Seketsu Fukuzawa

Regioselective Carbon–Carbon Bond Formation in Proteins with Palladium Catalysis; New Protein Chemistry by Organometallic Chemistry

Palladium‐catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki–Heck reaction for site‐specific carbon–carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His6‐fused Ras protein containing 4‐iodo‐L‐phenylalanine at position 32 (iF32‐Ras‐His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32‐Ras‐His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography–mass spectrometry (LC‐MS). The regioselectivity of the Mizoroki–Heck reaction was furthermore confirmed by LC‐MS/MS analysis. However, in addition to the biotinylated product (bF32‐Ras‐His), a dehalogenated product (F32‐Ras‐His) was detected by LC‐MS/MS. This dehalogenation resulted from the undesired termination of the Mizoroki–Heck reaction due to steric and electrostatic hindrance around residue 32. The biotinylated Ras showed binding activity for the Ras‐binding domain as its downstream target, Raf‐1, with no sign of decomposition. This study is the first report of an application of organometallic chemistry in protein chemistry.

Site‐Specific Functionalization of Proteins by Organopalladium Reactions

A new carbon–carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki–Heck and Sonogashira reactions). Reaction conditions were screened by using a model peptide, and the stability of the Ras protein under the reaction conditions was examined by using the wild‐type Ras protein. Finally, the iF–Ras proteins containing a 4‐iodo‐L‐phenylalanine residue were subjected to organopalladium reactions with vinylated or propargylated biotin. Site‐specific biotinylations of the Ras protein were confirmed by Western blot and LC‐MS/MS.

TEN MORE RITTERAZINES, CYTOTOXIC STEROIDAL ALKALOIDS FROM THE TUNICATE RITTERELLA TOKIOKA

Abstract Ritterazines D-M ( 4–13 ) have been isolated from the tunicate Ritterella tokioka and their structures elucidated by spectral data. Ritterazines D-M showed potent cytotoxicity against P388 murine leukemia cells with IC 50 values of 16, 3.5, 0.73, 0.73, 16, 14, 13, 9.5, 10, and 15 ng/mL, respectively.

N-terminal labeling of proteins by the Pictet–Spengler reaction

The Pictet-Spengler reaction was applied to the N-terminal labeling of horse heart myoglobin. This was performed in the following two steps: (1) conversion of the N-terminal glycine residue to an alpha-keto aldehyde by a transamination reaction and (2) condensation of the resulting activated myoglobin with tryptamine analogues by the Pictet-Spengler reaction. Ultraviolet (UV)/visible (vis) absorption and circular dichroism (CD) spectral data revealed that the tertiary structure of myoglobin was not altered by the Pictet-Spengler reaction.

Two new indole alkaloids, 2-(3,3-dimethylprop-1-ene)-costaclavine and 2-(3,3-dimethylprop-1-ene)-epicostaclavine, from the marine-derived fungus Aspergillus fumigatus.

Two new indole alkaloids, 2-(3,3-dimethylprop-1-ene)-costaclavine (1) and 2-(3,3-dimethylprop-1-ene)-epicostaclavine (2), together with the known compounds costaclavine (3), fumgaclavine A (4) and C (5), were isolated from the marine-derived fungus Aspergillus fumigatus. The planar structures of the two new compounds were elucidated on the basis of chemical and physicochemical evidence including MS, UV, IR and NMR spectra. Their stereochemistry was studied by NOESY, 1H–1H coupling constant and CD spectra. The compounds 1, 2, 3 and 5 showed weak cytotoxicity against a mouse leukemia cell line (P388).

Skeletal protein protection: the mode of action of an anti-osteoporotic marine alkaloid, norzoanthamine

Bone is composed of mineralized collagen fibrils. A marine alkaloid, norzoanthamine, accelerates the formation of a collagen–hydroxyapatite composite and enhances collagen release from an immobilized matrix vesicle model. Norzoanthamine recognizes a peptide chain nonspecifically and stabilizes its secondary structure, and collagen has polyvalent binding sites for norzoanthamine. This collagen–norzoanthamine supramolecular association is considered to be one of the most significant modes of action for enhancement of bone formation. The facts that norzoanthamine is nontoxic and that it has a collagen protective activity indicate that it may provide significant therapeutic benefits. In particular, it may be a promising drug candidate for osteoporosis treatment and prevention. Interestingly, norzoanthamine suppressed the proteolysis of not only collagen but also elastin and bovine serum albumin, so it apparently has a universal protective effect of guarding extracellular matrix proteins from degradation. This result suggests that norzoanthamine protect skeletal proteins in the host animal body from external stresses and possibly enhance survival.

Use of 15N-HMBC NMR techniques to determine the orientation of the steroidal units in ritterazine A1

Orientation of the two steroidal units about the pyrazine ring in ritterazine A, a cytotoxic dimeric steroid from the tunicate Ritterella tokioka, was determined by 15N-HMBC spectroscopy.

Distribution and Possible Function of the Marine Alkaloid, Norzoanthamine, in the Zoanthid Zoanthus sp. Using MALDI Imaging Mass Spectrometry

The role of the marine alkaloid, norzoanthamine, in the colonial zoanthid Zoanthus sp. was previously unknown. High concentrations of norzoanthamine are present in the epidermal tissue of Zoanthus sp., as determined using protonated molecular ion peak mapping of norzoanthamine by matrix-assisted laser desorption/ionization mass spectrometry and high-performance liquid chromatography quantification. Sodium dodecylsulfate polyacrylamide gel electrophoresis experiments indicate that norzoanthamine increases the resistance of collagen to damage from UV light, probably not via UV light absorption, but by strengthening collagen itself, thus suggesting that collagen strengthening may be the function of norzoanthamine in Zoanthus sp.

Simple and accurate single base resolution analysis of 5-hydroxymethylcytosine by catalytic oxidative bisulfite sequencing using micelle incarcerated oxidants

Oxidation of 5-methylcytosine (5mC) is catalyzed by ten-eleven translocation (TET) enzymes to produce 5-hydroxymethylcytosine (5hmC) and following oxidative products. The oxidized nucleotides were shown to be the intermediates for DNA demethylation, as the nucleotides are removed by base excision repair system initiated by thymine DNA glycosylase. A simple and accurate method to determine initial oxidation product 5hmC at single base resolution in genomic DNA is necessary to understand demethylation mechanism. Recently, we have developed a new catalytic oxidation reaction using micelle-incarcerated oxidants to oxidize 5hmC to form 5-formylcytosine (5fC), and subsequent bisulfite sequencing can determine the positions of 5hmC in DNA. In the present study, we described the optimization of the catalytic oxidative bisulfite sequencing (coBS-seq), and its application to the analysis of 5hmC in genomic DNA at single base resolution in a quantitative manner. As the oxidation step showed quite low damage on genomic DNA, the method allows us to down scale the sample to be analyzed.

A new protein engineering approach combining chemistry and biology, part I; site-specific incorporation of 4-iodo-L-phenylalanine in vitro by using misacylated suppressor tRNAPhe.

An Escherichia coli suppressor tRNAPhe (tRNAPheCUA) was misacylated with 4‐iodo‐L‐phenylalanine by using the A294G phenylalanyl–tRNA synthetase mutant (G294‐PheRS) from E. coli at a high magnesium‐ion concentration. The preacylated tRNA was added to an E. coli cell‐free system and a Ras protein that contained the 4‐iodo‐L‐phenylalanine residue at a specific target position was synthesized. Site‐specific incorporation of 4‐iodo‐L‐phenylalanine was confirmed by using LC–MS/MS. Free tRNAPheCUA was not aminoacylated by aminoacyl–tRNA synthetases (aaRSs) present in the E. coli cell‐free system. Our approach will find wide application in protein engineering since an aryl iodide tag on proteins can be used for site‐specific functionalization of proteins.