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C G Fields

Publications and source records attributed to C G Fields.

14 recordsLinked to original sources
PubMed1997

Backbone and side-chain dynamics of residues in a partially folded beta-sheet peptide from platelet factor-4.

Structurally characterizing partially folded states is problematic given the nature of these transient species. A peptide 20mer, T38AQLIATLKNGRKISLDLQA57 (P20), which has been shown to partially fold in a relatively stable turn/loop conformation (LKNGR) and transient beta-sheet structure, is a good model for studying backbone and side-chain mobilities in a transiently folded peptide by using 13C-NMR relaxation. Here, four residues in P20, A43, T44, G48, and 151, chosen for their positions in or near the loop conformation and for compositional variety, have been selectively 13C-enriched. Proton-coupled and decoupled 13C-NMR relaxation experiments have been performed to obtain the temperature dependencies (278 K to 343 K) of auto- and cross-correlation motional order parameters and correlation times. In order to differentiate sequence-neighbor effects from folding effects, two shorter peptides derived from P20, IATLK (P5) and NGRKIS (P6), were similarly 13C-enriched and investigated. For A43, T44, G48, and 151 residues in P20 relative to those in P5/P6, several observations are consistent with partial folding in P20: (1) C alpha H motional tendencies are all about the same, vary less with temperature, and are relatively more restricted, (2) G48 C alpha H2 phi (t) psi (t) rotations are more correlated, and (3) methyl group rotations are slower and yield lower activation energies consistent with formation of hydrophobic "pockets." In addition, T44 and 151 C beta H mobilities in P20 are more restricted at lower temperature than those of their C alpha H and display significantly greater sensitivity to temperature suggesting a larger enthalpic contribution to side-chain mobility. Moreover, at higher temperatures, side-chain methyls and methylenes in P20 are more motionally restricted than those in P5/P6, suggesting that some type of "folded" or "collapsed" structure remains in P20 for what normally would be considered an "unfolded" state.

Amino Acid Sequence↗
PubMed1995

Purification and analysis of synthetic, triple-helical "minicollagens" by reversed-phase high-performance liquid chromatography.

To better study collagen-mediated cellular and enzymatic activities, a generally applicable solid-phase methodology has been developed by which aligned triple-helical peptides (designated THPs or "minicollagens") ranging from 79 to 124 residues can be assembled. Reversed-phase HPLC is typically the purification method of choice following chemical synthesis of small proteins of this size, as well as one of the analytical techniques used to verify product purity. We have thus compared the effects of different stationary phases (C18, C4, or diphenyl), organic modifiers (acetonitrile or isopropanol), support pore sizes (120 angstroms, 300 angstroms, or nonporous), and counterions for the reversed-phase HPLC analysis of THPs. Large pore C18 or C4 reversed-phase HPLC gave broad peaks, resulting in poor resolution of the desired THP from synthetic impurities. Broad peaks were presumably due to conformational instability of THPs to reversed-phase conditions and subsequent slow cis-trans isomerization of the peptide bonds. Peak sharpness was improved greatly by use large-pore diphenyl reversed-phase HPCL. We found that THPs can be best resolved from synthetic impurities by diphenyl or non-porous C18 reversed-phase HPLC using water-acetonitrile gradients. These results most likely reflect conditions which maintain the native conformation of collagen-like triple-helices.

Amino Acid Sequence↗
PubMed1994

Generally applicable, convenient solid-phase synthesis and receptor affinities of octreotide analogs.

Octreotide, an analogue of the hormone somatostatin, has applications as a therapeutic and imaging agent for somatostatin-positive tumors. We have developed a generally applicable, convenient stepwise solid-phase synthetic protocol for octreotide (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-threoninol). [Cys(Acm)2,D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6,Cys(Acm)7, des(threoninol)]-octreotide was assembled by Fmoc solid-phase synthesis and the intramolecular disulfide bond formed by treatment of the resin-bound peptide with thallium trifluoroacetate [Tl(Tfa)3]. Side-chain protection of Trp by the Boc group was found to preserve Trp integrity during Tl(Tfa)3 treatment. The protected peptide was cleaved from the resin by aminolysis with threoninol and purified by semipreparative RP-HPLC. Isolated [D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6]octreotide had the correct molecular mass ([M+H]+ = 1275 Da) and sequence and was obtained in 14% yield at > 98% purity. [D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6]octreotide was utilized for the solution-phase synthesis of CPTA-D-Phe1-octreotide, where CPTA is 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoic acid. Cyclic dianhydride of diethylenetriaminepentaacetic acid (DTPA) was coupled to a portion of the protected peptide-resin following disulfide bond formation. The DTPA-conjugated, side-chain-protected peptide was cleaved from the resin by aminolysis with threoninol, side-chain deprotected with trifluoroacetic acid, and purified by semipreparative RP-HPLC. The isolated DTPA-D-Phe1-octreotide had the correct molecular mass ([M+H]+ = 1395 Da) and was obtained in 5% yield at > 90% purity. The efficiency of aminolysis was partially dependent upon the linkage between 4-(hydroxymethyl)phenoxy (HMP) handles and the resin and/or resin particle size. The somatostatin receptor binding affinities of synthetic DTPA-D-Phe1-octreotide and CPTA-D-Phe1-octreotide to AtT-20 mouse pituitary carcinoma cell membranes were examined by labeling with 111In and 64Cu, respectively, and performing Scatchard analyses. The dissociation constant (Kd) for our synthetic [111In]DTPA-D-Phe1-octreotide was 4.31 nM, which is comparable to a Kd = 5.57 nM obtained with commercially available DTPA-D-Phe1-octreotide. The Kd for [64Cu]CPTA-D-Phe1-octreotide was 78.5 pM. On the basis of the criteria of molecular mass, RP-HPLC elution time, sequence analysis, and somatostatin receptor binding affinity, our synthetic octreotide is identical to commercially available octreotide. The aminolysis protocol used here has distinct advantages over either reductive cleavage or preformed linker methods described previously for the preparation of octreotide.

Amines↗
PubMed1994

Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3).

Members of the matrix metalloproteinase (MMP) family have been implicated in disease states such as arthritis, periodontal disease, and tumor cell invasion and metastasis. Stromelysin 1 (MMP-3) has a broad substrate specificity and participates in the activation of several MMP zymogens. We examined known sequences of MMP-3 cleavage sites in natural peptides and proteins and compared sequence specificities of MMP-3 and interstitial collagenase (MMP-1) in order to design fluorogenic substrates that (i) would be hydrolyzed rapidly by MMP-3, (ii) would discriminate between MMP-3 and MMP-1, and (iii) could be monitored continuously without interference from MMP amino acid residues. Designed substrates were then screened for activity toward MMP-1, gelatinase A (MMP-2), MMP-3, and gelatinase B (MMP-9). The first of these substrates, NFF-1 (Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys-(Dnp)-Gly, where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is 2,4-dinitrophenyl), was hydrolyzed equally well by MMP-3 and MMP-2 (kcat/Km approximately 11,000 s-1 M-1). MMP-1 had 25% of the activity of MMP-3 toward NFF-1. The second substrate, NFF-2 (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2, where Nva is norvaline), was hydrolyzed 60 times more rapidly by MMP-3 (kcat/Km = 59,400 s-1 M-1) than MMP-1. Unfortunately, NFF-2 showed little discrimination between MMP-3, MMP-2 (kcat/Km = 54,000 s-1 M-1), and MMP-9 (kcat/Km = 55,300 s-1 M-1). The third substrate, NFF-3 (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2), was hydrolyzed rapidly by MMP-3 (kcat/Km = 218,000 s-1 M-1) and very slowly by MMP-9 (kcat/Km = 10,100 s-1 M-1), but there was no significant hydrolysis by MMP-1 and MMP-2. NFF-3 is the first documented synthetic substrate hydrolyzed by only certain members of the MMP family and thus has important application for the discrimination of MMP-3 activity from that of other MMPs. Although NFF-3 was designed by assuming that substrate subsites were independent and hence free energy changes derived from single mutation experiments were additive, we found discrepancies between predicted and experimental kcat/Km values, one on the order of 2000-5000. Thus, the design of additional discriminatory MMP substrates may require approaches other than assuming additive free energy changes, such as screening synthetic libraries and consideration of secondary and tertiary structures of substrates and the enzyme.

Amino Acid Sequence↗
PubMed1994

Promotion of human platelet adhesion and aggregation by a synthetic, triple-helical "mini-collagen".

Platelet activation and aggregation by fibrillar collagens are based on substrate primary, secondary, tertiary, and quaternary structure. Although several peptides incorporating sequences from the triple-helical domains of types I and III collagen inhibit collagen-mediated platelet aggregation, none independently promote platelet activation and aggregation. It is believed that the absence of these platelet activities is due to the lack of proper substrate tertiary and quaternary structures. We have utilized a synthetic, triple-helical "mini-collagen" that incorporates a known cell adhesion site (alpha 1(IV)1263-1277) to better understand the relationship between substrate primary, secondary, tertiary, and quaternary structure and platelet activation and aggregation. The promotion of platelet adhesion, activation, and aggregation was compared for this triple-helical polypeptide (THP), fibrillar and type IV collagens, and a single-stranded peptide (SSP) incorporating the alpha 1(IV)1263-1277 sequence. Glass-coated fibrillar and type IV collagens and the THP supported platelet adhesion at substrate concentrations of 0.33 nM, 0.20 nM, and 0.89 microM, respectively. When platelets were stirred with 10 micrograms/ml of fibrillar (0.33 nM) and type IV (0.20 nM) collagen, SSP (2.1 microM), and THP (0.89 microM), only the fibrillar collagen caused 3H-labeled arachidonic acid release, elevation of cytosolic calcium, irreversible aggregation, and secretion of granule contents. The THP (0.45-1.8 microM) effectively inhibited fibrillar collagen-mediated platelet aggregation, while the SSP did not. At a substrate concentration of 40 micrograms/ml, the THP (3.6 microM) was as effective as fibrillar collagen (1.3 nM) at inducing 3H-labeled oleic acid-labeled platelet activation and microaggregate formation, while the SSP and type IV collagen were relatively ineffective. Rotary shadowing images indicated that aggregates of the THP could form distinct quaternary structures, while the type IV collagen used here could not. These results are the first demonstrations of a synthetic peptide promoting platelet adhesion, activation, and aggregation and suggest that the combination of THP primary, secondary, tertiary, and quaternary structural features are required for platelet aggregation.

Adult↗
PubMed1993

Melanoma cell adhesion and spreading activities of a synthetic 124-residue triple-helical "mini-collagen".

A great variety of cells, such as melanoma cells, fibroblasts, platelets, keratinocytes, and epithelial cells, adhere to and migrate on specific regions within the triple-helical domains of types I, III, and IV collagen. The relative importance of collagen primary, secondary, and tertiary structures on these cellular activities has not been ascertained, as no general synthetic methodology exists to allow for the study of peptides incorporating biologically active sequences in triple-helical conformation. We have thus developed a novel, generally applicable solid-phase branching methodology for the synthesis of aligned, triple-helical collagen-model polypeptides (i.e. "mini-collagens"). Three nascent peptide chains are carboxyl-terminally linked through one N alpha-amino and two N epsilon-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. A homotrimeric triple-helical polypeptide (THP) of 124 amino acids, incorporating residues 1263-1277 of alpha 1 (IV) collagen, was synthesized. Highly metastatic mouse melanoma cells showed a profound preference for adhesion to this THP as compared with a single-stranded peptide (SSP) incorporating the same type IV collagen sequence or a branched peptide containing eight repeats of Gly-Pro-Hyp (designated GPP*). Specifically, 50% cell adhesion occurred at a THP concentration of 1.12 microM, while comparable levels of adhesion required [SSP] = 170 microM or [GPP*] > 100 microM. Melanoma cells also spread on the THP to a greater extent than on the SSP or GPP*. These results are the first direct demonstrations of the significance of triple helicity for cell adhesion to and spreading on a specific collagen sequence and support earlier conclusions of conformational dependency for cell adhesion to and migration on types I and IV collagen. In addition, the melanoma cell THP activities support the concept that tumor cell adhesion and spreading on type IV collagen involves multiple, distinct domains in triple-helical conformation. The triple-helical peptide synthetic protocol developed here will allow eventually for the study of both structure and biological activity of specific, glycosylated collagen sequences in homotrimeric and heterotrimeric forms.

Amino Acid Sequence↗
PubMed1993

Solid-phase synthesis and stability of triple-helical peptides incorporating native collagen sequences.

A generally applicable solid-phase methodology has been developed for the synthesis of triple-helical polypeptides incorporating native collagen sequences. Three nascent peptide chains are C-terminal linked through one N alpha-amino and two N epsilon-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. Different protecting group strategies, including several three-dimensionally orthogonal schemes, have been utilized for the synthesis of four homotrimeric triple-helical polypeptides (THPs) of 79-124 residues, three of which incorporate native type IV collagen sequences. Highly efficient assemblies were achieved by 9-fluorenylmethoxycarbonyl (Fmoc) N alpha-amino group protection, in situ 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate mediated couplings, and 1,8-diazabicyclo [5.4.0] undec-7-ene mediated Fmoc group removal. THPs were characterized by Edman degradation sequencing, size-exclusion chromatography, mass spectrometry, reversed-phase high performance liquid chromatography, and CD spectroscopy. THP thermal stabilities ranged from 35 to 59 degrees C, with chain length and Hyp content being the influential factors. Melting temperatures and van't Hoff enthalpies for peptide triple-helical denaturation could be correlated well to Hyp content. The THP synthetic protocol developed here will allow for the study of both structure and biological activity of specific collagen sequences in homotrimeric and heterotrimeric forms.

Amino Acid Sequence↗
PubMed1992

The development of high-performance liquid chromatographic analysis of allyl and allyloxycarbonyl side-chain-protected phenylthiohydantoin amino acids.

Ten phenylthiohydantoin (PTH) amino acids possessing allyl (Al) or allyloxycarbonyl (Aloc) side-chain-protecting groups have been characterized by high-performance liquid chromatography for use in Edman degradation sequence analysis. Optimized separation of side-chain-protected and -unprotected PTH amino acids was achieved on a C-18 reversed-phase column with a two-step gradient spanning 32 min. Five of the side-chain-protected amino acids [Cys(Al), Cys(Aloc), Lys(Aloc), Thr(Aloc), Tyr(Al)] were completely stable to the conditions of PTH derivatization, four [Asp(OAl), Arg(Aloc)2, Glu(OAl), Ser(Aloc)] were partially deprotected during PTH derivatization, and one [His(Aloc)] was completely deprotected during PTH derivatization. All allyl-based derivatives were well resolved from their side-chain-unprotected counterparts. Studies on the stability to piperidine treatment showed Asp(OAl), Cys(Al), Glu(OAl), Lys(Aloc), Thr(Aloc), and Tyr(Al), and possibly Arg(Aloc)2 and Ser(Aloc), to be suitable for peptide synthesis by 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry. Edman degradation of Al and Aloc side-chain-protected Conus geographus Lys9-alpha-conotoxin GI synthesized on 4-methylbenzhydrylamine-copoly(styrene-1%-DVB)-resin demonstrated the usefulness of these derivatives for solid-phase preview sequence analysis.

Amino Acid Sequence↗
PubMed1990

A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis.

The success of solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl (Fmoc) amino acids is often limited by deleterious side reactions which occur during TFA peptide-resin cleavage and side-chain deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and Cys, with TFA-liberated side-chain protecting groups and linkers. The purpose of this study was to assess the relative effectiveness of various scavengers in suppressing these side reactions. We found that the cleavage mixture 82.5% TFA : 5% phenol : 5% H2O : 5% thioanisole : 2.5% EDT (Reagent K) was maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each containing 20-50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides.

Amino Acid Sequence↗
PubMed1989

Solid phase peptide synthesis of 15N-gramicidins A, B, and C and high performance liquid chromatographic purification.

Four single-site 15N-labeled molecules of gramicidin have been synthesized using the 9-fluorenylmethoxycarbonyl method of solid phase peptide synthesis. Formylvaline was coupled as the N-terminal amino acid, and the peptide was cleaved from the resin with ethanolamine. Each synthesized gramicidin was purified in one step by semipreparative reverse phase high performance liquid chromatography and obtained in overall yields as high as 86%. The peptide was characterized by comparison with natural gramicidin using amino acid analysis, u.v. spectroscopy, and analytical high performance liquid chromatography.

Chromatography, High Pressure Liquid↗
PubMed1988

Solid-phase peptide synthesis and solid-state NMR spectroscopy of [Ala3-15N][Val1]gramicidin A.

[Ala3-15N][Val1]Gramicidin A has been prepared by solid-phase peptide synthesis and studied by solid-state 15N nuclear magnetic resonance spectroscopy. The synthesis of desformyl[Ala3-15N][Val1]gramicidin A employed N-hydroxysuccinimide esters of 9-fluorenylmethoxycarbonyl-N alpha-amino acids and completely avoided the use of acid. Since deblocking was done with piperidine and the peptide was removed from the resin by treatment with ethanolamine, this synthetic protocol prevented oxidation of the indole rings of this tryptophan-rich peptide and reduced truncations produced by acid hydrolysis. After formylation and purification by anion-exchange and high-pressure liquid chromatography, the peptide was obtained in an overall yield of 30%. Solid-state 15N nuclear magnetic resonance spectra of this peptide and uniformly labeled [15N]gramicidin A' oriented in hydrated lipid bilayers have been obtained, allowing unambiguous assignment of the [15N]Ala3 resonance in the latter. The solid-state 15N nuclear magnetic resonance experiments provide evidence that [Val1]gramicidin A is rotating about an axis that is perpendicular to the plane of the lipid bilayer and that the N--H axis is nearly parallel with the rotational axis. This study demonstrates that site-specifically labeled [15N]gramicidin A analogs prepared by solid-phase peptide synthesis are valuable tools in the study of the solid-state nuclear magnetic resonance spectra of samples in oriented lipid bilayers.

Amino Acid Sequence↗
PubMedDate not supplied

HBTU activation for automated Fmoc solid-phase peptide synthesis.

Excellent results have been obtained for the Fmoc solid-phase syntheses of peptides using the activating reagent 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU). Activation occurs very rapidly in N,N-dimethylformamide and N-methyl-pyrrolidone, optimal solvents for peptide-resin solvation. It has been observed that complete coupling reactions occur in only 10-30 min. Residues such as Arg, Ile, Leu and Val, which often require double coupling by other activation methods, react with high efficiency by single coupling when HBTU is used. The Fmoc/HBTU chemistry has recently been applied to the peptide synthesizers. The incorporation of trityl side-chain protection for Fmoc-Asn and Fmoc-Gln further enhances coupling efficiencies in difficult sequences.

Amino Acid Sequence↗
PubMedDate not supplied

Edman degradation sequence analysis of resin-bound peptides synthesized by 9-fluorenylmethoxycarbonyl chemistry.

The efficacy of Edman degradation sequence analysis for evaluating the synthetic efficiency of peptide-resin assembly by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase methodology has been studied. Prior researchers have described the use of solid-phase "preview" sequence analysis for peptides synthesized by tertiary-butyloxycarbonyl (Boc) chemistry, where benzyl-based side-chain protecting groups and peptide-resin linkers are stable to the conditions of Edman chemistry. We have successfully sequenced a variety of resin-bound peptides synthesized by Fmoc chemistry, where tertiary-butyl-based side-chain protecting groups and peptide-resin linkers are labile to the conditions of Edman chemistry. Crude peptides are liberated from trifluoroacetic acid-labile linkers during the first cycle of Edman degradation and subsequently "embedded" in membranes. For peptides up to 20 residues, embedded sequencing repetitive yields were comparable to those of solid-phase sequencing. Preview sequencing of resin-bound Fmoc-synthesized peptides proved to be advantageous compared to other analytical methods, in that synthetic failures were detected and quantitated at the point of occurrence, regardless of whether incomplete Fmoc deprotection or incomplete coupling was responsible, and without interference from by-products formed during peptide-resin cleavage. Quantitative ninhydrin analysis, which previously has been found to give false positive results due to removal of the Fmoc group by a combination of reagents and high temperature, gave false negative results in this study, most probably due to incomplete removal of the Fmoc group prior to coupling. Quantitative sequence analysis results were supported by high-performance liquid chromatographic, amino acid and electrospray mass spectrometric analyses of the crude and purified peptides.

Amino Acid Sequence↗