Repeat conformation heterogeneity in cerebellar ataxia, neuropathy, vestibular areflexia syndrome.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is a late-onset, slow-progressing multisystem neurodegenerative disorder. Biallelic AAGGG repeat expansion in RFC1 has been identified as causative of this disease, and repeat conformation heterogeneity (ACAGG repeat) was also recently implied. To molecularly characterize this disease in Japanese patients with adult-onset ataxia, we accumulated and screened 212 candidate families by an integrated approach consisting of flanking PCR, repeat-primed PCR, Southern blotting and long-read sequencing using Sequel II, GridION or PromethION. We identified 16 patients from 11 families, of whom seven had ACAGG expansions [(ACAGG)exp/(ACAGG)exp] (ACAGG homozygotes), two had ACAGG and AAGGG expansions [(ACAGG)exp/(AAGGG)exp] (ACAGG/AAGGG compound heterozygotes) and seven had AAGGG expansions [(AAGGG)exp/(AAGGG)exp] (AAGGG homozygotes). The overall detection rate was 5.2% (11/212 families including one family having two expansion genotypes). Long-read sequencers revealed the entire sequence of both AAGGG and ACAGG repeat expansions at the nucleotide level of resolution. Clinical assessment and neuropathology results suggested that patients with ACAGG expansions have similar clinical features to previously reported patients with homozygous AAGGG expansions, although motor neuron involvement was more notable in patients with ACAGG expansions (even if one allele was involved). Furthermore, a later age of onset and slower clinical progression were implied in patients with ACAGG/AAGGG compound heterozygous expansions compared with either ACAGG or AAGGG homozygotes in our very limited cohort. Our study clearly shows the occurrence of repeat conformation heterogeneity, with possible different impacts on the affected nervous systems. The difference in disease onset and progression between compound heterozygotes and homozygotes might also be suspected but with very limited certainty due to the small sample number of cases in our study. Studies of additional patients are needed to confirm this.

Magnesium methoxide-induced chemiluminescent decomposition of bicyclic dioxetanes bearing a 2'-alkoxy-2-hydroxy-1,1'-binaphthyl-7-yl moiety.

Bicyclic dioxetanes 2a-c bearing a 2'-alkoxy-2-hydroxy-1,1'-binaphthyl-7-yl moiety were effectively synthesized and their base-induced chemiluminescent decomposition was investigated by the use of alkaline metal (Na(+) and K(+) ) or Mg(2+) alkoxide in MeOH. When 2a-c were treated with tetrabutylammonium fluoride (TBAF) in dimethyl sulfoxide (DMSO) as a reference system, they showed chemiluminescence as a flash of orange light (maximum wavelength λmax (CL) = 573-577 nm) with efficiency Φ(CL) = 6-8 × 10(-2) . On the other hand, for an alkaline metal (Na(+) or K(+) ) alkoxide/MeOH system, 2a-c decomposed slowly to emit a glow of chemiluminescence, the spectra of which were shifted slightly toward red from the TBAF/DMSO system, and Φ(CL) (= 1.4-2.3 × 10(-3) ) was considerably decreased. In addition, Mg(OMe)2 was found to play a characteristic role as a base for the chemiluminescent decomposition of 2a-c through coordination to the intermediary oxidoaryl-substituted dioxetanes 13. Thus, Mg(2+) increased Φ(CL) to more than twice those with Na(+) or K(+) , while it shifted λmax (CL) considerably toward blue (λmax (CL) = 550-566 nm).

Base-induced chemiluminescent decomposition of bicyclic dioxetanes bearing a (benzothiazol-2-yl)-3-hydroxyphenyl group: a radiationless pathway leading to marked decline of chemiluminescence efficiency.

Charge-transfer-induced decomposition (CTID) of bicyclic dioxetanes 1b-d bearing a 3-hydroxylphenyl moiety substituted with a benzothiazol-2-yl group at the 2-, 6-, or 5-position was investigated, and their chemiluminescence properties were compared to each other, based on those for a 4-benzothiazolyl analogue 1a. Dioxetanes 1c and 1d underwent CTID to give the corresponding oxido anions of keto esters 8c or 8d in the singlet excited state with high efficiencies similarly to the case of 1a. On the other hand, 1b showed chemiluminescence with quite low efficiency, though it gave exclusively keto ester 2b. The marked decline of chemiluminescence efficiency for 1b was attributed to 1b mainly being decomposed to 8b through a radiationless pathway, in which intramolecular nucleophilic attack of nitrogen in the benzothiazolyl group to dioxetane O-O took place to give cyclic intermediate cis-11.

Diphenylparabanic acid as a synthon for the synthesis of α-diketones and α-ketocarboxylic acids.

Diphenylparabanic acid was found to react with >2 equiv of organolithiums at -78 °C to effectively give the corresponding symmetrical α-diketones. However, upon treatment with 1 equiv of organolithium, the parabanic acid gave mainly 5-substituted 5-hydroxyimidazolidine-2,4-diones. On the other hand, Grignard reagents were less reactive toward the parabanic acid at low temperature, and selectively gave the corresponding 5-hydroxyimidazolidine-2,4-diones even if more than 1 equiv of the reagents was used. A tandem process in which the parabanic acid was first reacted with a Grignard reagent and then reacted in one-pot with an organolithium effectively gave the unsymmetrical α-diketone. 5-Substituted 5-hydroxyimidazolidine-2,4-diones were useful as versatile precursors for preparing α-ketocarboxylic acids as well as unsymmetrical α-diketones.

Intramolecular charge-transfer-induced decomposition promoted by an aprotic polar solvent for bicyclic dioxetanes bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety.

Bicyclic dioxetanes 3a-d bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl group decomposed to give the corresponding keto esters 4a-d accompanied by the emission of bright light when simply dissolved in an aprotic polar solvent such as N-methylpyrrolidone (NMP) or DMF at 50-100 °C. This solvent-promoted decomposition (SPD) was effectively a chemiluminescence process caused by the hydrogen bonding of a phenolic OH with a solvent molecule(s). The characteristics of the chemiluminescence in SPD resembled those in base-induced decomposition (BID), which occurs through an oxidoaryl-substituted dioxetane 5 by an intramolecular charge-transfer-induced decomposition (CTID) mechanism. Both free energies of activation, ΔG(doubledagger)(SPD) and ΔG(doubledagger)(BID), increased in the order 3a < 3b < 3c < 3d, and were linearly correlated with each other. However, SPD showed features different from those of BID in terms of enthalpy of activation and entropy of activation. SPD had large negative values for ΔS(doubledagger) (ca. -71 J mol(-1) K(-1)) regardless of the substituent R at the 5-position for 3a-d, while the ΔS(doubledagger) values for BID changed from 0.5 to -22 J mol(-1) K(-1) as R became smaller. The enthalpy of activation ΔH(doubledagger) for SPD was 14-21 kJ mol(-1) smaller than that for BID.

Crucial dependence of chemiluminescence efficiency on the syn/anti conformation for intramolecular charge-transfer-induced decomposition of bicyclic dioxetanes bearing an oxidoaryl group.

Thermally stable rotamers of bicyclic dioxetanes bearing 6-hydroxynaphthalen-1-yl (anti-5a and syn-5a), 3-hydroxynaphthalen-1-yl (anti-5b and syn-5b), and 5-hydroxy-2-methylphenyl groups (anti-5c and syn-5c) were synthesized. These dioxetanes underwent TBAF (tetrabutylammonium fluoride)-induced decomposition accompanied by the emission of light in DMSO and in acetonitrile at 25 °C. For all three pairs of rotamers, the chemiluminescence efficiency Φ(CL) for anti-5 was 8-19 times higher than that for syn-5, and the rate of CTID (charge-transfer-induced decomposition) for anti-5 was faster than that for syn-5. The chemiluminescence spectra of the rotamers for 5a and 5c, respectively, were different. This discrepancy in the chemiluminescence spectra between rotamers can presumably be attributed to the difference in the structures of de novo keto imide anti-14 and syn-14 in an excited state, which inherit the structures of the corresponding intermediary anionic dioxetanes anti-13 and syn-13. The important difference in chemiluminescence efficiency between anti-5 and syn-5 is discussed from the viewpoint of a chemiexcitation mechanism for CTID of oxidophenyl-substituted dioxetane.

Thermodynamic aspects of thermal decomposition and charge-transfer-induced chemiluminescent decomposition for bicyclic dioxetanes bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety.

Uncatalyzed thermal decomposition (TD) and charge-transfer-induced decomposition (CTID) of dioxetanes were investigated to determine their thermodynamic characteristics. The dioxetanes examined were a series of 1-[4-(benzothiazol-2-yl)-3-hydroxyphenyl]-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]heptanes, 1a-d, bearing an alkyl substituent R, e.g., methyl, ethyl, isopropyl, or tert-butyl, at the 5-position and a parent dioxetane 1e with R = H. X-ray single crystallographic analysis was achieved for 1a-d. Both free energies of activation, DeltaG(double dagger)(TD) and DeltaG(double dagger)(CTID), increased in the order 1a < or = 1b < 1c < 1d. The free energy difference DeltaDeltaG(double dagger) = DeltaG(double dagger)(TD) - DeltaG(double dagger)(CTID) was ca. 27 kJ mol(-1) regardless of the substituent R. However, the use of Taft's dual-substituent parameter suggested that CTID was more sensitive to the polarity of the substituent R than TD. The entropy term for CTID, DeltaS(double dagger)(CTID), decreased from zero to a large negative value in the order of tert-butyl, isopropyl, ethyl, and methyl, whereas DeltaS(double dagger)(TD) did not show a similar tendency.

Synthesis of thermally stable acylamino-substituted bicyclic dioxetanes and their base-induced chemiluminescent decomposition.

Hydroxyaryl-substituted dioxetanes 2-4 fused with a pyrrolidine ring were selectively synthesized by singlet oxygenation of the corresponding dihydropyrroles 5-7. These N-acylamino-substituted bicyclic dioxetanes were quite stable thermally, and 2a and 2b were estimated to possess half-lives of 32 and 34 y at 25 degrees C. When treated with TBAF (tetrabutylammonium fluoride) in DMSO, these dioxetanes underwent charge-transfer-induced decomposition (CTID) to emit yellow-orange light. The chemiluminescence efficiencies Phi(CL) for dioxetanes 2 ranged from 10(-6) to 10(-2). On the other hand, dioxetane 4 bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety showed chemiluminescent decomposition with high efficiency (Phi(CL) = 0.15) comparable to its oxy-analogue 26. Prominent characteristics for CTID of the present dioxetanes were that the N-acylamino-group influenced the color of chemiluminescence as well as the rate of decomposition k(CTID), and furthermore an N-acyl substituent could decisively affect the singlet-chemiexcitation efficiency, as observed for the case of 2b.

Synthesis of sulfanyl-, sulfinyl-, and sulfonyl-substituted bicyclic dioxetanes and their base-induced chemiluminescence.

The singlet oxygenation of 4-tert-butyl-3,3-dimethyl-5-(3-oxyphenyl)-2,3-dihydrothiophenes 5c-e bearing an acetoxy or methoxy group at the 2-position exclusively gave the corresponding sulfanyl-substituted bicyclic dioxetanes 2c-e, while that of 5a without 2-substituent mainly gave sulfoxide 11 along with a small amount of dioxetane 2a. These dioxetanes were sufficiently stable thermally to permit handling at room temperature. Sulfanyl-substituted dioxetanes, 2c and 2e, were further oxidized with m-chloroperbenzoic acid to afford the corresponding sulfinyl-substituted dioxetanes 3c, 3e and sulfonyl-substituted dioxetanes 4c, 4e. X-ray single crystallographic analysis was performed for 2c and 4e. Base-induced decomposition of the dioxetanes in DMSO gave light with a maximum wavelength lambda(max)(CL) at 554 nm for 2a and 565 nm for 2e in moderate light yields, while sulfinyl-derivative 3e gave weak light with lambda(max)(CL) = 795 nm and sulfonyl-derivative 4e gave very weak light with lambda(max)(CL) = 848 nm.

Marked difference in fragmentation between collision-induced excitation and chemi-excitation of keto esters produced from dioxetanes bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety in negative-mode matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry.

A dioxetane bearing a hydroxyphenyl group produces an unstable oxidoaryl anion by deprotonation which rapidly decomposes with accompanying emission of light effectively by the intramolecular charge-transfer-induced decomposition (CTID) mechanism. Although several mechanisms have been proposed to explain chemi-excitation in CTID, strong experimental evidence is still lacking. In the course of our investigation to clarify the chemi-excitation process, negative-mode matrix-assisted laser desorption/ionization time-of-flight collision-induced dissociation tandem mass spectrometry (MALDI-TOF-CID-MS/MS) was used to investigate the decomposition of bicyclic dioxetanes bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety 1 and their related keto esters 2 in a gas phase. Dioxetanes 1 decomposed to give 2 in an electronically excited state, which underwent alpha-cleavage of a ketone moiety, while authentic 2 (ground state) hardly showed fragmentation in MS. On the other hand, 2 displayed fragment ions in CID-MS/MS, though the fragmentation pattern was significantly different between 2a (R = tert-butyl) and 2b-2d (R = isopropyl, ethyl and methyl, respectively): 2a exhibited mainly alpha-cleavage of a ketone moiety, while 2b-2d showed beta-cleavage of the aromatic ester moiety. The marked difference in fragmentation between 2 (electronically excited state) produced directly from 1 and authentic 2 under CID was most likely due to the difference in the excitation processes: chemi-excitation of 2 by CTID of 1 versus vibrational excitation of 2 induced by collision in MS/MS.

Color modulation for intramolecular charge-transfer-induced chemiluminescence of 1,2-dioxetanes.

Dioxetanes bearing an aromatic electron donor decompose with an accompanying emission of light by an intramolecular charge-transfer-induced chemiluminescence (CTICL) mechanism. The color of the chemiluminescence from CTICL-active aryl-substituted dioxetanes is controlled by the design of the aromatic moiety and its substitution pattern, and the substituent on the carbon of the dioxetane ring. In addition to these color modulations, a change in the conformation of dioxetanes bearing a biaryl group was also found to cause a change in the color of CTICL in the coordination sphere of a crown ether complex. This new color modulation system was further developed for optically active dioxetanes bearing 2-hydroxy-1,1'-binaphthyl, the CTICL of which took place under the chiral recognition of optically active crown ether complexes. The spectra of the chemiluminescence from biaryl-type dioxetanes did not coincide with the fluorescence spectrum from the authentic emitter in the coordination sphere, but they did coincide with each other in a completely homogeneous system. This finding suggests that the emitter of CTICL possesses a transient structure, which retains the afterimage of the conformation of the dioxetane.

Electron-transfer-induced decomposition of 1,2-dioxetanes in negative-mode matrix- assisted laser desorption/ionization time-of-flight mass spectrometry.

1,2-Dioxetanes bearing an aromatic electron donor undergo intramolecular charge-transfer-induced chemiluminescence (CTICL). Although there has been some controversy regarding the mechanisms involved, there is little experimental evidence to strongly support any of the proposed mechanisms. In the course of our investigations, to clarify these mechanisms, we tried to effectively ionize dioxetanes bearing a phenolic group and found that poly(3-octylthiophene-2,5-diyl) was a promising matrix for negative-mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). Electron-transfer ionization was found to take place for dioxetanes bearing a hydroxyphenyl moiety that had been further substituted with an aromatic group, which acted as an antenna to catch an electron from the matrix. Furthermore, the characteristic fragmentation of dioxetanes 3c-3d was thought to occur by the elimination of 2-methyl-1-propene (56 u) and pivalaldehyde (86 u) from deprotonated ion [M - H](-) of dioxetanes, based on the results of muliple mass spectrometry measurements of dioxetanes using MALDI quadrupole ion trap ToF-MS. Based on a comparison of fragmentation in dioxetanes and the corresponding keto esters, dioxetanes were presumed to initially generate excited keto esters from which fragmentation took place.

Colour change of chemiluminescence for base-induced decomposition of dioxetane bearing a 4-(4-cyanophenyl)iminomethyl-3-hydroxyphenyl group.

A bicyclic dioxetane 1 bearing a 4-(4-cyanophenyl)iminomethyl-3-hydroxyphenyl group was found to undergo base-induced decomposition with the accompanying emission of light, the colour of which changed depending on the base used and its concentration. When 1 was triggered with tetrabutylammonium fluoride (TBAF), 1 displayed an emission of glowing orange light. On the other hand, on treatment with a high concentration of potassium t-butoxide complexed with 18-crown-6 ether, 1 afforded a flash of blue light. The mechanistic study of this unprecedented phenomenon revealed that the emission of glowing orange light was due to the normal oxido anion of keto ester 11, whereas the emission of a flash of blue light was attributed to another species that was produced by addition of a nucleophile to an iminomethyl of unstable oxido anion of dioxetane 10.

[Amounts of sweat and salt loss due to sweating during a three-hour badminton practice in summer].

In 7 men and 5 women, we measured the amounts of sweat and fluid intake, and the ionic composition of sweat during a 3-hour badminton practice in summer. The amount of sweat was calculated as follows; body weight before practice (g)--body weight after practice (g)--urine volume (ml) +fluid intake (ml). We collected sweat by covering the non-dominant forearm with a plastic bag. The amounts of sweat and fluid intake during the 3-hour practice were 1809 +/- 715ml (mean +/- SD) and 658 +/- 344ml, respectively. Weight loss after the practice was 2.0 +/- 0.9% of their weight before the practice. The Na(+) and Cl(-) levels of the sweat about 30 min after the start of practice were 66 +/- 34 mEq/l and 54 +/- 32mEq/l, respectively. There was no significant difference between those ionic levels of the sweat about 30 min after the start of practice and those about 30 min before the end of practice. The sum of Na(+) and Cl(-) loss into sweat during a 3-hour practice session was supposed to be 6.9 +/- 5.3g, and to be above 10 g in 4 of 7 men, assuming that there were no regional differences in the ionic composition of sweat. The findings suggested that most of the participants should take more fluid and some of them might need salt intake during the practice.

Entomogenous fungi that produce 2,6-pyridine dicarboxylic acid (dipicolinic acid).

An inhibitor of the prophenoloxidase activation using extract from a silkworm pupa was isolated from a culture filtrate of Cordyceps militaris and identified as dipicolinic acid (DPA). The production of DPA in Clavicipitaceae fungi was examined. Entomogenous fungi that produce DPA were integrated into one group by a phylogenetic analysis based on 18S rDNA. It is suggested that the group acquired an ability to produce DPA during its evolution from plant pathogenic fungi to entomogenous fungi.

Reversible 1,4-cycloaddition of singlet oxygen to N-substituted 2-pyridones: 1,4-endoperoxide as a versatile chemical source of singlet oxygen.

N-substituted pyridones (1) easily undergo singlet oxygenation to give exclusively the corresponding endoperoxides (2), which decompose to give pyridones again while liberating 1O2 in high yield.

Chemiluminescence in molecular recognition: base-induced decomposition of optically active dioxetanes bearing a bisnaphthol moiety with a complex of optically active crown ether-potassium tert-butoxide.

Four optically pure isomers of dioxetane (1) were decomposed by the action of the complex of an optically active crown ether with potassium tert-butoxide to afford light with lambda(max)CL and gave four different spectrum shapes.

Fluoride-induced chemiluminescent decomposition of 1,2-dioxetanes bearing a phenyl moiety substituted with a methyl having an electron-withdrawing group.

A new type of dioxetane bearing a 3-(cyanomethyl)phenyl (1a), 3-(methoxycarbonylmethyl)phenyl (1b), 3-(benzoylmethyl)phenyl (1c), or 3-[cyano(methoxycarbonyl)methyl]phenyl group (1d) decomposed through an intermediary dioxetane bearing a benzylic carbanion to afford crimson to yellow light on treatment with TBAF in DMSO.

[Quantification of urinary hemoglobin by an immunological method].

The quantification method of urinary hemoglobin has not been established. We examined whether a reagent (Eiken, Tokyo) used to immunologically assay fecal hemoglobin could be utilized to quantify urinary hemoglobin. The coefficients of variation were 2.2-3.0% in the reproducibility of one-run assays using urine and 4.3-5.7% in that of multiple-run assays using the standard sample of hemoglobin. Urinary hemoglobin was unstable and decreased in a time- and temperature-dependent manner. However, addition of the hemoglobin stabilization buffer (50 mM phosphate buffer, pH 6.4) (Eiken) to urine made urinary hemoglobin stable. Urinary hemoglobin levels did not change significantly when stored at 4 degrees C for 7 days or at -80 degrees C for 30 days. The hemoglobin concentration (mean +/- SD) of urine showing 1+, 2+ and 3+ with a test strip was 385 +/- 165(n = 30), 1070 +/- 499(n = 40) and 4130 +/- 2770 ng/ml (n = 20), respectively. Urinary hemoglobin did not correlate with urinary albumin, transferrin, immunoglobulin G, N-acetyl-beta-D-glucosaminidase nor alpha 1 microglobulin. These results suggest that this immunological method using the hemoglobin stabilization buffer can be utilized for quantification of urinary hemoglobin, which may provide clinically important information.