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Contribution

Structural Change of Dimeric SOD Enzyme and Amyotrophic Lateral Sclerosis (ALS)

Yuzo NishidaYamagata University

1. Amyotrophic Lateral Sclerosis (ALS) andmutant Superoxide Dismutase (SOD)

Amyotrophic lateral sclerosis (ALS) is a progressiveparalytic disease characterized by selective degenerationof the upper and lower motor neurons.1,2) Although ALS ispredominantly a sporadic diseases, ~10% cases areinherited in an autosomal dominant manner (familial ALS(fALS)) and a subset of the fALS cases are caused bymutations in the SOD1 gene.3,4) The gene product of SOD1,cytoplasmic Cu,Zn-superoxide dismutase (SOD1), is aubiquitously expressed enzyme that catalyzes thedisproportionation reaction of superoxide radicals.5)

O2 + Cu(II) O2 + Cu(I) (1)

O2+Cu(I) O2H2 + Cu(II) (2)

H

The crystal structure of the SOD(Cu/Zn) has beendetermined,8) its dimeric structure being illustrated in Fig.1 (PDB, 1spd_x). The copper and zinc ions are bridged byanionic form of imidazole ring of histidine.

There are several lines of evidence that suggests thatSOD1 mutations result in a gain, rather than loss offunction that causes ALS. For instance, some fALS-associated mutant SOD1s retain full enzymatic activity.6)

In addition, SOD1 knock-out mice lack ALS symptoms,whereas transgenic mice expressing the fALS-associatedmutant G93A SOD1 develop ALS-like symptoms despiteexpression of endogenous mouse SOD1.7) Lastly,

overexpression of human wild type SOD1 fails to alleviatesymptoms in this transgenic mouse model for ALS.7)

One hypothesis explaining the gain of function of SOD1is that misfolding of the mutant alters the catalyticmechanism to allow production of oxidants such asperoxynitrite9) and possibly hydrogen peroxide.10) Anothermajor hypothesis suggests that toxicity caused byintracellular aggregation of SOD1. SOD1 inclusion bodies,which also react with anti-ubiquitous antibodies, are acommon pathological finding in motor neurons andneighboring astrocytes of ALS patients.11)

These two hypotheses, however, are not mutuallyexclusive when considering that oxidative modification ofproteins may contribute to aggregation and proteaseresistance. Protein aggregation is a common pathologicalfeatures of many neurodegenerative disorders,12)

including Huntington’s, Alzheimer’s, and Parkinson’sdiseases. In each case, misfolding and aggregationpropensity of mutant SOD1s may be the mechanism bywhich over 100 disparate mutations cause a common ALSphenotype. Although SOD1 aggregates may be inherentlytoxic or cause motor neuron toxicity by sequesteringchaperons and blocking proper functioning of theproteasome, origin of toxicity by SOD1 aggregates has notbeen elucidated.

To understand ALS pathogenesis, we must clarify howaltering SOD molecule can induce cell injury. To carry outsuch an investigation, we have started to clarify the originof the gain-of-funct ion, and mechanism of SODaggregation in solution.

The reaction mechanism of SOD1 enzyme has beeninvestigated by many authors. Very recently Nishida et al.have a postulated new mechanism for this enzyme based

Fig. 1 Dimeric structure of SOD molecule. Two copper and zinc ions are illustratedas colored circles.

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number 135

Cu(II)O

OH

on the results used by the model compounds.13) We havepointed out the importance of formation of a copper(II)-OOHspecies as an intermediate (see Scheme-I ) in the secondstep (2) above, and this hydrogen peroxide produced isimmediately removed from the wild-type enzyme becauseof the negligible interaction between hydrogen peroxide andthe copper(II) ion and the surrounding organic groups.

the human SOD enzymes. They reported that the free-radical generating activity of the mutant, as measured bythe spin-trapping method at low H2O2 concentration, isenhanced relative to that of the wild-type and G93A,wild-type < G93A < A4V, but the reason for the aboveobservation has not been clarified.

Scheme-I

Siddique et al.7) have determined the crystal structuresof human SOD, along with two other SOD structures, andhave established that the fALS mutations do not changeany active-site residues involved in the electrostaticrecognition of the substrate, the ligation of the metal ionsor the formation of the active-site channel, but only the slightchange in the neighborhood around the copper(II) ion isdetected. On the basis of rigorous studies defining thestructural and energetic effects of conserved hydrophobicpacking interactions in proteins, six of the fALS mutationswould be expected to destabilize the subunit fold or thedimer contact. But, I do not think that these destabilizingeffects on the subunit fold and the dimmer contact areenough to explain the all fALS pathogenesis.

In 1997, Yim et al. reported that a fALS mutant (Gly93Ala= G93A) exhibits an enhanced free radical-generatingactivity, while its dismutation activity is identical to that ofthe wild-type enzyme.14) In Figure 2, ESR spectra of DMPO-OH radical adducts formed in solution containing H2O2 and

Fig. 2. ESR spectra of DMPO-OH radical adducts formed in solutions containing H2O2 and the human SOD(Cu/Zn).A; wild-type SOD(Cu/Zn), B; G93A mutant SOD, C; A4V mutant SOD, D; heat-inactivated SOD (Yim et al., J. Biol.Chem., 1997, 272, 8861).

2. Origin of “Gain-of Function” in Mutant SODEnzyme and the Unique Reactivity of Copper(II)-hydroperoxide Adduct

In order to obtain a comprehensive solution for thecorrelation between the structural change in mutant SODand pathogenesis of fALS, we have studied the reactivityof a copper(I I)-OOH, proposed as an importantintermediate in the SOD reaction. For this purpose, we havesynthesized many copper(II) compounds with ligands thatcontain N,N-bis(2-picolylmethyl)amine moiety, as illustratedin Figure 3.15) The structural features of all the copper(II)compounds are very similar to each other (as an example,crystal structure of [Cu(bdpg)Cl]+ is illustrated in Fig. 4).In the presence of hydrogen peroxide, formation of aadduct formation as shown at the right side of Fig. 4 isanticipated, and this was consistent with the results on thereaction with cyclohexane, etc, and we have found that thereactivity of a peroxide adduct of the copper(II) compoundsis highly dependent on the R of the ligand system, whichshould be due to the slight structural change around thecopper(II) ion due to the different R in the ligand system.

We have measured the ESR spectra of the solutioncontaining a copper (II) complex and spin-trapping reagent,such as PBN (α-phenyl-N-tert-butylnitrone) and TMPN(N,N,N',N'- tetramethyl-4-piperidinol), specific reagents forOH• radical and singlet oxygen (1∆g) (Scheme-II ),respectively.16)

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Fig. 3. Chemical structures of the ligands used in our study.

O

Cu O

O

HC

NH2C

H2C

NH2

Scheme-II

NH

OH

MeMe

MeMe

N

OH

MeMe

MeMe

O

1O2

TMPN

Fig. 4. Left side: crystal structure of [Cu(bdpg)Cl]+; Right side:assumed structure of a peroxide adduct [Cu(bdpg)(OOH)]+.

R N CH2N 2

R= -CH2CH2C(=O)NH2 (bdpg)

(dpgs)R= -CH2C(=O)NHCH3

[H(dpal)]R= -CH2CH2C(=O)OH

(dpgt)R= -CH2C(=O)NHCH2C(=O)NHCH2COOH

(G-bdpg)R= -CH2CH2C(=O)NHCH2COOCH3

(bdpg-His)R= CH2CH2 CO

NH CH

CH2

CO

OCH3

R= -CH2CH2C(=O)NHCH3 (Me-bdpg)

R= CH2

HO

[H(Hphpy)]

N

NH

No ESR signal due to the formation of radical of PBNwas detected when the copper (II) complexes with (tpa)(=tris(2-pyridylmethyl)amine) or (bdpg), was mixed withH2O2 and PBN. However, strong peaks due to nitronradical formation of the corresponding TMPN (Scheme-II )was detected in the solution with Cu(tpa) complex, but notwith the Cu(bdpg) complex. Especially comparison betweenthe Cu(pipy)Cl+ and the Cu(mopy)Cl+ is very interesting.15)

Structural features of the two compounds are very closelyrelated, apart from the difference of the oxygen atom onthe morphorin ring of Cu(mopy)Cl+ complex is replaced bythe -CH2 in the Cu(pipy)Cl+ complex. (see the figurebelow)

In the case of Cu(pipy)Cl+, no formation of the nitroneradical was observed in the presence of hydrogenperoxide. Incontrast, high activity for the radical formationby the Cu(mopy)Cl+ complex was detected as illustrated inFigure 5. The similar high activity for radical formation ofTMPN was also observed for the copper(II) complex,[Cu(Hphpy)Cl]+. In this case, similar to the Cu(mopy)Cl+

complex, the addition of the H2O2 to the copper(II) solutiondoes not induce the change in ESR spectrum due to thecopper(II) ion; but the addition of TMPN leads to thedramatic change in the ESR signals attributed to thecopper(II) species (i.e., the change of hyperfine structurevalues due to copper atom). These are all comprehensively

(mopy) (pipy)

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elucidated on the assumption that the complex formationof copper (II), hydrogen peroxide, and TMPN occurs onlywhen three reagents are present in the solution, (see theFig. 6), and unique reactivity of the hydrogen peroxideobserved is detected only when the intermediate is formedin the solution.

Above facts are indicating that the reactivity of the Cu(II)-OOH is determined by the structural properties of theintermediate (see Fig. 6) i.e., by the chemical interactionsamong copper(II)-OOH species, peripheral groups andsubstrate.13) It should be noted here that althoughhydrogen peroxide has been believed to be relatively inertand not toxic to cells, our present results clearly show thatsome copper (II) chelates can activate the hydrogenperoxide to exhibit high reactivity similar to that of thesinglet oxygen (1∆g).

Fig. 5. ESR spectra of the solution containing[Cu(mopy)Cl]+, H2O2, and TMPN.

Fig. 6. Assumed intermediate among copper(II) chelate,H2O2 and TMPN.

We also found that some copper(II) complexes exhibithigh activity in the oxygenation of the methionine residueof amyloid beta-peptide(1-40) at sulfur atom18), and thedecomposition of several proteins in the presence ofhydrogen peroxide.19) All these facts may suggest that the“gain-of-function” of the mutant SOD is due to formation ofa long-lived highly reactive copper(II)-OOH as anintermediate in the process of SOD reaction. The chemicalstructures around the copper (II) in the mutant SOD isslightly changed, and this gives an unexpected effect onthe reactivity of a copper(II)-OOH as observed in ourpapers. In the mutant SOD C-N bond cleavage by the Cu(II)-OOH may give great changes in the surface of SOD,leading to destabilizing of the dimer contact of the SODenzyme.20) Thus, it is quite likely that formation andexistence of a highly reactive Cu(II)-OOH species is anintrinsic origin for oxidative stress in the pathogenesis offALS, which may be consistent with the recent studies onthe destabilizing of the dimer contact of the SODenzyme.21,22)

Cu(II) OO

H

TMPN

peripheral group of the ligand system

In order to get further information on the reactivity of acopper (II)-OOH species, we have measured the ESI-Massspectra of the solutions of copper (II) compounds andhydrogen peroxide. When hydrogen peroxide was addedto the Cu(Me-bdpg)Cl solution (see Figure 3), theformation of [Cu(bdpg)Cl], not [Cu(dpal)], was detected byESI-Mass spectra.17) These are clearly indicate that Cu(II)-OOH species can cleave the peptide at the C-N bondoxidatively, not hydrolytically, because the hydrolyticcleavage may give Cu(dpal) species from the Cu(Me-bdpg)compound.

O

CuN O

OH

C

HN CH3

O

CuN OH2

CNH2

3. Dissociation of Dimeric SOD molecule intoMonomers

As stated before, it is widely recognized that proteinaggregation are a common pathological features of manyneurological disorders, including Huntington’s, Alzheimer’s,and Parkinson’s diseases and that SOD1 aggregates maybe inherently toxic or cause motor neuron toxicity bysequestering chaperons and blocking proper functioningof the proteasome.

In 2004, Rakhit et al. reported that SOD1, normally adimeric enzyme, dissociates to monomers prior toaggregation for both wild type and mutant proteins.23) Theyused the “Dynamic Light Scattering (DLS)” method todetect the dissociation of dimeric SOD to monomers. Veryrecen t l y we have repor ted tha t the cap i l l a ryelectrophoresis method (CE) is very suitable to investigatethe conformat ional change of the proteins andaggregation states of the proteins in solution.24) As anexample, two CE profiles of SOD and transferrin areillustrated in Fig. 6.25) Although the concentrations of thetwo enzymes are the same, the peak intensities are quitedifferent from each other, and this has been rationalizedon the fact that SOD has a rigid dimeric structure insolution, but dimeric structure of apo-transferrin is moreflexible.26)

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We have observed that the drastic decrease of the peakstrength due to the dimeric SOD molecule occurs whenthe copper(II)/ascorbic acid solution was added to the SODmolecule as shown in Fig. 7.; our experimental system wassame as that reported by Rakhit et al. This clearly showsthat the dissociation of the dimeric SOD molecule can bereadily detected by the CE method.

We also have found that the presence of excesshydrogen peroxide induces the loosening or dissociation

Figure 6. CE profiles of the solutions (protein 2 mg/1ml).A: SOD; B: Apo-transferrin, and C; Fe(ida) complex wasadded to solution B.

Fig. 7. CE profiles of the solutions containing SOD (SOD3 mg/1ml). red: SOD only. Green and blue: copper(II)/ascorbate solution was added to SOD solution. (green,measured immediately after addition, and blue, at after60 min.)

of dimeric structure of SOD molecule.26) As the origin ofthe dissociation of the dimeric SOD in the presence ofhydrogen peroxide is clear,26) it seems quite likely that theoxidant in the system used by Kakhit et al. should behydrogen peroxide, and that sporadic ALS may be relatedwith the presence of hydrogen peroxide, and the samediscussion may be applied to the elucidation of sporadicprion diseases (see alter).

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By using antibody methods to rapidly purified SOD1 andcoupling this with mass spectrometry, Sato et al. havemeasured the relative accumulated levels of wild-type andmutant SOD1 in erythrocytes of 29 SOD1-mutated fALSpatients.22) They observed that the patients withundetectable SOD1 mutant had the shortest diseasedurations. Although age at disease onset was found to beuncorrelated with the amount of mutant SOD1, the evidenceconvincingly shows a strong inverse correlation betweendisease duration and mutant accumulation. In other words,an accelerated disease course is found for mutants thatare less stable. This surprising discovery implys that it isthe misfolded unstable forms of SOD1 mutants thatcontribute to toxicity underlying disease progression, andthat despite its apparent importance for progression, SOD1mutant stability is not correlated with disease onset. Thusdissociation of the dimeric SOD1 molecule to misfoldedmonomers should be an essential important process forAPS pathogenesis. As it has become apparent thathydrogen peroxide plays an important role in the formationof SOD1 monomers,26) we should pay attention to theformation of excess hydrogen peroxide in the human body,especially due to the reaction between a dimeric iron (III)species and glutathione cycle and other related systems.27)

4. Copper(II)-OOH in Sporadic Prion Diseases

Between 1980 and roughly 1996, about 750,000 cattleinfected with BSE (bovine spongiform encephalopathy, oneof TSEs) were slaughtered for human consumption in GreatBritain, and at present it is accepted that the central eventin TSEs is the post-translational conversion of the normalcellular prion protein (PrPC) into an abnormal isoform ofcalled scrapie PrP (PrPSc) that has a high-β-sheet contentand is associated with transmissible disease.28) It isgenerally recognized that PrPC is a copper-containingprotein (at most 4 copper ions are present within theoctarepeat region located in the unstructured N-terminus).

Analysis of recombinant mouse and chicken PrPC has leadto the discovery of an important “gain-of-function”following the formation of the PrPC copper complex; PrPC

has been shown to contribute directly to cellular SODactivity.

The misfolded prions (PrPSc) ultimately kills neurons andleaves the brain riddled with holes, like a sponge. Inaddition to PrPSc, another protease-resistant PrP of 27-30kDa, which is called as PrP27-30 was extracted fromaffected brains. It should be noted here that PrP27-30 isderived from only PrPSc (not from PrPC), and no differencein amino acid sequence between PrPC and PrPSc have beenidentified. Based on these facts we may assume that thechemical environment around the copper ion in the PrPSc

should be different from those in the PrPC; this situation issimilar to the difference observed between the those aroundcopper(II) ions in the wild-type and mutant SOD enzyme.Thus, it is most likely that the “gain-of-function” in the PrPSc

due to a “highly reactive” Cu(II)-OOH formation may occuras described for the mutant SOD molecule, which leads tothe cleavage of the peptide bonds around the copper ion(near at about 90 site), giving dangerous PrP27-30; thelatter protein may behave as like the misfolded SODmonomer. In addition to this, it seems quite likely that thecopper(II) ions in PrPC and also PrPSc may react withhydrogen peroxide to yield a Cu(II)-OOH species, whichmay give serious effects toward the PrPC such asoxygenation at methionine residue, conformational change(i.e., formation of PrPSc), and degradation of protein in thepresence of hydrogen peroxide (see Scheme-III ). Severalexperimental facts observed for the native prionproteins29-32) seem to be consistent with our discussions.All these findings support our proposal that hydrogenperoxide, which may derive from the SOD function of PrPSc

and abnormal metabolism of iron ions27) is likely to be thereal origin of oxidative stress in sporadic prion diseases.

Scheme-III

Cu(II) chelates near the surface of synapse

Cleavage, degradation, and conformational change of PrPC

H2O2

Misfolding of the protainsFormation of aggregates

Formation of PrPSc

Formation of PrP27-30

H2O2

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number 135

References

[1] R. G. Smith and S. H. Appel, Annu. Rev. Med., 1995, 46,133.

[2] R. H. Brown, Jr., Cell, 1995, 80, 687.[3] H.-X. Deng, et al., Science, 1993, 261, 1047.[4] M. W.-Pazos, et al., Science, 1996, 271, 515.[5] I. Fridovich, Annu. Rev. Biochem., 1995, 64, 97.[6] D. R. Borchelt, et al., Proc. Natl. Acad. Sci. USA., 1994, 91,

8292-8296.[7] L. I. Brujin et al., Science, 1998, 281, 1851-1854.[8] J. A. Tainer, E. D. Getzoff, J. S. Richardson, and D. C.

Richardson, Nature, 1983, 306, 284.[9] A. G. Estevez et al., Science, 1999, 286, 2498-2500.[10] M. B. Yim et al., Proc. Natl. Acad. Sci. USA., 1996, 93, 5709-

5714.[11] M. Watanabe et al., Neurobiol. Dis., 2001, 8, 933-941.[12] B. Caughey and P. T. Lansbury, Annu. Rev. Neurosci., 2003,

26, 267-298.[13] Y. Nishida and S. Nishino, Z. Naturforsch., 2001, 56c, 144-

153; Y. Nishida, Med. Hypothesis Res., 2004, 1, 227.[14] M. B. Yim et al., J. Biol. Chem., 1997, 272, 8861.[15] T. Kobayashi, T. Okuno, T. Suzuki, M. Kunita, S. Ohba, and

Y. Nishida, Polyhedron, 1998, 17, 1553.[16] S. Nishino, T. Kobayashi, M. Kunita, S. Ito, and Y. Nishida, Z.

Naturforsch., 1999, 54c, 94.[17] S. Nishino, M. Kunita, Y. Kani, S. Ohba, H. Matsushima, T.

Tokii, and Y. Nishida, Inorg. Chem. Communications, 1999,3, 145.

[18] S. Nishino and Y. Nishida, Inorg. Chem. Communications,2001, 4, 86; Synth. Reac. Inorg. Metal-Org. Nano-MetalChem., 2005, 35, 677.

[19] S. Nishino, A. Kishita, and Y. Nishida, Z. Naturforsch. J.Biosciences, 2001, 56c, 1144.

[20] P. Cioni, A. Pesce, B. Morozzo, S. Castelli, M. Falconi, L.Parrilli, M. Bolognesi, G. Strambini, and A. Desideri, J. Mol.Biol., 2003, 326, 1351.

[21] O. Matsumoto and Fridovich, Proc. Natl. Acad. Sci. USA.,2002, 99, 9010.

[22] K. Yamanaka and D. W. Cleveland, Neurology, 2005, 65,1859.

[23] R. Rakhit et al., J. Biol. Chem., 2004, 279, 15499-1550.[24] A. Kishita and Y. Nishida, Annu. Report CIN, 2004, 1; Nishida

et al., Synth. Reac. Inorg. Metal-Org. Nano-Metal Chem.,2005, 35, 379; Nishida et al., Z. Naturforsch., 2007, 62b, 205.

[25] Y. Sutoh et al., Chem. Lett., 2005, 34, 141.[26] Y. Nishida et al., Z. Naturforsch., 2006, 61c, 273.[27] Y. Nishida, et al., Z. Naturforsch., 2007, 62c, 608; Y. Nishida,

Recent Res. Devel. Pure & Appl. Chem., 1999, 3, 103; Med.Hypothesis Res., 2004, 1, 227.

[28] B. Caughey, Trends Biochemical Sciences, 2001, 25, 235.[29] J. R. Requena, N. D. Dimitrova, G. Legname, S. Teijira, S. B.

Prusiner, and R. L. Levine, Arch. Biochem. Biophys., 2004,432, 188.

[30] H. E. M. MaMahon, A. Mange, N. Nishida, C. Creminon, D.Casanova and S. Lehmann, J. Biol. Chem., 2001, 276, 2286.

[31] R. Requena, D. Groth, G. Legname, E. R. Sradtman, S. B.Prusiner, and R. L. Revine, Proc. Natl. Acad. Sci. USA., 2001,98, 7170.

[32] N. T. Watt, D. R. Taylor, A. Gillott, D. A. Thomas, W. S. Perera,and N. M. Hooper, J. Biol. Chem., 2005, 280, 35914; B. J.Tabler, S. Turnbull, N. J. Fullwood, M. German, and D. Allsop,Biochem. Soc. Trans., 2005, 33, 548; B. J. Tabler, O. M. A.E.-Agnaf, S. Turnbull, M. J. German, K. E. Paleologou, Y.Hayashi, L. J. Kooper, N. J. Fullwood, and D. Allsop, J. Biol.Chem., 2005, 280, 35789; N. T. Watt and N. M. Hopper,Biochem. Soc. Trans., 2005, 33, 1123; S. Fernaeus, K. Reis,K. Bedecs, and T. Land, Neuroscience Lett., 2005, 389, 133.

(Received Feb. 2008)

Introduction of authors

Yuzo NishidaProfessor, Faculty of Science, Yamagata University

[Brief career history] Ph. D from Kyushu University. 1969, Assistant Professor of Faculty of Science, Kyushu University.1987, Associate Professor of Faculty of Science, Yamagata University, 1991, Professor of Faculty of Science, Yamagata

University, 1998, Professor of Institute for Molecular Science, since 2000, Present post.[Specialty] Coodination chemistry, Biological inorganic chemistry, Brain disease chemistry.

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number 135

Sugar Building Blocks

OHO

NO

O O

OO

Ph

OCH3

OBnO

NO

O O

OO

Ph

OCH3 OOBzHO

BzOBzO

OCH3

OO

NO

O O

OO

Ph

OCH3

O

AcOAcHN

AcO OAc

AcO

SPh

COOCH3O

OAcAcO

AcOAcO

O C

NH

CCl3

OOHHO

BnOHO

O OCH3

OOAcAcO

OOAc

SPhO

OBnBnO

OOBn

SPh

OO

N3

O

OO

Ph

OCH3

OHO

OO

Ph

N3

O OCH3

OOBnHO

HOBnO

O OCH3

OOBn

BnOBnO

O

OOBnHO

HOBnO

OOCH3

OOBn

BnOBnO

O

OOBnHO

BnOBnO

OOCH3

OOBn

BnOBnO

O

OOBnBnO

HOBnO

OOCH3

[M1479] [M1609] [M1933]

[M1598] [M1706] [T2295]

[M1725] [P1680] [P1660]

[M1643]

[M1637] [M1634]

[M1726]

[M1686] [M1727]

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number 135

Benzyne Precursors

T2089 2-(Trimethylsilyl)phenyl Trifluoromethanesulfonate (1) 1g, 5g

M1882 4-Methyl-2-(trimethylsilyl)phenyl Trifluoromethanesulfonate (2) 1g, 5g

M1883 2-Methyl-6-(trimethylsilyl)phenyl Trifluoromethanesulfonate (3) 1g, 5g

M1884 3-Methoxy-2-(trimethylsilyl)phenyl Trifluoromethanesulfonate (4) 1g, 5g

M1885 4-Methoxy-2-(trimethylsilyl)phenyl Trifluoromethanesulfonate (5) 1g, 5g

T2465 1-(Trimethylsilyl)-2-naphthyl Trifluoromethanesulfonate (6) 1g, 5g

T2466 3-(Trimethylsilyl)-2-naphthyl Trifluoromethanesulfonate (7) 1g, 5gB3047 3,3'-Bis(trimethylsilyl)biphenyl-4,4'-diyl Bis(trifluoromethane-

sulfonate) (8) 1g, 5gT2467 1,3,5-Tris[4-(trifluoromethanesulfonyloxy)-3-(trimethylsilyl)-

phenyl]benzene (9) 1g

In general, generation of benzyne requires the addition of strong base or high temperature.However, benzyne precursors that can be used in a milder condition have been developed. 2-TMS-phenyltriflate (1) and its analogs are some of the excellent benzyne precursors, and react with fluoride ion toproduce benzyne under mild conditions. Moreover, since these precursors don’t generate iodobenzenewhich derives from the elimination group of iodonium-type benzyne precursors, it is possible to use it forthe palladium catalyzed reaction. Therefore, these precursors are being applied to the efficient syntheses ofpolyaromatic compounds.

1 2 3 4

5 6 7 89

TMS

OTf TMSTfO

OTfTMS

TfO

TMS

TMSOTf

TMSOTf

TMS

OTf

MeOTf

TMS

MeTMS

OTf

OMe

TMS

OTf

MeOOTf

TMS TMS

OTf

(cont.)

R

TMS

OTfRR

R1 R2

R

R'

R'

R

R

R

R

R'TMS

TfO

R1 R2

CsF,

Lewis acid

CsF, Pd catalyst

TBAF

CsFPd catalyst

1)

2)

3)

4)

5), 6), 7)

CsF, Pd catalystOR

O

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number 135

References1) Y. Himeshima, T. Sonoda, H. Kobayashi, Chem. Lett., 1983, 1211.2) T. T. Jayanth, M. Jeganmohan, C.-H. Cheng, J. Org. Chem., 2004, 69, 8445.3) A. Cobas, E. Guitián, L. Castedo, J. Org. Chem., 1997, 62, 4896.4) D. Peña, S. Escudero, D. Pérez, E. Guitián, L. Castedo, Angew. Chem. Int. Ed., 1998, 37, 2659.5) D. Peña, D. Pérez, E. Guitián, L. Castedo, J. Am. Chem. Soc., 1999, 121, 5827.6) D. Peña, D. Pérez, E. Guitián, L. Castedo, J. Org. Chem., 2000, 65, 6944.7) E. Yoshikawa, K. V. Radhakrishnan, Y. Yamamoto, J. Am. Chem. Soc., 2000, 122, 7280.8) Z. Liu, R. C. Larock, J. Org. Chem., 2006, 71, 3198.9) U. K. Tambar, B. M. Stoltz, J. Am. Chem. Soc., 2005, 127, 5340.

10) J. Zhao, R. C. Larock, Org. Lett., 2005, 7, 4273; H. Yoshida, H. Hukushima, J. Ohshita, A. Kunai, Angew. Chem. Int.Ed., 2004, 43, 3935.

11) TCIMAIL, 2006, number 129, 16.

Related CompoundsA1464 1-Aminobenzotriazole 1g, 5gD2503 Diphenyliodonium-2-carboxylate Monohydrate 5gP1620 Phenyl[2-(trimethylsilyl)phenyl]iodonium Trifluoromethanesulfonate 1g

(cont.)

I0643 (Isocyanoimino)triphenylphosphorane (1) 5g

Synthesis of α-Diazoketones

Compound 1 is a useful reagent for the synthesis of diazoketones. The compound 1 was reactedwith acid chlorides followed by hydrolysis and then treated with triethylamine and a catalytic amount ofp-toluenesulfonyl chloride to give diazoketones 2.1) Moreover, the compound 1 has been used for thesynthesis of heterocyclic compounds 3 and 4.2)

References1) Synthesis of α-diazoketones

a) E. Aller, P. Molina, Á. Lorenzo, Synlett, 2000, 526.b) M. M. Bio, G. Javadi, Z. J. Song, Synthesis, 2005, 19.

2) Synthesis of heterocyclic compoundsa) A. Souldozi, A. Ramazani, Tetrahedron Lett., 2007, 48, 1549.b) A. Souldozi, A. Ramazani, N. Bouslimani, R. Welter, Tetrahedron Lett., 2007, 48, 2617.

1

i) RCOCl

iii) Et3N, TsCl (cat.)

O

RN2

2+ Ph3PO

1)

NN

O + Ph3PO

2a)

+ Ph3PO

2b)

CH2Cl2, r.t.

CH2Cl2, r.t.

,

3

4

ON

N

PhPh

CO2MeMeO2C

H

Y. 92%

Y. 91%

only Z stereoisomers

R=BnR=ClCH2

ii) H2O

H

CO2Me

CO2Me Ph Ph

OH O

OH

O

Y. 74%Y. 68%

P N N C

12

number 135

P Chiral Ligand

B3035 (R,R)-DIPAMP [=(R,R)-1,2-Bis[(2-methoxyphenyl)phenylphosphino]ethane] (1a) 100mg, 1g

B3036 (S,S)-DIPAMP [=(S,S)-1,2-Bis[(2-methoxyphenyl)phenyl phosphino]ethane] (1b) 100mg, 1g

Compound 1 are P-chiral ligands developed by Knowles et al. This compound 1 forms a stablecomplex with rhodium, and the complex has been used as a catalyst for an asymmetric hydrogenationreaction. Particularly, the asymmetric hydrogenation reaction of α-acetylamino cinnamic acid derivative 2is well known, and it provides L-amino acid derivative 3 with high optical purity. When this compound 3is de-protected, L-DOPA, which is used for the treatment of Parkinsonism, can be obtained. Knowles wonthe Nobel Prize in Chemistry for his achievement of developing this asymmetric synthesis of L-DOPA in2001.

References1) Asymmetric hydrogenation with a complex of Rh and a chiral bisphosphine

a) W. S. Knowles, M. J. Sabacky, B. D. Vineyard, D. J. Weinkauff, J. Am. Chem. Soc., 1975, 97, 2567.b) B. D. Vineyard, W. S. Knowles, M. J. Sabacky, G. L. Bachman, D. J. Weinkauff, J. Am. Chem. Soc., 1977, 99, 5946

Building Block of a Conducting Polymer

B2934 3-Bromothiophene-2-carboxaldehyde (1) 1g, 5g, 25g

Compound 1 is useful as a material for synthesis of condensed heterocyclic compounds such asthienothiophene derivatives. The thienothiophene derivatives have been expected to be applied to conduct-ing polymers.

References1) Synthesis of thieno[3,2-b]thiophene derivatives

L. S. Fuller, B. Iddon, K. A. Smith, J. Chem. Soc., Perkin Trans. 1, 1997, 3465.2) Synthesis of fused thieno-azepine derivatives

X. Beebe, V. Gracias, S. W. Djuric, Tetrahedron Lett., 2006, 47, 3225.

CH

OCOR

R'O

C C OH

NHCOCH3

O

CH2 C

NHCOCH3

C

H

O

OH

R'O

OCOR

CH2 C

NH2

C

H

O

OH

HO

OH

, [Rh(COD)Cl]2 (cat.), H2

PP

Ph

PhOCH3

CH3O

1a (cat.)

L-DOPA2 3

S

S

Br

Br

S

S

R

R

S

S

R

R n

S

S

Br

Br

S

S

Br

S

SCO2H Br Br

i) HSCH2CO2Et, K2CO3, DMF

ii) LiOH, THF

iii) Br2, AcOH aq.

iv) LDA, THF

v) Br2

vi) Zn, AcOH

1S CHO

Br

BrBr

13

number 135

Sulfene Equivalent

M1669 Methanedisulfonyl Dichloride (1) 5g

Compound 1 reacts with bases such as Et3N and produces disulfene 2. This compound 2 easilyreacts with diene compounds to form [4+2] cycloadduct 3. Therefore, the compound 2 can be used as atrapping reagent for diene compounds because of this property.1) In addition, the compound 2 also reactswith α-olefins to form γ-chloroalkanesulfonyl chloride.2)

References1) [4+2] Cycloaddition reaction of dienes with sulfenes

a) G. Opitz, M. Deissler, K. Rieth, R. Wegner, H. Irngartinger, B. Nuber, Liebigs Ann., 1995, 2151.b) G. Opitz, M. Deissler, T. Ehlis, K. Rieth, H. Irngartinger, M. L. Ziegler, B. Nuber, Liebigs Ann., 1995, 2137.

2) Sulfonylation of alkenesH. Goldwhite, M. S. Gibson, C. Harris, Tetrahedron, 1965, 21, 2743.

O2S

Me

Me

Me

Me

MeMe

Me

Me

Me Me

O2S

Y. 69%

Et3N

CH3CN

SO2SO2Cl

Cl

Cl

Cl

Bz2O2

+ +

Y. 50% Y. 26%

1

OS S

O

OO

Cl S

O

O

CH2 S

O

O

Cl

MeMe

Me

MeMe

2 3

1)

2)

SNH2

O

SN

O

R1S

NH

O

R1

R2

H2N R1

R2

HCl.

1a 2 3 4

R1CHO

Lewis acid

R2MgBr HCl

MeOH

R1=Et, R2=Me Y.96%, dr 93 : 7R1=Ph, R2=Et Y.98%, dr 92 : 8

1a)

CH2Cl2

Asymmetric Synthesis of α-Branched Amines

B2907 (R)-(+)-tert-Butylsulfinamide (1a) 1g

B2908 (S)-(-)-tert-Butylsulfinamide (1b) 1g

Compounds 1a and 1b are chiral auxiliary supplements and can be reacted with aldehydes orketones to give sulfinyl imines 2. Then the addition reaction between compound 2 and a Grignard reagent1)

is carried out to obtain the compounds 3 with high diastereoselectivities. The optically active amines 4 isobtained in high yields by treatment of compounds 3 with hydrochloric acid. Moreover, a convenientsynthetic method of optically active β-amino acids using the compounds 1 has been reported.2)

References1) Asymmetric synthesis of α-branched amines

a) G. Liu, D. A. Cogan, J. A. Ellman, J. Am. Chem. Soc., 1997, 119, 9913.b) Y. Bolshan, R. A. Batey, Org. Lett., 2005, 7, 1481.

2) Asymmetric synthesis of β-amino acidsT. P. Tang, J. A. Ellman, J. Org. Chem., 1999, 64, 12.

14

number 135

Asymmetric Two-center Phase-transfer Catalyst

D3476 (S,S)-TaDiAS-2nd [= 6,10-Dibenzyl-N,N'-dimethyl-N,N,N',N'-tetrakis- (4-methylbenzyl)-1,4-dioxaspiro[4.5]decane-(2S,3S)-diylbis(methylammonium) Tetrafluoroborate] (1a) 200mg, 1g

D3475 (R,R)-TaDiAS-2nd [= 6,10-Dibenzyl-N,N'-dimethyl-N,N,N',N'-tetrakis- (4-methylbenzyl)-1,4-dioxaspiro[4.5]decane-(2R,3R)-diylbis(methylammonium) Tetrafluoroborate] (1b) 200mg, 1g

TaDiAS (Tartrate-derived Diammonium Salt) is an asymmetric phase transfer catalyst developedby Shibasaki et al. TaDiAS has two ammonium ions in the molecule and its ions cooperatively act togetherto hold anions in an asymmetric space. Previously, TaDiAS has been achieved in high asymmetric yields.More recently, TaDiAS-2nd has been developed in which an alkyl chain of the acetal site of TaDiAS issubstituted with a cyclohexane ring in order to improve the reactivity and enantioselectivity of thesecatalysts. Tandem synthesis of alkaloid (+)-Cylindricine C has been reported using TaDiAS-2nd as anasymmetric Michael reaction catalyst.

* In the literature, arrangement of two benzyl groups in TaDiAS-2nd is in the (S,S)-form. However, ourproduct here is a mixture of diastereomers. Although there may be slight differences in their reactivity andenantioselectivity, it is confirmed that the reaction proceeds without any problem using our product.

References1) Short synthesis of (+)-cylindricine C by using TaDiAS-2nd

a) T. Shibuguchi, H. Mihara, A. Kuramochi, S. Sakuraba, T. Ohshima, M. Shibasaki, Angew. Chem. Int. Ed., 2006, 45,4635.

b) H. Mihara, T. Shibuguchi, A. Kuramochi, T. Ohshima, M. Shibasaki, Heterocycles, 2007, 72, 421.

Ph

Ph

O

O N

N

Me

Me

C6H4-4-Me

C6H4-4-Me

C6H4-4-Me

C6H4-4-Me

2BF4

(S,S)-TaDiAS-2nd (1a)

O

O N

N

R3

R3

Ar

Ar

Ar

Ar

2X

TaDiAS

R1

R2

Ph N

Ph

CO2Bn O O

C6H135

+(S,S)-TaDiAS-2nd* (cat.)

O

N Ph

Ph

BnO2C

O

C6H135

catalytic asymmetricMichael reaction

tandem cyclization N

BnO2C C6H13

N

C6H13

O O

HO(+)-cylindricine C

total (6 steps)

15

number 135

Endo-α, an Enzyme that transfers a whole sugar

A1844 endo-α-N-Acetylgalactosaminidase (= Endo-α), Recombinant from Bifidobacterium longum expressed in Escherichia coli (EC 3.2.1.97) 100 munits*

* 1 unit will hydrolyze 1µmol of Galβ1-3GalNAcα-pNP to Galβ1-3GalNAc and pNPper minute at pH 5.0 and 37 °C

Yamamoto et al. have recently purified and isolated endo-α-N-acetylgalactosaminidase (Endo-α)found in the culture fluid of Bifidobacterium longum.1a) Endo-α can recognize the structure of theGalβ1-3GalNAc disaccharide α-linked with a hydroxyl group. It releases Galβ1-3GalNAc by hydrolysis.When a compound possessing a hydroxyl group coexists as an acceptor, the released Galβ1-3GalNAc istransferred to the acceptor.1b)

Discovered by Yamamoto et al., Endo-α can transfer Galβ1-3GalNAc to various compounds suchas oligosaccharides, peptides, and proteins, using core 1 contained in mucin-type oligosaccharide chains asa donor. As a tool for the enzymatic synthesis of glycoconjugates, it is expected that many applications forEndo-α will be found in a wide range of fields.

This Endo-α was merchandised as the fruition of NEDO project.

References1) Endo-α-N-acetylgalactosaminidase from bifidobacterium longum

a) K. Fujita, F. Oura, N. Nagamine, T. Katayama, J. Hiratake, K. Sakata, H. Kumagai, K. Yamamoto, J. Biol. Chem.,2005, 280, 37415.

b) H. Ashida, K. Yamamoto, T. Murata, T. Usui, H. Kumagai, Arch. Biochem. Biophys., 2000, 373, 394.c) T. Katayama, K. Fujita, K. Yamamoto, J. Biosci. Bioeng., 2005, 99, 457.

This product is not available for purchase in the U.S.

Endo-α+

Donor Acceptor

O

HO

O

OH

AcHNO

O

HO

HO

OH

OH

O

HO

O

OH

AcHNO

O

HO

HO

OH

OHOH

OH

Acceptor: compounds prossessing hydroxyl groups e.g. oligosaccharides, peptides, proteins, etc.