed using collagen sequence-based peptides and MMP family members. Results of these studies have been valuable for defining the differences in MMPs and for creating fluorogenic substrates that can continuously monitor MMP activity. However, these studies have also demonstrated that these peptides may not be very good models of native MMP substrates, and that the additivity principle is not always applicable for designing synthetic MMP substrates.

Proteinlike molecular architecture: Biomaterial applications for inducing cellular receptor binding and signal transduction

The development of biomaterials with desirable biocompatibility has presented a difficult challenge for tissue engineering researchers. First and foremost, materials themselves tend to be hydrophobic and/or thrombogenic in nature, and face compatibility problems upon implantation. To mediate this problem, researchers have attempted to graft proteins or protein fragments onto biomaterial surfaces to promote endothelial cell attachment and minimize thrombosis. We envisioned a novel approach, based on the capability of biomolecules to self-assemble into well-defined and intricate structures, for creating biomimetic biomaterials that promote cell adhesion and proliferation. One of the most intriguing self-assembly processes is the folding of peptide chains into native protein structures. We have developed a method for building proteinlike structural motifs that incorporate sequences of biological interest. A lipophilic moiety is attached onto an Nα-amino group of a peptide chain, resulting in a “peptide-amphiphile.” The alignment of amphiphilic compounds at the lipid-solvent interface is used to facilitate peptide alignment and structure initiation and propagation, while the lipophilic region adsorbs to hydrophobic surfaces. Peptide-amphiphiles containing potentially triple-helical or α-helical structural motifs have been synthesized. The resultant head group structures have been characterized by CD spectroscopy and found to be thermally stable over physiological temperature ranges. Triple-helical peptide-amphiphiles have been applied to studies of surface modification and cell receptor binding. Cell adhesion and spreading was promoted by triple-helical peptide-amphiphiles. Cellular interaction with the type IV collagen sequence α1(IV) 1263–1277 increased signal transduction, with both the time and level of induction dependent upon triple-helical conformation. Collectively, these results suggest that peptide-amphiphiles may be used to form stable molecular structures on biomaterial surfaces that promote cellular activities and improve biocompatibility.

Micropatterning gradients and controlling surface densities of photoactivatable biomolecules on self-assembled monolayers of oligo(ethylene glycol) alkanethiolates

BACKGROUND Bioactive molecules that are covalently immobilized in patterns on surfaces have previously been used to control or study cell behavior such as adhesion, spreading, movement or differentiation. Photoimmobilization techniques can be used, however, to control not only the spatial pattern of molecular immobilization, termed the micropattern, but also the surface density of the molecules--a characteristic that has not been previously exploited. RESULTS Oligopeptides containing the bioactive Arg-Gly-Asp cell-adhesion sequence were immobilized upon self-assembled monolayers of an oligo(ethylene glycol) alkanethiolate in patterns that were visualized and quantified by autoradiography. The amount and pattern of immobilized peptide were controlled by manipulating the exposure of the sample to a UV lamp or a laser beam. Patterns of peptides, including a density gradient, were used to control the location and number of adherent cells and also the cell shape. CONCLUSIONS A photoimmobilization technique for decorating surfaces with micropatterns that consist of variable densities of bioactive molecules is described. The efficacy of the patterns for controlling cell adhesion and shape has been demonstrated. This technique is useful for the study of cell behavior on micropatterns.

Sequence dependence of aspartimide formation during 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis

We have examined the sequence dependence of aspartimide formation during Fmoc-based solid-phase synthesis of the peptide Val-Lys-Asp-X-Tyr-Ile. The extent of aspartimide formation and subsequent conversion to the α- or β-piperidide was characterized and quantitated by analytical reversed-phase high-performance liquid chromatography and fast atom bombardment mass spectrometry. Aspartimide formation occurred for X=Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Acm), Gly, Ser, Thr and Thr(tBu). No single approach was found that could inhibit this side reaction for all sequences. The most effective combinations, in general, for minimization of aspartimide formation were (i) tert-butyl side-chain protection of aspartate, piperidine for removal of the Fmoc group, and either 1-hydroxybenzotriazole or 2,4-dinitrophenol as an additive to the piperidine solution; or (ii) 1-adamantyl side-chain protection of aspartate and 1,8-diazabicyclo[5.4.0]undec-7-ene for removal of the Fmoc group.

Minimization of tryptophan alkylation following 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis

Abstract We have examined Pmc and Pbf side-chain protection of Arg and Boc side-chain protection of Trp in an attempt to minimize side-chain protecting group “scavenger” use following Fmoc-based solid-phase synthesis. The extent of Trp alkylation was characterized and quantified by analytical RP-HPLC. Edman degradation sequence analysis, and ESMS. The Pbf group offered lower TFA-induced Trp alkylation than the Pmc group. The combination of Trp(Boc) and Arg(Pbf) resulted in extremely low levels of Trp alkylation during TFA treatment of the peptide-resin in the absence of scavengers.

Trifluoroacetic acid cleavage and deprotection of resin-bound peptides following synthesis by Fmoc chemistry.

Publisher Summary The successful assembly of a peptide sequence represents only half the challenge of solid-phase peptide synthesis (SPPS). The side-chain protecting groups and linkers designed for fluorenylmethyloxycarbonyl (Fmoc) protocols are labile to trifluoroacetic acid (TFA). The procedure described is suitable for the various linkers cleaved by TFA, whether producing C-terminal acids or amides. Commercially available and suggested linker-resins for generating peptide acids are 4-alkoxybenzyl alcohol, 4-hydroxymethylphenoxyacetic acid (HMPA/PAB), and 2 chlorotrityl chloride. Under certain circumstances, TFA can cleave both at the linker-peptide bond and at the attachment point of the linker to the resin. Consequently, two events may occur. The free carbonium ion of the linker may alkylate Trp, with the resulting peptide exhibiting a more hydrophobic high-performance liquid chromatography (HPLC) elution behavior and a higher mass than the desired peptide. In addition, the cleavage yield may be noticeably reduced due to a reaction between a Trp or Cys residue and the linker ion while still attached to the resin. In a study of the correlation between the proximity of Trp to Arg in a sequence and extent of modification of Trp, it was found that this side reaction is greatest when Trp and Arg are separated by one residue.

Solid-Phase Peptide Synthesis

B. Merrifield, Concept and Early Development of Solid-Phase Peptide Synthesis. Methods for Solid-Phase Assembly of Peptides: P.F. Alewood, D. Alewood, L. Miranda, S. Love, W. Meutermans, and D. Wilson, Rapid in Situ Neutralization Protocols for Boc and Fmoc Solid-Phase Chemistries. J.M. Stewart, Cleavage Methods Following Boc-Based Solid-Phase Peptide Synthesis. D.A. Wellings and E. Atherton, Standard Fmoc Protocols. C.G. Fields and G.B. Fields, Trifluoroacetic Acid Cleavage and Deprotection of Resin-Bound Peptides Following Synthesis by Fmoc Chemistry. M. Meldal, Properties of Solid Supports. F. Albericio and L.A. Carpino, Coupling Reagents and Activation. M.F. Songster and G. Barany, Handles in Solid-Phase Peptide Synthesis. C. Blackburn and S.A. Kates, Solid-Phase Synthesis of Cyclic Homodetic Peptides. I. Annis, B. Hargittai, and G. Barany, Disulfide Bond Formation in Peptides. J. Kihlberg, M. Elofsson, and L.A. Salvador, Direct Synthesis of Glycosylated Amino Acids from Carbohydrate Peracetates and Fmoc Amino Acids: Solid-Phase Synthesis of Biomedicinally Interesting Glycopeptides. J.W. Perich, Synthesis of Phosphopeptides Using Modern Chemical Approaches. S.B.H. Kent, Chemoselective Ligation. A.C. Braisted, J.K. Judice, and J.A. Wells, Synthesis of Proteins by Subtiligase. F. Albericio, P. Lloyd-Williams, and E. Giralt, Convergent Solid-Phase Synthesis. M. Lebl and V.Krchnak, Peptide Libraries. Analytical Techniques: G.A. Grant, M.W. Crankshaw, and J. Gorka, Edman Sequencing as Tool for Characterization of Synthetic Peptides. A.J. Smith, Amino Acid Analysis. C.T. Mant, L.H. Kondejewski, P.J. Cachia, O.D. Monera, and R.S. Hodges, Analysis of Synthetic Peptides by High-performance Liquid Chromatography. A. Sanchez and A.J. Smith, Capillary Electrophoresis. S. Beranova-Giorgianni and D.M. Desiderio, Fast Atom Bombardment Mass Spectrometry of Synthetic Peptides. D.J. Burdick and J.T. Stults, Analysis of Peptide Synthesis Products by Electrospary Ionization Mass Spectrometry. W.T. Moore, Laser Desorption. Specialized Applications: T.W. Muir, P.E. Dawson, M.C. Fitzgerald, and S.B.H. Kent, Protein Signature Analysis for Studying Structure-Activity Relationships in Peptides and Proteins. J.L. Lauer and G.B. Fields, In Vitro Incorporation of Synthetic Peptides into Cells. Y-C. Yu, T. Pakalns, Y.Dori, J.B. McCarthy, M. Tirrell, and G.B. Fields, Construction of Biologically Active Protein Molecular Architecture Using Self-Assembling Peptide-Amphiphiles. E. Barbar, C. Woodward, and G. Barany, Nuclear Magnetic Resonance Characterization of Synthetically Derived Partially Folded Proteins. J.P. Tam and J.C. Spetzler, Multiple Antigen Peptide System. J.D. Wade and G.W. Tregear, Relaxin. K.H. Mayo, Solution Nuclear Magnetic Resonance Characterization of Peptide Folding. T.A. Cross, Solid-State NMR Characterization of the Gramicidin Channel Structure. R.H. Angeletti, L.F. Bonewald, G.B. Fields, Six-Year Study of Peptide Synthesis. Index.

CONSTRUCTION OF BIOLOGICALLY ACTIVE PROTEIN MOLECULAR ARCHITECTURE USING SELF-ASSEMBLING PEPTIDE-AMPHIPHILES

The peptide-amphiphiles described here provide a simple approach for building stable protein structural motifs using peptide head groups. One of the most intriguing features of this system is the possible formation of stable lipid films on solid substrates, or the use of the novel amphiphiles in bilayer membrane systems, where the lipid tail serves not only as a peptide structure-inducing agent but also as an anchor of the functional head group in the lipid assembly. The peptide-amphiphile system potentially offers great versatility with regard to head and tail group composition and overall geometries and macromolecular structures. For building materials with molecular and cellular recognition capacity, it is essential to have a wide repertoire of tools to produce characteristic supersecondary structures at surfaces and interfaces.

Solid-phase synthesis of triple-helical collagen-model peptides*

The triple-helical conformation of collagen has been proposed to be important for mediation of cellular activities, such as adhesion and activation, extracellular matrix assembly, and enzyme function. We have developed synthetic protocols that allow for the study of biological activities of specific collagen sequences in triple-helical conformation. These methods primarily involve solid-phase assembly and covalent linkage of three peptide chains. The resultant triple-helical peptides have sufficient thermal stabilities to permit structural and biological characterization under physiological conditions. The present article critically reviews the various approaches for constructing synthetic triple-helices.

Formation of a Disulfide Bond in an Octreotide-Like Peptide: A Multicenter Study

Publisher Summary This chapter describes the formation of a disulfide bond in an octreotide-like peptide. Producing cyclized peptides with the correct structure can be achieved readily by either on-resin or post-cleavage techniques. Post-cleavage techniques are less expensive and provide reasonable yields of the desired product. However, on-resin techniques produce greater yields of the final product, but are more expensive to perform. It is best to use a combination of analytical techniques which complement each other in their ability to detect the correct product and unwanted byproducts. Coded samples of unpurified peptides can be characterized by amino acid analysis (AAA), high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted, laser desorption ionization mass spectrometry (MALDI-MS), Edman sequencing, and spectrophotometric analysis following treatment with Ellmans reagent. These methods permit quantitation and evaluation of purity, and a measure of the amount of oxidized peptide.