116
PRODUCTION, DETECTION, AND CHARACTERIZATION
[6]
in Table I. Published versions of the data have appeared,l,2,7 and periodic updates of the online version are carried out. Direct dial access 9,1° to the database is available. The database is integrated with a bibliographic database (RCDCbib) and a registry file (RCDCreg) which contains substance information for the chemical species. Acknowledgments This work was carried out under contract with the U.S. Department of Energy and supported by the Division of Chemical Sciences, Officeof Basic Energy Sciences, at the Notre Dame Radiation Laboratory (A.B.R.) (Contract DE-AC02-76ER00038) and at BrookhavenNational Laboratory(B.H.J.B.) (ContractDE-AC02-76CH00016). The Radiation ChemistryData Center is also supported by the Officeof Standard ReferenceData of the National Institute of Standards and Technology. 9 W. P. Helman, G. L. Hug, I. Carmichael, and A. B. Ross, Radiat. Phys. Chem. 32, 89 (1988). t0 For information on access to the RATES database, write to the author (A.B.R.) at the Radiation Chemistry Data Center, Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556. Some tables can be purchased from the Radiation Chemistry Data Center.
[6] U s e o f P o l y a m i n o c a r b o x y l a t e s as M e t a l C h e l a t o r s By DIANE E. CABELLI and BENON H. J. BIELSm Introduction The polyaminocarboxylates (PACs) discussed here all contain at least one basic unit of NCH2COO-. In particular, they include iminodiacetate, IDA [HN(CH2COO-)2]; nitrilotriacetate, NTA [N(CH2COO-)3]; ethylenediaminetetraacetate, EDTA {[CH2N(CH2COO-)2]2}; diethylenetriaminepentaacetate, DTPA [(-OOCCH2)2NCH2CH2N(CH2COO-)CH2 CH2N(CH2COO-)2]; and variants of the basic EDTA structure where some of the acetate groups are substituted or the ethylene bridge is modified. The common characteristics of all of these species are that they bind metals strongly and are commonly used in in vitro and in vivo biological studies as metal (particularly iron and copper) chelators. The assumption is often made that by chelation these ligands will remove metals and thus render them inactive in relation to the mechanism under study. The purpose of this chapter is to discuss the chemistry of these metal-PAC complexes in the presence of O2-/HO2, H202, and OH radicals, point out METHODSIN ENZYMOLOGY,VOL. 186
Copyright© 1990by AcademicPress,Inc. All riots of reproductionin any formreserved.
[6]
POLYAMINOCARBOXYLATES AS M E T A L CHELATORS
1 17
some of the relevant reaction rates, and note where the chemistry of these metal chelates becomes important relative to the chemistry of oxy radical/ peroxide systems. The importance of using very pure metal-free chelators cannot be overemphasized. The strong chelating ability of PACs results in the tendency for metals to be introduced into a system along with the chelator. Even if the metal complex reacts with O2-/HO2 with a rate constant of only l0 5 M -~ sec -~, 10/xM of a metal is competitive with the nanomolar concentrations of superoxide dismutase (SOD) that are commonly used. Recrystallization of the PACs is a convenient method of purification as they all tend to be insoluble in aqueous solution as acids and are solubilized as the pH is raised. It should be noted that recrystallizations carried out without ultrapure acid, base, water are pointless. Fortunately, ultrapure acids and bases are now commercially available from a number of sources. OH/O- Radicals and Polyaminocarboxylates It has been convincingly demonstrated that superoxide/perhydroxyl radicals and peroxide do not react with PACs at any significant or measurable rate. O H / O - radicals, however, react very efficiently with all PACs. O H . ~.~ O ~ + H +
(pK, = 11.9)
(1)
The detailed mechanism of these reactions is still uncertain, but it is well established that OH radical attack on PACs ultimately leads to degradation (i.e., decarboxylation, etc.). It is relevant to note that when Fentontype reactions are carried out in the presence of metal-PAC complexes, the PAC must be considered as a competitor for the OH radical in the same fashion as any other OH radical scavenger. The rate constants of OH with a number of PACs are given in Table I. As can be seen, PACs react very efficiently with O H / O - with rate constants that, at neutral pH, range from 2 x 108 to 5 × 109 M -I sec -l. Mn+/M ~+l)+ Polyaminocarboxylates and HO2/O2-/H202 The reactions of the three metals of interest, manganese, copper, and iron, with superoxide/perhydroxyl and peroxide are each discussed separately.
Manganese The overall mechanism by which Mn2+/Mn3+-PAC complexes react with HO2/O2- [where PAC is EDTA, NTA, or 1,2-cyclohexanediamine-
118
[6]
PRODUCTION, DETECTION, AND CHARACTERIZATION TABLE I REACTIVITY OF O H / O - RADICALS WITH POLYAMINOCARBOXYLATES
Reaction
pH
OH + glycine
OH + IDA O- + IDA OH + N T A OH + EDTA O- + EDTA OH + DTPA O H + trans-CyDTA C
p K of PAC a
1-6 9.5-9.7 10 1 7 13 0-9 4.0 9.0 Alkaline 5-11
2.36, 9.57
1.8, 2.6, 9.3
0.8, 1.8, 2.5, 9.3 1.5, 2.0, 2.7, 6.1, 10.2
1.8, 2.7, 4.3, 8.5, 10.5 2.4, 3.5, 6.1, 12.4
0
k, M -~ sec -~ b 1.5 2 5 5 2 9.1 2.3 4 2 1 5 1.2
× 107 x 109 X 109 X 107 X 108 x 108 x 109 x 108 x 109 × 10s × 109 × 10 ~°
Data from A. E. Martell and R. M. Smith, "Critical Stability Constants, Volume I: A m i n o A c i d s . " Plenum, N e w York, 1974. b Data from G. V. B u x t o n , C. V. G r e e n s t o c k , W. P. H e l m a n , and A. B. Ross, J. Phys. Chem. Ref. Data 17, 513 (1988).
c trans-CyDTA, trans-l,2-cyclohexanediamine-N,N,N',N'-tetraacetate.
N,N,N',N'-tetraacetate (CyDTA)] occurs by the following mechanism1,2: kl M n 2+ + 02 MnO2 + M n 3+ + 0 2 -
.
MnO2 +
k-i
2 H+, k2 ) Mn3+ + H202 k3
,
Mn2+
+ o:
(2) (3) (4)
Although reaction (2) has been shown to be reversible in studies involving simple inorganic ligands, 3 no such studies have been reported using PAC chelators. The reaction between superoxide and manganic complexes is written as a simple reaction yielding Mn 2+ and Oz ; however, this does not preclude a mechanism similar to that shown by equilibrium (2) and reaction (3). The respective rates and pH values reported for these systems are given in Table II. Although both Mn3ff and MnOH 2÷ have been shown to react with H202 in acidic solution with rate constants of 7 × 104 and 3 × 10 4 M -1 sec -l, respectively, 4 Mn 2÷- and Mn3+-EDTA complexes have 1 j. Lati and D. Meyerstein, J. Chem. Soc. Dalton Trans., 1185 (1978). J. Stein, J. P. Fackler, Jr., G. J. McClune, J. A. Fee, and L. T. Chan, lnorg. Chem. 18, 3511 (1979). 3 D. E. Cabelli and B. H. J. Bielski, J. Phys. Chem. 88, 3111, 6291 (1984). 4 G. Davies, L. J. Kirschenbaum, and K. Kustin, lnorg. Chem. 7, 146 (1968).
[6]
POLYAMINOCARBOXYLATES AS METAL CHELATORS
1 19
T A B L E II REACTIVITY OF Mn2+/Mn3+-POLYAMINOCARBOXYLATE COMPLEXES WITH H02/O2-
PAC
k2, M -1 sec -t (pH)
k3, M -I sec -1 (pH)
k4, M -1 sec -1 (pH)
NTA EDTA CyDTA
4 x 108 (4.5); 1.2 × 10s (5.5) a 3 × 107 (4.5); 7.5 x 106 (5.5) a --
1.8 x 103 (4.5); 9 x 10: (5.5) a 3 x 103 (4.5, 5.5) a --
1.2 x 107 (6.0) b 5 x 104 (10.0)* 7.2 x 105 (9.2) c
a Data from Ref. 1. b Data from W. H. Koppenol, F. Levine, T. L. Hatmaker, J. Epp, and J. D. Rush, Arch. Biochem. Biophys. 251, 594 (1986). c Data from Ref. 2.
been shown to react very slowly, if at all, with peroxide2: Mu3+-EDTA + H202 ~ very slow Mn2+-EDTA + H202 ~ no reaction
(pH 10.0) (pH 10.0)
(5) (6)
In contrast, the complex of Mn(II) with EDDA (ethylenediaminediacetate) acts as a catalase for the disproportionation of H 2 0 2 t o H 2 0 and 02 ; the kcat value is 5.4 M-: sec -1, where d[H202]/dt = kcat[Mn(II)EDDAT[H202]. The essential difference between this and other Mn(II)PAC systems is that the manganic EDDA intermediate is binuclear and acts as a catalyst for the two-electron reduction of peroxide. 5
Copper The use of PAC chelators appears to render Cu 2÷ complexs unreactive toward HO2/O2-. As Cu+-PAC complexes are very reactive toward 02, the reverse step of the cycle (Cu ÷ + HO2/O2-) can be neglected. The products of the reaction between H202 and Cu ÷ complexed with phenanthroline and simple inorganic ligands is currently the subject of much research. However, no rates for the reaction of PAC complexes of Cu ÷ with peroxide have been reported.
Iron The reactivity of Fe2+/Fe3+-PAC complexes toward H O 2 / O 2 - / H 2 0 2 has received the most attention because of the wealth of chemistry, Fenton and otherwise, that occurs in these systems. The most extensive studies that have been reported involve EDTA as the PAC ligand and suggested the following mechanism6: 5 j. D. Rush, private communication, 1989. 6 C. Bull, G. J. McClune, and J. A. Fee, J. Chem. Soc. 105, 5290 (1983).
120
PRODUCTION, DETECTION, AND CHARACTERIZATION
[6]
T A B L E III REACTIVITY OF Fe2+-Fe3+-POLYAMINOCARBOXYLATE COMPLEXES WITH H O 2 / O 2- AND H202 a
PAC
k , , M -1 s e c -1
k9, M -1 s e c -1
EDTA EDDA NTA CyDTA DTPA H20 HEDTA EHPGJ
2 x 104 a, 7 x 103 d 7.8 x 104 e 3 x 104 e 1.3 X 103f 1.4 x 103 e; 5.1 x 102 d 57.8 h 4.2 × 104 d --
(2.5-3.5) x 102 c __ __ -__ -__ --
ks,
M -1 s e c -I b
kT, M -1 s e c -! b
__ _ __ --
1.9 × 106
2 × 10 7 -__ 1.4 × 107
10~ 7.6 × 105
All reported values are bimolecular rates at the specified pH. b Data from B. H. J. Bielski, D. E. Cabelli, R, L. Arudi, and A. B. Ross, J. Phys. Chem. Ref. Data 14, 1041 (1985). c Data from Ref. 7, pH 8-10. a Data from J. D. R u s h and W. H. Koppenol, J. Inorg. Biochem. 29, 199 (1987), pH 7.4. e Data from J. D. R u s h and W. H. Koppenol, J. Am. Chem. Soc. 110, 4957 (1988), pH 7.2. I Data from S. Rahhal and H. W. Richter, Radiat. Phys. Chem. 12, 129 (1988), pH 7.0. Data from S. Rahhal and H. W. Richter, J. Am. Chem. Soc. 110, 3126 (1988), p H 7.0. h Data from H. N. Po and N. Sutin, lnorg. Chem. 7, 621 (1968), 1.0 M HCIO4. i p H 7.0. J E H P G , Ethylenediamine-N,N'-bis[2-(2-hydroxyphenyl)acetate]; pH 7.0; data from G. R. Buettner, J. Biol. Chem. 262, 11995 (1987).
Fe 3+ + 02- --~ Fe 2+ + 02 Fe z+ + 02- --} Fe3+-O22Fe 3÷ + H202 ~ Fe3+-O22- + 2 H + Fe 2+ + Oz- + 2 H ~ Fe 2+ + H202 ~ product(s)
(7) (8) (9) (10) (11)
where the product(s) of reaction (11) are controversial (see Table III). Acknowledgments This work was supported by National Instituteof Health Grant R01GM23658-12 and was carried out at the Brookhaven National Laboratory under Contract DE-AC02-76CH00016 with the U.S. Department of Energy, and supported by its Division of Chemical Sciences, Office of Basic Sciences.
PRODUCTION, DETECTION, AND CHARACTERIZATION
[6]
in Table I. Published versions of the data have appeared,l,2,7 and periodic updates of the online version are carried out. Direct dial access 9,1° to the database is available. The database is integrated with a bibliographic database (RCDCbib) and a registry file (RCDCreg) which contains substance information for the chemical species. Acknowledgments This work was carried out under contract with the U.S. Department of Energy and supported by the Division of Chemical Sciences, Officeof Basic Energy Sciences, at the Notre Dame Radiation Laboratory (A.B.R.) (Contract DE-AC02-76ER00038) and at BrookhavenNational Laboratory(B.H.J.B.) (ContractDE-AC02-76CH00016). The Radiation ChemistryData Center is also supported by the Officeof Standard ReferenceData of the National Institute of Standards and Technology. 9 W. P. Helman, G. L. Hug, I. Carmichael, and A. B. Ross, Radiat. Phys. Chem. 32, 89 (1988). t0 For information on access to the RATES database, write to the author (A.B.R.) at the Radiation Chemistry Data Center, Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556. Some tables can be purchased from the Radiation Chemistry Data Center.
[6] U s e o f P o l y a m i n o c a r b o x y l a t e s as M e t a l C h e l a t o r s By DIANE E. CABELLI and BENON H. J. BIELSm Introduction The polyaminocarboxylates (PACs) discussed here all contain at least one basic unit of NCH2COO-. In particular, they include iminodiacetate, IDA [HN(CH2COO-)2]; nitrilotriacetate, NTA [N(CH2COO-)3]; ethylenediaminetetraacetate, EDTA {[CH2N(CH2COO-)2]2}; diethylenetriaminepentaacetate, DTPA [(-OOCCH2)2NCH2CH2N(CH2COO-)CH2 CH2N(CH2COO-)2]; and variants of the basic EDTA structure where some of the acetate groups are substituted or the ethylene bridge is modified. The common characteristics of all of these species are that they bind metals strongly and are commonly used in in vitro and in vivo biological studies as metal (particularly iron and copper) chelators. The assumption is often made that by chelation these ligands will remove metals and thus render them inactive in relation to the mechanism under study. The purpose of this chapter is to discuss the chemistry of these metal-PAC complexes in the presence of O2-/HO2, H202, and OH radicals, point out METHODSIN ENZYMOLOGY,VOL. 186
Copyright© 1990by AcademicPress,Inc. All riots of reproductionin any formreserved.
[6]
POLYAMINOCARBOXYLATES AS M E T A L CHELATORS
1 17
some of the relevant reaction rates, and note where the chemistry of these metal chelates becomes important relative to the chemistry of oxy radical/ peroxide systems. The importance of using very pure metal-free chelators cannot be overemphasized. The strong chelating ability of PACs results in the tendency for metals to be introduced into a system along with the chelator. Even if the metal complex reacts with O2-/HO2 with a rate constant of only l0 5 M -~ sec -~, 10/xM of a metal is competitive with the nanomolar concentrations of superoxide dismutase (SOD) that are commonly used. Recrystallization of the PACs is a convenient method of purification as they all tend to be insoluble in aqueous solution as acids and are solubilized as the pH is raised. It should be noted that recrystallizations carried out without ultrapure acid, base, water are pointless. Fortunately, ultrapure acids and bases are now commercially available from a number of sources. OH/O- Radicals and Polyaminocarboxylates It has been convincingly demonstrated that superoxide/perhydroxyl radicals and peroxide do not react with PACs at any significant or measurable rate. O H / O - radicals, however, react very efficiently with all PACs. O H . ~.~ O ~ + H +
(pK, = 11.9)
(1)
The detailed mechanism of these reactions is still uncertain, but it is well established that OH radical attack on PACs ultimately leads to degradation (i.e., decarboxylation, etc.). It is relevant to note that when Fentontype reactions are carried out in the presence of metal-PAC complexes, the PAC must be considered as a competitor for the OH radical in the same fashion as any other OH radical scavenger. The rate constants of OH with a number of PACs are given in Table I. As can be seen, PACs react very efficiently with O H / O - with rate constants that, at neutral pH, range from 2 x 108 to 5 × 109 M -I sec -l. Mn+/M ~+l)+ Polyaminocarboxylates and HO2/O2-/H202 The reactions of the three metals of interest, manganese, copper, and iron, with superoxide/perhydroxyl and peroxide are each discussed separately.
Manganese The overall mechanism by which Mn2+/Mn3+-PAC complexes react with HO2/O2- [where PAC is EDTA, NTA, or 1,2-cyclohexanediamine-
118
[6]
PRODUCTION, DETECTION, AND CHARACTERIZATION TABLE I REACTIVITY OF O H / O - RADICALS WITH POLYAMINOCARBOXYLATES
Reaction
pH
OH + glycine
OH + IDA O- + IDA OH + N T A OH + EDTA O- + EDTA OH + DTPA O H + trans-CyDTA C
p K of PAC a
1-6 9.5-9.7 10 1 7 13 0-9 4.0 9.0 Alkaline 5-11
2.36, 9.57
1.8, 2.6, 9.3
0.8, 1.8, 2.5, 9.3 1.5, 2.0, 2.7, 6.1, 10.2
1.8, 2.7, 4.3, 8.5, 10.5 2.4, 3.5, 6.1, 12.4
0
k, M -~ sec -~ b 1.5 2 5 5 2 9.1 2.3 4 2 1 5 1.2
× 107 x 109 X 109 X 107 X 108 x 108 x 109 x 108 x 109 × 10s × 109 × 10 ~°
Data from A. E. Martell and R. M. Smith, "Critical Stability Constants, Volume I: A m i n o A c i d s . " Plenum, N e w York, 1974. b Data from G. V. B u x t o n , C. V. G r e e n s t o c k , W. P. H e l m a n , and A. B. Ross, J. Phys. Chem. Ref. Data 17, 513 (1988).
c trans-CyDTA, trans-l,2-cyclohexanediamine-N,N,N',N'-tetraacetate.
N,N,N',N'-tetraacetate (CyDTA)] occurs by the following mechanism1,2: kl M n 2+ + 02 MnO2 + M n 3+ + 0 2 -
.
MnO2 +
k-i
2 H+, k2 ) Mn3+ + H202 k3
,
Mn2+
+ o:
(2) (3) (4)
Although reaction (2) has been shown to be reversible in studies involving simple inorganic ligands, 3 no such studies have been reported using PAC chelators. The reaction between superoxide and manganic complexes is written as a simple reaction yielding Mn 2+ and Oz ; however, this does not preclude a mechanism similar to that shown by equilibrium (2) and reaction (3). The respective rates and pH values reported for these systems are given in Table II. Although both Mn3ff and MnOH 2÷ have been shown to react with H202 in acidic solution with rate constants of 7 × 104 and 3 × 10 4 M -1 sec -l, respectively, 4 Mn 2÷- and Mn3+-EDTA complexes have 1 j. Lati and D. Meyerstein, J. Chem. Soc. Dalton Trans., 1185 (1978). J. Stein, J. P. Fackler, Jr., G. J. McClune, J. A. Fee, and L. T. Chan, lnorg. Chem. 18, 3511 (1979). 3 D. E. Cabelli and B. H. J. Bielski, J. Phys. Chem. 88, 3111, 6291 (1984). 4 G. Davies, L. J. Kirschenbaum, and K. Kustin, lnorg. Chem. 7, 146 (1968).
[6]
POLYAMINOCARBOXYLATES AS METAL CHELATORS
1 19
T A B L E II REACTIVITY OF Mn2+/Mn3+-POLYAMINOCARBOXYLATE COMPLEXES WITH H02/O2-
PAC
k2, M -1 sec -t (pH)
k3, M -I sec -1 (pH)
k4, M -1 sec -1 (pH)
NTA EDTA CyDTA
4 x 108 (4.5); 1.2 × 10s (5.5) a 3 × 107 (4.5); 7.5 x 106 (5.5) a --
1.8 x 103 (4.5); 9 x 10: (5.5) a 3 x 103 (4.5, 5.5) a --
1.2 x 107 (6.0) b 5 x 104 (10.0)* 7.2 x 105 (9.2) c
a Data from Ref. 1. b Data from W. H. Koppenol, F. Levine, T. L. Hatmaker, J. Epp, and J. D. Rush, Arch. Biochem. Biophys. 251, 594 (1986). c Data from Ref. 2.
been shown to react very slowly, if at all, with peroxide2: Mu3+-EDTA + H202 ~ very slow Mn2+-EDTA + H202 ~ no reaction
(pH 10.0) (pH 10.0)
(5) (6)
In contrast, the complex of Mn(II) with EDDA (ethylenediaminediacetate) acts as a catalase for the disproportionation of H 2 0 2 t o H 2 0 and 02 ; the kcat value is 5.4 M-: sec -1, where d[H202]/dt = kcat[Mn(II)EDDAT[H202]. The essential difference between this and other Mn(II)PAC systems is that the manganic EDDA intermediate is binuclear and acts as a catalyst for the two-electron reduction of peroxide. 5
Copper The use of PAC chelators appears to render Cu 2÷ complexs unreactive toward HO2/O2-. As Cu+-PAC complexes are very reactive toward 02, the reverse step of the cycle (Cu ÷ + HO2/O2-) can be neglected. The products of the reaction between H202 and Cu ÷ complexed with phenanthroline and simple inorganic ligands is currently the subject of much research. However, no rates for the reaction of PAC complexes of Cu ÷ with peroxide have been reported.
Iron The reactivity of Fe2+/Fe3+-PAC complexes toward H O 2 / O 2 - / H 2 0 2 has received the most attention because of the wealth of chemistry, Fenton and otherwise, that occurs in these systems. The most extensive studies that have been reported involve EDTA as the PAC ligand and suggested the following mechanism6: 5 j. D. Rush, private communication, 1989. 6 C. Bull, G. J. McClune, and J. A. Fee, J. Chem. Soc. 105, 5290 (1983).
120
PRODUCTION, DETECTION, AND CHARACTERIZATION
[6]
T A B L E III REACTIVITY OF Fe2+-Fe3+-POLYAMINOCARBOXYLATE COMPLEXES WITH H O 2 / O 2- AND H202 a
PAC
k , , M -1 s e c -1
k9, M -1 s e c -1
EDTA EDDA NTA CyDTA DTPA H20 HEDTA EHPGJ
2 x 104 a, 7 x 103 d 7.8 x 104 e 3 x 104 e 1.3 X 103f 1.4 x 103 e; 5.1 x 102 d 57.8 h 4.2 × 104 d --
(2.5-3.5) x 102 c __ __ -__ -__ --
ks,
M -1 s e c -I b
kT, M -1 s e c -! b
__ _ __ --
1.9 × 106
2 × 10 7 -__ 1.4 × 107
10~ 7.6 × 105
All reported values are bimolecular rates at the specified pH. b Data from B. H. J. Bielski, D. E. Cabelli, R, L. Arudi, and A. B. Ross, J. Phys. Chem. Ref. Data 14, 1041 (1985). c Data from Ref. 7, pH 8-10. a Data from J. D. R u s h and W. H. Koppenol, J. Inorg. Biochem. 29, 199 (1987), pH 7.4. e Data from J. D. R u s h and W. H. Koppenol, J. Am. Chem. Soc. 110, 4957 (1988), pH 7.2. I Data from S. Rahhal and H. W. Richter, Radiat. Phys. Chem. 12, 129 (1988), pH 7.0. Data from S. Rahhal and H. W. Richter, J. Am. Chem. Soc. 110, 3126 (1988), p H 7.0. h Data from H. N. Po and N. Sutin, lnorg. Chem. 7, 621 (1968), 1.0 M HCIO4. i p H 7.0. J E H P G , Ethylenediamine-N,N'-bis[2-(2-hydroxyphenyl)acetate]; pH 7.0; data from G. R. Buettner, J. Biol. Chem. 262, 11995 (1987).
Fe 3+ + 02- --~ Fe 2+ + 02 Fe z+ + 02- --} Fe3+-O22Fe 3÷ + H202 ~ Fe3+-O22- + 2 H + Fe 2+ + Oz- + 2 H ~ Fe 2+ + H202 ~ product(s)
(7) (8) (9) (10) (11)
where the product(s) of reaction (11) are controversial (see Table III). Acknowledgments This work was supported by National Instituteof Health Grant R01GM23658-12 and was carried out at the Brookhaven National Laboratory under Contract DE-AC02-76CH00016 with the U.S. Department of Energy, and supported by its Division of Chemical Sciences, Office of Basic Sciences.
