Patai S., Rappoport Z. 1997 The chemistry of functional groups. The chemistry of double-bonded functional groups / 0909 110

17. Syntheses and uses of isotopically labelled compounds

1059

in the (py)2 HOAc system proceed with a D KIE of 1.7 1.8. No TS structures have been assigned to the above D KIEs determined from the product ratios. According to the reaction pathways542 outlined in equation 281a the C D bond rupture takes place in the course of interaction of 471 with the saturated hydrocarbon C6D12 to produce the carbon radical C6D11 that is trapped by the bound dioxygen to give 472-C6D11, which in turn rapidly transfers the second D atom to the nearest oxygen and stabilizes as ketone C6D10(O) (equation 283).

O2

−

OOH

+

c-C6

H12

(1M)

OH

470

L2 FeIII

OO.

+ pyH

-py,H2 O

L2 FeIV

OOC6 H11 -c

		(281a)
(471)	(472)
FeIIL2
c-C6 H10 (O) + 2FeIII(DPA)(DPAH) + 2H2 O
472 C R1H ! R2(O) C R1OH C H2O C FeII(PA)2	or FeII(DPAH)2	282
D
O		O
O
471		(283)
	+ DO
.
−D

D12

D KIEs in the free radical splitting of the C H/C D bonds in the RDS caused by the energy difference of C H/C D bonds should have (at 25 °C) the value of 7. Cyclohexane- D12 contained 99.5 atom % D at the beginning of reaction, but the yields of the products have been of 2 8 mM only, although the initial concentration of substrates was 0.5 M c-C6H12/0.5 M c-C6D12. In the initial periods of reaction at degrees of oxidation less than 0.5% the deuteriated ketone might be formed by free radical splitting of the residual C H bonds in a not completely deuteriated C6D11H compound. The interpretation of the kH/kD of 2 in terms of specific interaction of 471 with c-C6D12 at very low degrees of conversion of substrate should thus be postponed.

18. Brief review of isotope effect studies in catalytic reactions

Substantial KIEs have been observed in the direct dissociative chemisorption of CD4/CH4 and C2D6/C2H6 on the Ir (110) surface546 associated with the asymmetric CD and CH stretching modes.

Oxidative coupling of CH4/CD4 mixtures over natural Mn mineral catalysts was found to be comparable with that carried out over a synthetic Mn oxide catalyst547. Theoretical predictions of secondary D IEs for ring opening in the reactions cis, cis, cis-1,3,5-cyclooctatriene has been made using ab initio MO theory540. A secondary DIE of 1.3 at 99.5 °C has been observed in the extrusion of ethylene-D4 from rhenium(V)

1060 Mieczysław Ziełinski´ and Marianna Kanska´

diolates, ( -C5(Me)5)Re(O)(OCHRCHRO), and the kinetics of oxidation of norbornene, norbornadiene and trans-cyclooctene by ( -C5(Me)5)ReO3 has been determined549. The isomerization and metathesis of 1-butene over molybdenum-aluminia catalyst has been studied550 with the use of 1-butene-D8.

The noticeable inverse DIEs in formation of hydrocarbons and of oxygenates (including MeOH, EtOH, MeCHO) have been observed by performing CO H2 and CO D2 reactions over Rh catalyst promoted with vanadium551. The inverse DIE for the ethene formation through a late TS has been observed552 in ethanol dehydration over Nb dimers on a SiO2 surface. The dimers have an oxygen-bridged dimeric structure [Nb O (surface) D 0.193 nm, Nb Si D 0.328 nm, Nb Nb D 0.303 nm] by EXAFS. The Nb monomers possess the dehydrogenation ability. The shift of the catalyst from dehydrogenation (basic property) to dehydration (acidic property) has been activated by the nucleation of one atom to two Nb atoms in active structures. The mechanism of catalytic dehydrogenation of alcohols by the 2,2-bipyridine-copper(I) chloride dioxygen system in acetonitrile has been studied with the use of deuterium isotope effect determinations553. Deuterium IE study of the dehydrogenation of cyclooctane with IrCl(CO)(PR3)2 indicated the substantial H H bonding in the TS of this reaction554. The decomposition of (Me2CD)2 Te in helium in the presence or absence of mercury and/or Me2Cd showed that the all-hydrogen abstraction reactions occur from the Me groups since D1-propene and D1- propane are the only observable products555. Deuterium isotopic labelling showed that catalytic hydrogenation of ˛-methylstyrene by 9,10-dihydroantracene proceeds via a stepwise radical mechanism induced by bimolecular formation of radicals556. The inductive formation of H2, HD and D2 has been observed in the oxidation of ascorbic acid by nitrosylpentacyanoferrate(II) promoted by illumination with visible light and the use of excess oxidant557. The non-statistical distribution of deuterium has been interpreted as the indication of KIE. The mechanism of oxidation of H/D cyclohexane, toluene, adamantane, propane and ethane in the presence of t-Bu hydroperoxide and oxygen gas with methane monooxyenase (MMO) structural model, iron complex fFeO(OAC)[tris- ((1-methylimidazol-2-yl)methyl)amine]2g3C , has been investigated558. The deuterium isotope effect, kH/kD of 2.1 š 0.3, has been found for the oxidation of cyclohexane in the system MeCN/Zn/HOAc/O2/2-methyl-imidazole/Fe porphyrin/Me viologen (methyl viologen D 1,10-dimethyl-4,40-bipyridinium dichloride)559.

A substantial inverse solvent isotope effect kH2 O/kD2 O D 0.18 š 0.02 has been

observed560 in the stepwise solvolysis of trans-[RuVI(tpy)(O)2 (MeCN)]2C by PPh3, Ph2PCH2CH2PPh2 or by Ph2PCH2PPh2 to give the free diphoshine dioxides and [RuII(tpy)(MeCN)3]2C (tpy D 2,20 ,60 ,200 -terpyridine). An appreciable inverse deuterium isotope effect for the aldehyde formation has been observed561 in the hydroformylation of liquid olephins (1-hexene, 1-heptene, 1-octene and Me-10-undecenoate) by CO/D2 versus CO/H2, catalysed by SiO2-supported sulphonated-triphenylphosphine rhodium complexes. The H2/D2 coordination and hydrogenolysis of formyl species in the last step are considered as responsible561 for the overall inverse IE. Low values of DIEs have been observed in the reaction of R1R2CN2 with (MeO)2P(O)D catalysed with Cu(OAc)2(CuL2) complex and interpreted as consistent with rate-limiting attack of LCu:CR1R2 on the phosphoryl-group O, while the high value of the DIE observed in Cu(OTf)2 catalysed reaction of diazafluorene has been interpreted as consistent with intermediacy of a carbene CuOTf complex562 and rate-limiting D transfer. Solvent IEs in the oxidation of chloracetic acids by sodium-N-bromo-p-toluenesulphonamide (BAT, bromamine-T) catalysed by Ru(III) ion has been studied in D2O563. The primary DIE of 1.62 š 0.13 observed in the addition of catecholborane to the CpRu(PPh3)Me complex leading to formation of the corresponding ruthenium hydride and methylcatecholborane564 and other

17. Syntheses and uses of isotopically labelled compounds

1061

factors have been taken as indicating that the mechanism involves a four-centered TS with partial cleavage of the B D bond during formation of the B C bond. The mechanism of the synthesis of indoles from o-tolyl isocyanides catalysed by Ru(dmpe)2 (H) (naphthyl) and Ru(dmpe)2(H)2 has been studied565 using 4-t-butyl-2,6-xylyl-˛,˛,˛-D3 isocyanide. The observed D KIE in this case showed that the C H activation is faster than the C D activation in an intramolecular competition. No DIE was noted in a competitive isotope experiment in which the selection of the bond was intermolecular565.

The 13C-labelling experiments allowed one to determine the relative contributions of cyclic and of bond-shift mechanisms in the isomerization and cracking reactions of 2- methylpentane and hydrogenolysis of methylcyclopentane over Pt TiO2 catalysts prepared by different methods566.

Deuterium isotope effects in the photocatalytic and thermal dark dehydrogenation of 2-propanol-2-D (at 82.4 °C) with f[Ru SnCl3 6]3 ! [RuCl SnCl3 5]4 g complexes have been found to be 2.53 and 2.10, respectively, and interpreted as caused by C H bond splitting in the RDS566b.

C. Isotope Studies of Enzymatic Biochemical Reactions

1. Deuterium tracer and KIE studies with Cephalosporins

Deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS), the enzyme isolated from Cephalosporium acremonium catalysing587 the ring expansion of penicillin N, 473, to deacetoxycephalosporin (DAOC), 474, and the hydroxylation of 474 to deacetylcephalosporin C(DAC), 475, which in vivo is acetylated by a different enzyme to give cephalosporin C, 476 (equation 284), converts also the unnatural substrate exomethylene cephalosporin C, 477a, directly to DAC, 475 (equation 285).

D-AAHN	S	Me		S
			DA OC/ DA CS
O	N	Me	Fe2 +,O2 , α−KG	N
				CH3
	H	COOH
				COOH
	(473)		Fe2 +,O2 , α−KG	(474)
D-AA = δ (D-α-Aminoadipoy1)			DA OC/ DA CS	(284)
α-KG = α-Ketoglutarate
	S		D-AAHN	S
	N			N
		CH2 OH	O	CH2 OAc
	COOH			COOH
	(475)			(476)

The mechanism of reaction 285 proposed previously568 has been reinvestigated567 by synthesizing the [4-2H]exomethylene cephalesporin C, 477b, by electrolysis of 476 in a deuteriated buffer. Incubation of 477b with DAOC/DACS provided 475, and also the spiro-epoxide cepham 478. The ratio of 475 to 478 varied with the overall degree of

1062	Mieczysław Ziełinski´ and Marianna Kanska´

enzymic conversion from 1 : 0.4 (at approximately 25% conversion) to 1 : 0 (at 100% conversion of 477b). A conclusion from this and other isotopic experiments has been reached that 478 is a shunt metabolite formed through the operation of a DIE on an enzyme-bound intermediate. Aldehyde 479 was isolated from the post incubation mixture when 478 was treated by DAOC/DACS and cofactor (equation 286).

−		(CH2 )3
OOC				NH				S
									1
		+							2
	H
		NH3	O					N
									4
				O
									CH2
								R	COOH
(477a) R = H
(477b) R = D			Fe2 +,O2 ,α- KG					DA OC/ DA CS
−		(CH2 )3
OOC				NH				S
									1
	H	+							2		(285)
		NH3	O					N	4
				O					CH2 OH
								COOH
			(475)
		D-AAHN					S
							1
							2		O
		+			N		4
			O
						D		COOH
			(478)
			−OOC					(CH2 )3	NH		S
478	DA OC/ DA CS			H			+
							NH3		O	NH
	Fe2	+, O2 , α-KG
	(NH4 )2 SO4								O	NH2	CHO

COOH
(479)	(286)

Two competitive KIE experiments have been carried out subsequently in which a mixture of 477a and 477b have been incubated with DAOC/DACS. Samples of varying degrees of enzymic conversion were removed at different reaction times and the ratio 477a/477b determined by MS. No isotopic enrichment was detected during conversion. This indicates that the C 4 D bond is not involved in steps up to and including the first irreversible one569,570.

Formic acid treatment of a mixture of epoxide 478 with 475 provided the cephalosporin C lactone 480 together with 478. The detailed mechanism involving the addition of iron(IV)-oxene to the double bond shown in equation 285 has been proposed567.

17. Syntheses and uses of isotopically labelled compounds				1063
−
OOC	(CH2 )3	NH	S
H	+
	NH3
		O	N
		O
			O
		(480)	O

CONH(CH2 )2 OH

MeO

(483)

CONH(CH2 )2 OH

MeO

(484)

2. Deuterium solvent IE in the antibody catalysed hydrolysis of enol ethers

The enantioselective hydrolysis of alkyl enol ether 481 to the corresponding carbonyl compound 482, catalysed by the antibody 14D9 proceeding571 with very high enantioselectivity of protonation at the ˇ-carbon atom (equation 287), has been studied572 in both

H2O and D2O. The solvent KIE was kH/kD cat D 1.75 for the antibody catalysed reaction and 1.92 for the reaction of 481 with hydronium ion. The reduction of the isotope

effect in the antibody catalysed reaction is consistent with the side chain operating as a general acid in the rate-determining proton transfer to the ˇ-carbon of the enol/ether. The catalysis increases by a factor of 34 from the six-membered ring enol ether 484 to its five-membered ring analog 483.

H+
Me	+	Me
MeO	MeO	H

O

Me

H2 O,−H+	H
_MeOH

Ar	Ar		Ar
(481)		(482)	(287)

Ar =	NH
	OH
	O

1064	Mieczysław Ziełinski´ and Marianna Kanska´

3. Mechanistic study of aromatase (cytochrome P450 CYP19) in rat ovary and human placenta with the use of [1˛,2˛-3H]androstenedione and [1ˇ,2ˇ-3H]androstenedione

485

High estrogen levels are associated with a number of diseases including breast cancer and endometriosis, and those/these have been treated successfully by decreasing estrogen levels through inhibition of the CYP19 enzyme573. The three consecutive steps in aromatase oxidation of 485, requiring 3 equiv. of NADPH and molecular oxygen leading to aromatization of the steroid A-ring and loss of formic acids574,575, are shown in equation 288.

O O

OH

H H

T T

Me

H H

T T

O O

(485)

O O

T H O

H

T

O

HO

(486)

(288)

The tritium KIEs in estrogen formation by aromatase determined by comparing the rate constants associated with 7-[3H]androstanedione with those tritiated specifically at C 1 and C 2 , have been found to be insignificant576 and were interpreted as indicating that there exists an enzymatic step between the 19-al-androstenedione interme-

diate 486 and hydrogen abstraction or enolization575,577. No tritium KIEs have been detected576, in oxidations of 485, and the [1˛,2˛-3H2] and [1ˇ-3H] androstenediones by rat ovary microsomes (ROM). The distribution of tritium in the products in these oxidations showed that tritium is lost stereoselectively from the ˇ-face upon incubation with HPM (equation 288) and retained on the ˛-face following incubation. The aromatase located in ROM differs from aromatase in HPM by an inability to remove the 2ˇ-tritium from androstenodione. Aromatization of the steroid A-ring requires, besides the oxidative removal of the 1ˇ-hydrogen atom and deformylation, also the enolization of the C 3 carbonyl.

The retention of tritium at C 2 in the conversion of testosterone to estradiol is interpreted as the result of triitium IE associated with enolization of 4-dien-3-one intermediate. The enolization follows after the deformylation and 1ˇ-hydrogen abstraction steps and is

17. Syntheses and uses of isotopically labelled compounds

1065

assisted by the enzyme. Differences in accumulation of intermediates between aromatase in rat ovary and in human placenta suggest that these enzymes are structurally different576.

4. Deuterium isotope effect study of the metabolism of testosterone by CYP2C11

Cytochrome P450 system, CYP2C11, converts578 testesterone to 2˛-hydroxytestoste- rone, 488, and to 16˛-hydroxytestosterone, 489 and androstenedione, 490. Pathways of the testosterone-2,2,4,6,6-2H5 (487) metabolism by CYP2C11 are shown in equation 289. Deuteriated 488 is formed by the deuterium abstraction pathway via the active oxygen intermediate 491 (EOSw). The D-ring metabolites 489 and 490 are formed by nondeuterium abstraction pathways from the active oxygen intermediates 492 (EOSx). In competitive experiments the CYP2C11, incubated with mixtures of D0 T and D5 T catalyzed the formation of metabolites in the ratio 2˛ OHT H/ 2˛ OHT D D

5.10š 0.09, 16˛ OHT H/ 16˛ OHT D D 1.12š 0.06 and (Andro)H/(Andro)D D 1.23. The non-competitive experiments, combined with steady-state rate equations derived for

multimetabolite formation, indicated579 that testosterone is able to dissociate from the (EOS) complexes and then reassociate in the same or in different orientation. Thus the single title enzyme is able to hydroxylate opposite ends of substrate 487 providing metabolites 488, 489 and 491.

D	D	OH	HO	D	OH
O	D		O	D
	D			D
	D			D
	(487)			(488) (2 α -OHT)D
					(289)
	D	OH		D	O
D			D
O	D	OH	O	D
	D			D
	D			D
	(489) (16 α-OHT)D			(490) (Andro)D

5. Deuterium solvent IE in the androstenedione formation from progesterone

The pL (L D H or D) dependence of the solvent DIEs associated with progesterone 493 oxidation to 17˛-hydroxyprogesterone 494 and 17-O-acetyltestosterone 495 and 17˛-hydroxyprogesterone oxidation to androstenedione 496 has been determined in microcosms from pig testes580 (equation 290). The initial rate of oxidation of 493 to 494 has been associated with the pL-independent inverse solvent isotope effect (SIE) (kH/kD D 0.75 0.95 in 30% DOD) while the oxidation of 495 has been associated with the pL-independent positive SIE in 30% DOD (kH/kD of about 2), DOD inhibited the formation of 496 from 444 in noncompetitive in pL-dependent manner. Androgens are synthesized from progesterone in a two-step reaction involving the 17˛-hydroxylation

1066	Mieczysław Ziełinski´ and Marianna Kanska´
D	D
				OH
		D
	D			H
		O	H
	D
	O	N	O	N
	N		N
	Fe		Fe
N	N		N	N
	(491) EOSW		(492) EOSX

(equations 291a and 291b) and by cleavage of the C 17 side chain (via peroxide chemistry), (equation 292a, 292b), catalysed580 582 by the enzyme P450CYP17.

	O
	O
	17
	OH
O
(493) progesterone	(494)
∆4 -pregne-3,20-dione)	(290)
O Ac	O
OH

(495) C2 1H3 0 O3 (496)

Androstenedione

The pL-independent IEs have therefore been interpreted580 as indicating that DOD shifts the acid base equilibrium from (FeIII OO ) to the protonated intermediate (FeIII OOH) and increases in that way the rate of synthesis of products formed via oxene chemistry (inverse SDIE) and decreases the rate of products formed through the peroxide chemistry

	17. Syntheses and uses of isotopically labelled compounds						1067
(positive SDIE).
FeIII	O O H		[ Fe O ] 3 +	FeIII • O		FeIII
		Ac	H	Ac	H	Ac	OH
					•

					(hydroxylation mechanism)		(291a)
Fe3C -O-O C HA	! F3C -OOH C A and FeIII-OOH					! [Fe-O]3C C OH
							(291b)
	Me			Me
FeIII O O			O−			AcOH
	C				O		O
				C
					H

	O	O
	OH

	Heterolytic path						(292a)
	Me			•
FeIII	O O C	−	FeIII
		O		O
				•	OH	HO	OH
		OH
							(292b)
			AcOH
					O	H2 O
		Homolytic path

It has been suggested580 that 494 binds to an unprotonated form of the enzyme in a manner facilitating the C 17 side-chain cleavage via peroxide chemistry580.

1068	Mieczysław Ziełinski´ and Marianna Kanska´

6. D KIE associated with alkane hydroxylation by cytochromes P-450 and intermolecular D KIE in the alkane hydroxylations catalysed by manganese and iron porphyrin complexes

D KIE in the

hydroxylation of deuteriated norcamphors583, 497

499 (k /k

D

3.8),

584

of the compounds 501 (kH/kD

11)

H

D

in the hydroxylation

D

and 500

(kH/kD

D

585

586

598

12.8

14.0)

as

well

as in the hydroxylation of several

other alkynes

in

the cycytochrome P-450, porphyrin/PhlO(CH2Cl2) and in the FeTTPCl/Ph lO(CH2Cl2) oxidation systems and in other chemical model systems have been reviewed599 and partly elsewhere600,601. Intramolecular KIEs in the hydroxylation of 1,3-dideuterioadamantane catalysed by iron and manganese complexes of meso-tetrakis (2,6-dichlorophenyl) porphyrin and meso-tetramesitylporphyrin [MIII(TDCPP)Cl and MIII(TMP)Cl, MIII D

Fe or Mn], with

KHSO5, NaOCl

and PhlO (equation 293),

have

been found

to be

8.71š0.20 (20 °C) and 7.52š0.21 (20 °C) with Fe(TMP)Cl/NaOCl and Fe(TMP)Cl/PhlO,

respectively. KlEs

of 4.09 š 0.17

(20 °C) with Fe(TMP)Cl

and

4.74 š 0.17

with

Mn(TMP)Cl were obtained for KHSO5 oxidant in benzene solvent.

D

M(III)(Por)X

OH

D

H

D

(293)

H

H

OH

D

M(III)(Por)X

H

H

D

The (AH/AD) values less than 0.5 and [ Ea]DH larger than 1.15 kcal mol 1 observed in the oxidations with Fe(TMP)Cl/NaOCl and -/PlO systems, suggest the contribution of tunnelling symmetrical linear H-transfer transition states and a pure metal-oxo species in this case (502). The lower values of kH/kD observed when active metal-oxo-like species have been generated by KHSO5 regardless of the metalloporphyrin, have been rationalized by participation of a bent TS involving the leaving group of the oxidant (503).

7. Deuterium and tritium isotope effects in the lactate dehydrogenations by flavocytochrome b2

Flavocytochrome b2 catalyses the oxidation of lactate to pyruvate at the expense of cytochrome C. After reduction of flavin (FMN) by the substrate, reducing equivalents

are transferred to heme b2 and from there to cytochrome C602. The mechanism of this process has been studied603 at 5.0 °C by determining the D KIE in the FMN reduction

using L-[2-2H]lactate and wild-type enzyme and also with the Y143F mutant prepared from transformed Escherichia coli604. Tritium IE in the conversion of [2-3-H]lactate to