Metal complexes as hydrolytic catalysts
of phosphate diester cleavage
2020/5/14
Literature Seminar
M1 Fujiyoshi

Contents• Introduction• Examples of artificial catalyst ligand• Ligand with hydroxy group• DNA plasmid cleavage• Summary

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1Contents• Introduction• Examples of artificial catalyst ligand• Ligand with hydroxy group• DNA plasmid cleavage• Summary

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2Phosphate diester in nature

Nucleic acids
BaseO P OOOOHO P OOOO

DNA
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BaseHPhospholipids
BaseO P OOOOOHO P OOOORNABase

Phosphatidylcholine
MeNMeMeOOOPOH

OROO

ROOOH

COOHOOOH 2N
POHORORO

Phosphatidylserine
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3Enzymes cleave phosphate ester

Protein phosphatases
Remove phosphate group from Ser/Thr/Tyr

Nucleotidases
Nucleotide to nucleoside
DNA cleavage (DNase, Topoisomerase)

In gluconeogenesis
Remove phosphate group from six-carbon
phosphate intermediate

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Wikipedia phosphatase
https://en.wikipedia.org/wiki/Phosphatase

Calcineurin (PP2B) is a protein
phosphatase enzyme involved inimmune system function.
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4Artificial catalyst mimicking phosphatase
Artificial catalyst

LORO

POHORMLLH 2OORO

POH

ROH

HO

What is the motivation of studies in this field?
1. For a better understanding of the chemistry of the corresponding hydrolytic enzymes
2. For the detoxification of pesticides and chemical weapons (which often have phosphateester-like structures)
3. For employments as artificial restriction enzymes for molecular biology
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F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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5Hydrolysis catalyzed by a metal ion

1. Deprotonation of coordinated water
2. Ligand exchange between water and phosphate
diester
3. Intramolecular nucleophilic attack of OH–
4. Pentavalent intermediate
5. Departure of leaving group
6. Deprotonation of phosphate monoester
7. Ligand exchange between water and phosphatemonoester
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F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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DNA model substrate
DNADNA

Half-life time (t1/2): 100,000~100,000,000 years(in water, 25 ºC, pH = 7)
DNA is tremendously resistant to hydrolysis due to its polyanionicnature.

BaseO P OOOOHO P OOOO

Base

Bis para-nitrophenyl phosphate (BNP)

HBNPO2N

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OPO -OO

NO2

Rate of sponteneous hydrolysis: 1.6 10–11 s–1
t1/2: 1300 years(in water, 25 ºC, pH = 7)
→ rate-limiting step is nucleophile attack (not leaving groupdeparture)
F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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7RNA model substrate
RNABaseO P OOOOOHO P OOOORNABaseOH

2-hydroxypropyl-para-nitrophenyl phosphate(HPNP)

HPNPOH
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O
PO -OO

Half-life time: 110 years (in water, 25 ºC, pH = 7)
RNA is less resistant to hydrolysis than DNA due to the presence ofthe 2’-hydroxyl group on the ribose ring, which acts as internal
nucleophile in the cleavage reaction.

NO2The pseudo-first-order rate constant for the cleavage of HPNP:
2.0 10–8 s–1 (in water, at 25 ºC, pH = 7.0)
(Half-life time: about 1 year)

F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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8RNA cleavage reaction mechanism

Hydrolysis of RNADifferent mechanism from hydrolysis of DNA→ Intermolecular nucleophilic attack by hydroxyl group2020/5/14

F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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9What metal ion is suitable for this catalyst?
In Nature
Mg2+, Zn2+, Ca2+, Fe2+
In Artificial system
Zn2+, Cu2+, Co3+, Fe3+ and lanthanide ions (Eu3+, Ce4+)
better Lewis acid and better reactivity
Zn2+ is the only metal frequently encountered in both natural and artificial agents.
・ A good Lewis acid・ Exchange ligands rapidly
・ Not toxic
・ Not redox active
・ No ligand field stabilization energy
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F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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10Contents• Introduction• Examples of artificial catalyst ligand• Ligand with hydroxy group• DNA plasmid cleavage• Summary

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11The first study of Zn complex catalyst for hydrolysis of BNP

TREN (1):kBNP = 5.1 10–8 s–1( 2 over background reaction)

Bpy (2):

kBNP = 1.4 10–6 s–1 ( 53)

pH = 7.0, 75 ºC, 1 mM catalyst, 0.1 M NaNO3M. A. De Rosch and W. C. Trogler, Inorg. Chem., 1990, 29, 2409–2416.

The different activity of the two ligands→ The availability of free binding sites in the complexes

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F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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12The number of ligand bindings to a metal ion[12]aneN4 (3): kBNP = 2.8 10–9 s–1 ( 46)[12]aneN3 (4): kBNP = 3.3 10–8 s–1 ( 550)
pH = 7, 35 ºC, 1 mM catalyst, 0.2 M NaClO4
T. Koike and E. Kimura, J. Am. Chem. Soc., 1991, 113, 8935–8941.

Tridendate ligand > Tetradendate ligand→ The occupation of binding sites on the metal ion→ The acidity of the metal-bound water molecule

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F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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13Tetradentate ligand

L. Bonfa ́ , M. Gatos,
F. Mancin, P. Tecillaand U. Tonellato,Inorg. Chem., 2003,
42, 3943–3949.

[14]aneN4 (12) > TREN (1) (3-fold)Common: Tetradentate and pKa of water
TREN (1) wraps around the metal in a trigonal bipyramidal geometry
Less space for additional ligands than the facial coordination mode of [14]aneN4 (12)2020/5/14

F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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14Tridentate ligand

L. Bonfa ́ , M. Gatos,
F. Mancin, P. Tecillaand U. Tonellato,
Inorg. Chem., 2003,42, 3943–3949.

Tripodal ligand (5, 6, 7) > Linear ligand (9, 10, 11)
A linear correlation with a slope value (βnuc) of 0.20 (5, 6, 7)
1. A strongly acidic metal ion activates the substrate toward nucleophilic attack
2. The correlated increase in acidity of the metal-bound water decreases its nucleophilicity[12]aneN3 (4): the most reactive Zn-based monometallic agent at pH 7
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F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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Bimetallic complex for BNP hydrolysis
13kBNP = 6.4 10–6 s–1, ( 2000)( 5 over monometallic system)in water 20 % DMSO, 55 ºC, pH = 8.36
(13):

W. H. Chapman, Jr and R. Breslow, J. Am. Chem. Soc., 1995, 117, 5462–5469

substrate binds to one Zn2+, OH– to the other Zn2+
14

kBNP = 3.7 10–7 s–1 ( 9200)( 1.8 over monometallic system)in water 10 % DMSO, pH 7, 40 ºC
BPAN (14):N. V. Kaminskaia, C. He and S. J. Lippard, Inorg. Chem., 2000, 39, 3365–3373.

µ-hydroxo bridge → general base catalyst

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F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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16Plasmid DNA cleaving bimetallic system

15(15): cleavage of pBR322 DNA (12 µM bp)
k = 1.0 10–5 s–1 ( 20 over monometallic system)in 20 mM HEPES, pH 7.0, 37ºC, 3.6 µM (complex)
310-helical conformation→ bind to DNA by inserting the two macrocycles within three adjacent phosphate groups
C. Sissi, P. Rossi, F. Felluga, F. Formaggio, M. Palumbo, P. Tecilla, C. Toniolo and P. Scrimin, J. Am. Chem. Soc., 2001, 123,3169–3170.

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Cooperation between Zn2+ ions and organic functional groups

16(16): Zn2+ complex ( 5 over Bpy) (in MeCN/water 19 : 1)
E. Kovari and R. Krämer, Chem. Ber., 1994, 127, 2151–2157

Cu2+ complex is more reactive ( 4 107 over the background reaction) pH = 7, 20 ºC→ The square planar coordination geometry of this metal placed the substrate in a better
position to interact with the ammonium groups.
E. Kovari and R. Krämer, J. Am. Chem. Soc., 1996, 118, 12704–12709.
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F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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18Bimetallic complex for HPNP
13(13):

1072 for HPNP
39for UpU
5over the monometallic complex
One metal ion binds the phosphate oxygen→ Lewis acid activation
The second coordinates to the substrate hydroxylgroup→ Promote deprotonation and subsequent
nucleophilic attack
π–stacking between ligand and substrate
W. H. Chapman, Jr and R. Breslow, J. Am. Chem. Soc., 1995, 117,5462–5469

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F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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19Calixarene-based trimetallic complex
Calix[4]arene (17)HPNP transesterification 32000
35 over monometallic system
in 50% water/CH3CN, pH 7, 25 ºC
Hydrophobic pocket → the binding of the substrate
Flexibility → ajustment of the intermetallic distance17

P. Molenveld, et al., Chem. Soc. Rev., 2000, 29, 75–86;
P. Molenveld, et al., J. Org. Chem., 1999, 64, 3896–3906;
P. Molenveld, et al., Angew. Chem., Int. Ed., 1999, 38, 3189–3191;
P. Molenveld, et al., Eur. J. Org. Chem., 1999, 3269–3275;
P. Molenveld, et al. J. Am. Chem. Soc., 1997, 119, 2948–2949.
R. Cacciapaglia, et al., J. Am. Chem. Soc., 2006, 128, 12322–12330.
R. Cacciapaglia, et al., J. Org. Chem., 2005, 70, 624–630
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Not active for simple phosphate ester→ One Zn2+ activates phosphate and second
promotes deprotonation of hydroxyl group.
RNA UpU: 8600, UpU and GpG > ApA→ Third Zn2+ ion binds acidic amide of U or G

F. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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Guanidinium group promotes the reaction

The terpyridine-like ligand bearing two guanidinium groups (18)
Hydrolysis of RNA ApA
6 magnitude (pH = 7.4, 37 ºC)
3300 over complex without guanidium group
9 over complex substituted for ammonium group
18

H. Ait-Haddou, J. Sumaoka, S. L.
Wiskur, J. F. Folmer-Andersen and
E. V. Anslyn, Angew. Chem., Int.
Ed., 2002, 41, 4014–4016.

2020/5/14① Hydrogen bonding② Electrostatic interactions③ Acid catalysis by the two guanidinium groups→ additional substrate activation→ transition state stabilization→ possibly leaving group assistanceF. Mancin, et al. , New J. Chem., 2007, 31, 800–817

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Contents• Introduction• Examples of artificial catalyst ligand• Ligand with hydroxy group• DNA plasmid cleavage• Summary

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22Serine at the active site of alkaline phosphatase2020/5/14

Zhang, H., Yang, L., Ding, W., et al., J. Biol. Inorg. Chem., 2018, 23, 277–284

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Cleavage of phosphodiester catalysed by Cu complex with –OH group

Reactivity
CuL3
10030

over CuL2
over CuL1

CuL1: direct hydrolysis
CuL2: mainly hydrolysis& some transesterification
CuL3: mainly transesterification→ similar pKa (~ 8.8)
→ The difference between CuL3 and CuL2 isderived from the structure.

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M. J. Young, D. Wahnon, R. C. Hynes, J. Chin, J. Am. Chem. Soc., 1995, 117, 9441–9447.

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24Structual insight of Cu complex with –OH group

Bond angle
CuL1’(P):O–Cu–O 95.70º,
CuL2’:
O–Cu–Cl 102.75º,
CuL3(predict): O–Cu–O < 95.70º,

N–Cu–O 99.34º
N–Cu–O 79.20º
N–Cu–O > 99.34º

The distance between phosphate and alkoxide or hydroxide
CuL3 > CuL2 > CuL1 (= reactivity)
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×M. J. Young, D. Wahnon, R. C. Hynes, J. Chin, J. Am. Chem. Soc., 1995, 117, 9441–9447.

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Pyridine amino group promote hydrolysis

BNP cleavage: 4 > 5 ~ 3 > 2
Pyridine amino group promotes hydrolysis of phosphate diester.
→ Increase of the acidity of metal bound water
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M. Livieri, F. Mancin, G. Saielli, J. Chin and U. Tonellato, Chem. Eur. J., 2007, 13, 2246–2256.

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Structures of hydroxide (A) and alkoxide (B, C) complexes

Trigonal-bipyramid geometry
Equatorial:
the two pyridine nitrogen atoms and the hydroxyl group
Apical:the tertiary amino group and the water molecule
The energy of the hydroxide species is about 5–6 kcal mol–1 lower than that of the alkoxide.
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M. Livieri, F. Mancin, G. Saielli, J. Chin and U. Tonellato, Chem. Eur. J., 2007, 13, 2246–2256.

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Proposed mechanism for BNP cleavage by using Zn2+–4 complex.

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M. Livieri, F. Mancin, G. Saielli, J. Chin and U. Tonellato, Chem. Eur. J., 2007, 13, 2246–2256.

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Contents• Introduction• Examples of artificial catalyst ligand• Ligand with hydroxy group• DNA plasmid cleavage• Summary

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DNA cleavage by Cu complex catalyst: pBR 322 plasmid DNAkobs (s–1)
Tacn: 1.3×10–5
C1: 1.3×10–4
C2: 1.9×10–4
C3: 5.5×10–5

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pBR 322 plasmid DNA (38 μM bp), [Cu(tacn)(OH2)2]2+ and C1−C3 (150 μM),
in 40 mM HEPES buffer, pH 7.2, 37 °C

Linda Tjioe, Tanmaya Joshi, Craig M. Forsyth, Boujemaa Moubaraki, Keith S. Murray, JoëlBrugger, Bim Graham, and Leone Spiccia, Inorganic Chemistry, 2012 51 (2), 939-953

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DNA cleavage by Cu complex catalyst: Concentration and pH

pH = 7.0, 37 °C, 4 h

2020/5/14[Cu] 150 µM, 37 °C, 4 hLinda Tjioe, Tanmaya Joshi, Craig M. Forsyth, Boujemaa Moubaraki, Keith S. Murray, JoëlBrugger, Bim Graham, and Leone Spiccia, Inorganic Chemistry, 2012 51 (2), 939-953

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DNA cleavage by Cu complex catalyst: L1

2020/5/14

Linda Tjioe, Tanmaya Joshi, Craig M. Forsyth, Boujemaa Moubaraki, Keith S. Murray, JoëlBrugger, Bim Graham, and Leone Spiccia, Inorganic Chemistry, 2012 51 (2), 939-953

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DNA cleavage by Cu complex catalyst: L2 and L3

2020/5/14

Linda Tjioe, Tanmaya Joshi, Craig M. Forsyth, Boujemaa Moubaraki, Keith S. Murray, JoëlBrugger, Bim Graham, and Leone Spiccia, Inorganic Chemistry, 2012 51 (2), 939-953

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Contents• Introduction• Examples of artificial catalyst ligand• Ligand with hydroxy group• DNA plasmid cleavage• Summary

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34Summary•

This system is one of “nanozymes” and has been developed since 1990’s.

•

Zn2+ ion is one of the suitable metal ion for artificial catalyst of phosphate diester hydrolysis.

•Zn2+ complex reactivity depends not only on Lewis acidity but also on structure.

•Functional groups increase the reactivity of metal complexes.

•Cu-tacn ligand with guanidinium group can cleavage the phosphate diester of plasmid DNA.

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