William V. Sweeney

Aspartimide formation in base-driven 9-fluorenylmethoxycarbonyl chemistry

Abstract Aspartimide and its piperidine adduct formed between -Asp-Asn- has been found during an Fmoc-based synthesis of an EGF-like domain in a blood coagulation factor. Model studies with seven susceptible -Asp-X- pentapeptides show that -Asp(Ot-Bu)-Asn(Trt)- and -Asp(Ot-Bu)-Gly- are the most problematic sequences.

Spectroscopic studies of the seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus

The seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus have been investigated by low-temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies and room temperature ultraviolet-visible absorption spectroscopy. The results confirm the presence of one trinuclear and one tetranuclear iron-sulfur cluster in both ferredoxins and facilitate comparison of the electronic and magnetic properties of the oxidized and reduced [3Fe-xS] clusters. MCD magnetization data are consistent with an S = 2 ground state for both reduced [3Fe-xS] clusters, but indicate differences in the rhombicity of the zero-field splittings. The data permit rationalization of the absence of a delta M = 4 EPR transition for the reduced [3Fe-xS] cluster in A. vinelandii ferredoxin I. Spectroscopic studies of anaerobically isolated A. vinelandii ferredoxin I do not support the hypothesis that the [3Fe-xS] cluster arises as a result of aerial oxidative damage to a [4Fe-4S] cluster during isolation. The possibility that two distinct forms of [3Fe-xS] clusters can exist in A. vinelandii ferredoxin I was investigated by spectroscopic studies as a function of pH. The results reveal two distinct and interconvertible forms of the reduced [3Fe-xS] cluster, but do not permit rationalization of the inconsistencies in the structural data that have been reported for the oxidized clusters.

The electron paramagnetic resonance of oxidized clostridial ferredoxins

Summary A weak EPR resonance at g = 2.015 has been observed in five oxidized Clostridial-type ferredoxins. Oxidized Clostridium acidi-urici [ 57 Fe] ferredoxin exhibited a broadened resonance at g = 2.015 relative to the signal from native or reconstituted [ 56 Fe]ferredoxin. This experiment, in conjunction with other results, demonstrates that the signal exhibited by oxidized Clostridial ferredoxin either originates from the Fe 4 S 4 * clusters of the protein or from a paramagnetic species bound to the clusters. Oxidized C. acidi-urici ferredoxin, when treated with the oxidant potassium ferricyanide, was found to exhibit an approximate 100 fold increase in the intensity of the signal. Results of experiments performed on the ferricyanide treated ferredoxin are consistent with the existence of a third oxidation state of the protein.

Calcium binding and putative activity of the epidermal growth factor domain of blood coagulation factor IX

The first epidermal growth factor (EGF)-like domain of human Factor IX and two chimeric analogs of this domain and EGF were synthesized unambiguously and purified to homogeneity. The synthetic EGF-like domain and its analogs showed the correct mass ions by the fission ionization mass spectrometry and similar disulfide pairings as those found in EGF, but failed to exhibit any putative EGF activity in the receptor and mitogenic assays. However, in NMR titration experiments, the EGF-like domain and one of its analogs were found to bind Ca2+ but not Mg2+. Our results therefore show that the EGF-like domain of Factor IX has the ability to bind calcium ion, shares the structural motif of EGF but does not retain the active determinants responsible for the EGF activity.

13C-NMR of Clostridium pasteurianum ferredoxin after reductive methylation of the amines using [13C]formaldehyde

Clostridium pasteurianum 2(4Fe-4S) ferredoxin has been reductively methylated using [13C]formaldehyde and sodium cyanoborohydride. Lys3 and the N-terminal alanine, the only amines in the protein, are both dimethylated by this procedure. 13C-NMR titration of the apo, oxidized and reduced modified ferrodoxin indicate that the lysine pK is slightly over 10 in all three forms of the protein. In contrast, the N-terminal alanine shifts from a pK of 7.7 in the apoprotein to greater than 9 in both the oxidized and reduced modified ferredoxin. The unexpectedly high pK observed for the N-terminus is consistent with the presence of an ion pair in both the oxidized and reduced native forms of the protein. The methylated ferrodoxin is considerably less stable than the native protein, indicating an important role for the amines in protein stability.

The lack of a solvent accessible hydroxide or water ligand to iron at the 3Fe center of Azotobactervinelandii ferredoxin I

The X-ray crystal structure of Azotobacter vinelandii ferredoxin I (FdI) describes a planar 3Fe-3S center in which one of the iron atoms is ligated to a solvent accessible oxo ligand, presumably from water or hydroxide (Ghosh et al., (1982) J. Mol. Biol. 158, 73-109). Efforts to displace the proposed oxo ligand with cyanide were unsuccessful, even in 80% dimethylsulfoxide. In addition, comparison of the electron spin echo envelopes for H2O- and D2O-equilibrated samples of FdI showed only a slight deuterium modulation, far less than would be expected were water to be bound as an iron ligand. These results do not support the presence of a solvent accessible oxo ligand to the 3Fe center as described in the X-ray crystal structure.

Characterization of a side reaction using stepwise detection in peptide synthesis with Fmoc chemistry

Publisher Summary Solid phase peptide synthesis almost always employs either Boc or Fmoc chemistry; Boc chemistry requires acidic conditions for deblocking, and the potential side reactions have been extensively studied; however, in Fmoc chemistry repetitive basic conditions are required for deblocking, and only a few of the base-catalyzed side reactions have been characterized. This chapter describes a method used to demonstrate that a side reaction well known in Boc chemistry, but thought not to occur under the conditions of Fmoc. Stepwise analysis is an efficient and direct method to monitor the progress of side reactions in Fmoc chemistry. The analysis involves stepwise micro-scale TFA cleavage in conjunction with HPLC and MS. Micro-scale TFA cleavage can be conveniently carried out on a small amount of sample. MS analysis of peptide ladders provides a rapid method for monitoring and identification of side reactions in peptide synthesis.