P Diastereomerism in Organocobalt Corrinoids. Synthesis

Facile cr/P Diastereomerism in Organocobalt Corrinoids. Synthesis, Characterization, and Thermolysis of a-Neopentylcobalt Corrinoids. Xiang Zou and Ke...
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J. Am. Chem. SOC.1993, I 15, 6689-6698

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Facile cr/P Diastereomerism in Organocobalt Corrinoids. Synthesis, Characterization, and Thermolysis of a-Neopentylcobalt Corrinoids Xiang Zou and Kenneth L. Brown' Contribution from the Department of Chemistry, Box CH, Mississippi State University, Mississippi State, Mississippi 39762 Received February 8, I993

Abstract: Coenzyme Bl2 analogs a-neopentylcobinamide (a-NpCbi+) and a-neopentylcobalamin (a-NpCbl), where the bulky organic ligand is in the ylower* (a)axial ligand position, have been synthesized by reductive alkylation of cyanoaquocobinamideand aquocobalamin, respectively, with neopentyl bromide at -0 "C, and characterized by their UV-visible spectra, GC/MS analysis of their anaerobic solid-state pyrolysis products, and thermal and photolytic conversion to corresponding j3 diastereomers. The thermolyses of a-NpCbi+ and a-NpCbl were studied in aerobic aqueous solution, and the rate constants for Co-C bond homolysis were found to be 5.3 X lo3and 3.4 X 103times larger at 35 "C and 2.3 X 104 and 1.1 X 104 times larger at 5 "C than those for @-NpCbi+and base-off j3-NpCbl, respectively. As expected, product analysis under anaerobic conditions using HTEMPO as a free radical trap and under aerobic conditions using 0 2 as a free radical trap showed that thermolysis of these a-neopentylcobalt corrinoids involves homolytic fission of the Co-C bond. The activation parameters were determined from Eyring plots (a-NpCbi+, AH* = 21.4 f 0.4 kcal mol-', AS* = 0.9 f 1.3 eu; a-NpCbl, AH* = 22.5 f 0.2 kcal mol-', AS* = 3.1 f 0.1 eu). Both AH*and AS*are significantly smaller than the values previously obtained for O-NpCbi+and base-off j3-NpCbl. Thus, although steric interactions between the neopentyl group and the more numerous and larger downward projecting corrin side chains would be expected to weaken the a Co-C bond relative to the j3 Co-C bond, there also seems to be an electronic destabilization of the Co-C bond in the a diastereomers. This, in turn, suggests that the steric congestion at the a face may be relieved by an upward puckering of the corrin ring, leading to a weakened a Co-C bond. It is generally accepted that the unique Co-C bond homolysis in coenzyme B12 is the first step in the coenzyme B1z-dependent enzymatic rearrangement reactions.' One plausible hypothesis for Co-C bond activation in B12-dependent enzymes requires a flexible corrin ring that is upwardly distorted to labilize the carbon-cobalt bond in the enzyme-substrate-wenzyme complex? suggesting that the tremendous rate enhancement (- 10*2)3for the Co-C bond homolysis by such enzymes is mostly due to steric effects. This theory has received some support from studies of thermolysis of 8-5'-deoxyadenosylcobalamin (coenzyme BIZ, @-AdoCb1)3-6and numerous other model complexes, especially those with bulky organic ligands that have no j3 hydrogen atoms (1) (a) Halpern, J. Science 1985, 227, 869. (b) Dolphin, D., Ed. 8 1 2 ; Wiley: New York, 1982. (2) (a) Schrauzer, G. N.; Grate, J. H. J. Am. Chem. Soc. 1981,103,541. (b) Grate, J. H.; Schrauzer, G. N. J. Am. Chem. Soc. 1979, 101, 4601. (3) (a) Finke, R. G.; Martin, B. D. J. Inorg. Biochem. 1990,40, 19. (b) Hay, B. P.; Finke, R. G. Polyhedron 1988, 7, 1469. (c) Hay, B. P.; Finke, R. G. J. Am. Chem. Soc. 1986,108,4820. (d) Finke, R. G.; Hay, B. P. Inorg. Chem. 1984,23, 3041. (4) Abbreviations: Ado, S'-dwxyadenosyl; Np, neopentyl; HTEMPO, 4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy; NpHTEMPO, 1-(2,2-dimc thylpropoxy)-2,2,6,6tetramethyl-4-hydroxypiperidine,TEMPO, 2,2,6,6-tetramethylpiperidinyloxy;P-RCbl, 8-alkylcob(III)alamin;a-RCbl, a-alkylcob(1II)alamh @-RCbi,~-alkylcob(III)imide;a-RCbi, a-alkylcob(II1)inamide; HzOCbl, aquocob(II1)alamin;(HzO)zCbi,diaquocob(II1)inamide. In a-alkylcobalt corrinoids,the organic ligand occupies the "lower" axial ligand position, while in /3-alkylcobalt corrinoids, the organic ligand is in the "upper" ligand positiod. See Figure 1 for structures. (5) (a) Halpern, J.; Kim, S.-H.; hung, T. W . J. Am. Chem. SOC.1985, 107, 2199. (b) Halpern, J.; Kim. S.-H; Lcung, - T. W. J. Am. Chem. SOC. 1984,106, 8317. (6) Gjerde, H. B.; Espenson, J. H. Organometallics 1982, I, 435. (7) Brown, K. L.; Brooks, H. B. Inorr. Chem. 1991.30. 3420. (8) (a) Halpem. J. Bonding Energeth in Organometallic Compounds; ACS Symposium Series 428; Marks, T. J. Ed.; American Chemical Society: Washington, DC, 1990; p 100. (b) Halpem, J. Bull. Chem. Soc. Jpn. 1988, 61, 13. (c) Kim, S.-H.; Chen, H. L.; Feilchenfeld, N.; Halpern, J. J. Am. Chem. SOC.1988,110,3 120. (d) Geno, M. K.; Halpern, J. 1.Am. Chem. Soc. 1987,109,1238. (e) Tsou, T.-T.; Loots, M.; Halpern, J. J. Am. Chem. SOC. 1982,104,623. (f) Halpem, J.; Ng,F. T. T.; Rempel, G. L. J . Am. Chem. Soc. 1979, 101,1124.

and thus must undergo Co-C bond fission by homolysis.7-14 The latter complexes decompose more rapidly than those with sterically less demanding alkyl groups, and the activation parameters and bond dissociation energies for thermolytic Co-C bond homolysis of a number of these complexes have been determined in aqueous solution as well as in other solvents.7-14 Relatively little is known, however, about the stability of the Co-C bond in a-alkylcobalt corrinoids, in which the alkyl group is coordinated at the lower (or a) axial ligand position of the corrinoid (Figure 1).4 Sterically, the corrin ring is believed to be more crowded at the a face, which bears the b, d, and e propionamide side chains and the substituted propionamide f side chain (Le., the nucleotide loop side chain), than the 8 face, which bears only three acetamide side chains (Figure 1). Only with the recent synthesis and characterization of a series of stable a-alkylcobaltcorrinoidshas the formation of these a diastereomers been consideredto be a general For the thirteen known examples of a-alkylcobalt corrinoids that have been characterized so far, all but one (R = CHzCH3) are obtained by standard synthetic procedures (Le., reductive alkylation of a (9) Martinho S i m h , J. A.; Bcauchamp, J. L. Chem. Rw. 1990,90,629. (10) Nome, F.; Rezende, M. C.; Sabbia, C. M.; Da Silva, A. C. Can. J. Chem. 1987, 65, 2095. (11) Blau, R. J.; Espenson, J. H. J. Am. Chem. Soc. 1985,107, 3530. (12) Baldwin, D. A.; Betterton, E. A.; Chemaly, S. M.; Pratt, J. M. J . Chem. Soc., Dalton Trans. 1985, 1613. (13) (a) Martin, B. D.; Finke, R. G. J. Am. Chem. Soc. 1990,112,2419. (b) Hay, B. P.; Finkc, R. G. J. Am. Chem. Soc. 1987,109,8012. (c) Finke, R. G.; Smith, B. L.; Mayer, B. J.; Molinero, A. A. Inorg. Chem. 1983, 22, 3677. (14) (a) Ng, F. T. T.; Rempel, G. L. Inorg. Chim. Acta 1983, 77, L165. (b) Ng, F. T. T.; Rempel, G. L. J. Am. Chem. SOC.1982, 104, 621. (15) Alelyunas,Y. W.;Fleming,P.E.;Finke,R.G.;Pagano,T.G.;Marzilli, L. G . J. Am. Chem. Soc. 1991, 113, 3781. (16) (a) Brown, K. L.; Salmon, L.; Kirby, J. A. Organometallics 1992,II, 422. (b) Brown, K. L.; Zou, X . ; Salmon, L. Inorg. Chem. 1991,30, 1949. (c) Brown, K. L.; Evans, D. R. Inorg. Chem. 1990, 29, 2559. (17) Brown, K. L.; Zou, X . Inorg. Chem. 1992, 31, 2541. (18) Brown, K. L.; Zou, X . J. Am. Chem. SOC.1992,114,9643.

OOO2-7863/93/1515-6689$04.00/0 0 1993 American Chemical Society

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O1 h) was required to induce the interconversion at the same level of irradiation (3V tungsten lamp) that previously was found to induce the more rapid interconversion of other a-and /3-RCba’s.17.20 Similarly, a-NpCbl and B-NpCbl could also be interconverted photolytically in acidic anaerobic solution where the axial nucleotide is protonated. At the photolytic stationary state, approximately 5-10% a-NpCbl was obtained. Unfortunately, the exact stationary-state positions for these photolytic interconversions of the a-NpCbi’s and the a-NpCbl’s could not be determined because (29) Robinson, K. A.; Itabashi, E.; Mark, H. B. Inorg. Chem. 1982,21, 357 1. (30) Kim, M.-H.; Birke, R. L. J. Electroanal. Chem. Inrerfacial Elecrrochem. 1983,144, 331. (31) (a) Lexa, D.; Savhnt, J. M. Acc. Chem. Res. 1983,6,235. (b) Lexa, D.; Savhnt, J. M. J. Am. Chem. SOC.1970, 100, 3220. (32) Zhou, D.-L.; Tinembart, 0.;Scheffold, R.;Walder, L. Helu. Chim. Acra 1990, 73, 2225. (33) Shepherd, R. E.; Zhang, S.;Dowd, P.; Choi, G.; Wilk, B.;Choi, S.-C. Inorg. Chim. Acra 1990, 174, 249. (34) Brown, K. L.; Zou, X.; Richardson, M.; Henry, W. P. Inorg. Chem. 1991, 30, 4834.

J . Am. Chem. SOC.,Vol. 115, No. 15, 1993 6693

Organocobalt Corrinoids Table I. Rate Constants for the Thermal Homolysis of

a-Neopentylcobalt Corrinoids 104 S-I

0

T,OC

a-NpCbi+

a-NpCbl

30.1 25.0 24.96 20.0 14.9 10.0 5.0

35.3 & 1.4 19.4 & 0.3 18.8 & 0.6 9.61 & 0.11 5.28 & 0.32 2.50 & 0.08 1.34 & 0.09

18.9 & 0.1 10.3 & 0.4 9.8 & 0.2 5.03 & 0.25 2.57 & 0.05 1.27 & 0.05 0.60 & 0.01

0 In 0.1 M potassium phosphate buffer, ionic strength 1 M (KCl) with 3% acetonitrile. Anaerobic thermolysis in potassium phosphate buffer (0.1 M, pH 7.0) with HTEMPO (10 mM).

of the thermal labilities of these complexes. The anaerobic photolytic process could not be cleanly separated from simultaneous untrapped thermolysis in the time domain required to induce a complete reaction under the conditions used since the two processes occur on the same time scale. As is the case with other a-alkylcobalamin~,~~~~O a-NpCbl was converted completely to 8-NpCbl upon anaerobic thermolysis at ambient temperature and neutral pH where the latter is base-on. Anaerobic thermolysis of a-NpCbi+ at ambient temperature but in the absence of a radical trap also converted this material to its j3 diastereomer, but the rate (tip > 3 h) was extremely slow compared to that of a trapped thermolysis (tip 6 min, Table I). It has been recently shown that the cage efficiency for a radical pair in an alkylcorrinoid system can be very high (Fc 1 0.94),35 The slow anaerobic thermal conversion from a-NpCbi+ to @-NpCbi+compared to that of the trapped thermal decomposition of a-NpCbi+ may be a cage effect since the trap prevents the recombination of the caged radical pair. The implication of this finding is currently under further investigation. Product Analyses for Thermolysis of a-NeopentylcobaltCorrinoids. In order to confirm that the thermolysis of a-NpCbl follows a homolytic cleavage pathway as does that of B-NpCbl, the products of anaerobic thermolysis of a-NpCbl in the presence of HTEMPO (10 mM) were analyzed by spectrophotometric and gas chromatographic methods. Finke and co-worker~~~ have studied the dependence of the yield of the trapped alkyl radical on nitroxide trap concentration in ethylene glycol and found that as little as 1 mM of TEMPO was adequate to trap 98%of the free neopentyl radical in 8-NpCbl thermolysis and that 10 mM TEMPO trapped 100%of the alkyl radical. This is likely to be true for HTEMPO as well, and so 10 mM of HTEMPO in a reaction solution should be sufficient to trap the free alkyl radical formed from a-NpCbl thermolysis. As expected for thermal Co-C bond homolysis, anaerobic thermolysis of a-NpCbl in the presence of 10 mM HTEMPO produced nearly quantitative yields of Np-HTEMPO (92 f 6%) as analyzed by gas chromatography. Furthermore, cob(1I)alamin was produced in 102 2% yield by spectrophotometric determination. Similar experiments were also performed for a-NpCbi+. However, because of the thermal lability of a-NpCbi+, the amount of Np-HTEMPO being trapped by this method (see Experimental Section) may be expected to be somewhat depressed due to decomposition of a-NpCbi+ during the latter stage of the HPLC purification process and transfer to the reaction solution. In this case, the yields of NpHTEMPO and cob(I1)inamide were 70 f 3% and 75 f 1%, respectively, the other 25-30% being cob(III)inamide, formed by unavoidable premature aerobic decomposition prior to establishment of the anaerobic condition, as evidence by a peak at ca. 350 nm (due to contaminating cob(1II)inamide) in the cob(I1)inamide spectrophotometric quantitation. Nevertheless, the nearly quantitative yields of cob(I1)alamin and NpHTEMPO for a-NpCbl thermolysis, and the

-

*

(35) Garr, C. D.; Finke, R.G.J. Am. Chem. Soc. 1992, 224, 10440.

close to 75% and nearly 70% yields of cob(I1)inamide and N p HTEMPO for a-NpCbi+ thermolysis, respectively, strongly suggest that homolytic cleavage is the predominant mechanism for the a Co-C bond thermolysis, as is the case for the j3 diastereomers.7.8c Product analysis was also attempted for aerobic thermolysis of the a-neopentylcobalt corrinoids. In this case, the neopentyl radical is trapped by 0 2 to form the neopentylperoxy radical that subsequently decomposes to pivalaldehyde and neopentanol (Scheme I).7J1 To identify and quantitate these products, we attempted direct analysis by gas chromatography but failed, due to technical difficulties in separating the pivalaldehyde (bp 74 "C) and neopentanol from the solvent, given the small quantities of products involved. Consequently, pivalaldehyde was derivatized to form its Schiff s base with aniline in the presence of molecular sieves (4 A) as a drying agent. The Schiff's base is stable in organic solvents and can be easily quantitated by gas chromatography. By this method, the yields of pivalaldehydewere found to be 53 f 6%for a-NpCbl and 59 h 3%for a-NpCbi+thermolysis, in substantial agreement with the 50% yield predicted from trapping of the neopentyl radical by 02 (Scheme I). The Schiff s base product from thermolysis of a-neopentylcobalt corrinoids was also analyzed by GC/MS and was found to be identical to synthetic material. Both product analyses show that Scheme I is the apparent mechanism, Le., a homolytic Co-C bond cleavage leading to a neopentyl radical, for the thermal decomposition of a-neopentylcobalt corrinoids. The organic free radical product (neopentane free radical) from COX bond homolysis is either trapped by HTEMPO (anaerobic) or 0 2 (aerobic) after escaping from the solvent cage. Thermal Homolysis of a-NpCbi+. Repetitive UV-visible scans of an aerobic solution of a-NpCbi+ (5 X M) in potassium phosphate buffer (pH 7.0, 0.1 M) showed first-order spectral changes with isosbestic points at 482, 368, and 333 nm (Figure 3). The kinetics of this reaction were conveniently followed spectrophotometrically at 348 nm where the largest spectral change occurred. Rate constants for the thermolysis were determined at temperatures ranging from 5 to 30 OC at approximately 5 'C intervals. Above 30 OC, the reaction was too fast to reestablish the target temperature rapidly after addition of the a-NpCbi+ stock solution to provide accurate rate measurements. The rate constants thus obtained are collected in Table I, and an Eyring plot of these data is shown in Figure 4. The enthalpy and entropy of activation derived from the Eyring plot are 21.4 f 0.4 kcal mol-' and 0.9 f 1.3 cal mol-' K-1 (Table 11), respectively. Anaerobic thermolysis of a-NpCbi+ in the presence of 10 mM HTEMPO at 24.9 OC showed that the rates of thermolysis were almost identical (Table I), using HTEMPO (anaerobic) or 0 2 (aerobic) as the radical scavenger. Thermal Homolysis of a-NpCbl. Aerobic thermolysis of a-NpCbl was studied at pH 7.0 in potassium phosphate buffer. Repetitive scans of an a-NpCbl solution showed spectral changes consistent with loss of the alkyl group, forming a cobalt(II1) corrinoid (Le., after air oxidation of the cob(I1)alamin product). However, the thermolysis was somewhat more complicated than that of a-NpCbi+, and slow secondary spectral changes, though small, were observed toward the end of the reaction. These slow spectral changes were accompanied by a slow 7 band shift, after the primary thermolysis, from 356 to 358 nm with an isosbestic point at about 351.5 nm. The final spectrum was that of HzOCbl. We suspected that this slower reaction represented the formation of the base-on species of HzOCbl from the base-off cob(II1)alamin species formed after rapid air oxidation of the base-off cob(I1)alamin product (Scheme 11). In a-NpCbl, the alkyl group prevents the looped nucleotide from coordinating to the cobalt. After air oxidation of the initial cob(I1)alamin

6694 J. Am. Chem. SOC.,Vol. 115, No. 15, 1993

Zou and Brown

Scheme I

x*

=-+ OH

Table II. Enthalpy and Entropy of Activation for Thermolysis of Neopentylcobalt Corrinoids 1.0

0.8

compd

U P , kcal mol-'

AS*,cal mol-' K-1

22.5 0.2' 30.0 0.8 28 1' 26.7 1.2' 23.4 0.2d8 33 2, 32.2 0.6f 21.4 0.4' 29.7 1.3' 32.1 O.ld

*

ref

a-NpCbl 8-NpCbl, bw-off &NpCbl, base-onb

3.1 0.1' 11.5 2.6 21 1' 15 5' 2.6 0.1d8 35 l@' 33*2, 0.9 1.3' 10.8 3.90 17.3 0.4d

this work

** * * * *

2a 8c 25 this work

*

*

Abs 0.6 a-NpCbi+ &NpCbi+

0.4

* * *

7 7

8c

7

2a

a In 0.1 M potassium phosphate buffer, pH 7.0, ionic strength 1 M (KCl). Corrected values for base-on &NpCbl unless otherwise indicated. In potassium phosphate buffer, pH 6.8. In sodium phosphate buffer, pH 7.0. Not corrected for base-on/base-off equilibrium. /In ethylene glycol.

0.2

0

h (nm) Figure%Repetitivespectral scans (every 4 min) oftheaerobic thermolysis

Scheme II

of a-NpCbi+ in potassium phosphate buffer (pH 7.0) at 25 'C. The last two scans were at 12-min intervals.

5 3 -

3

-35

'

-36

*

-37

.

-38

.

-39

-

off species of j3-RCbl's (and 8-CNCbl) is f a ~ t , Hayward 3~ et aL3' reported from spectrophotometric observations in H2SO,/H20 mixtures that the equilibration of the base-on and base-off forms of HzOCbl is achieved "only slowly". Similarly, one of us36 also observed this slow equilibration process for base-on and base-off HzOCbl by3'PNMR. As ligand substitutionsat cobalt corrinoids are known to be dissociatively dominated,26*3wthe slow dissociation of water from the a-position of base-off HzOCbl is the rate-limiting step. In order to tell if slow formation of base-on HzOCbl might be the cause of the secondary spectral changes complicating the aerobic thermolysis of a-NpCbl, the spectrum of HzOCbl was (36) (a) Brown, K.L.; Hakimi, J. M.;Jacobsen, D. W. J. Am. Chem. Soc. 1984,106,7894. (b) Brown, K.L.; Hakimi, J. M.Inorg. Chem. 1984,23,

0 e

3.25

3.35

3.45

3.55

3.65

im (x 103) Figure4. Eyring plots for the thcrmolysisof u-neopentylcobalt corrinoids (ln(kob,h/kBT) versus 1/T, where h is Planck's constant and Boltzmann's constant): ( 0 )or-NpCbl; (A)a-NpCbi+.

kg

is

thermolysis product, formation of the base-on speciesof HzOCbl isstronglythermodynamicallyfavored (AGO =-lo5 kcalmol-').M While formation of the base-on species from the free base, base-

1756. (37) Hayward,G. C.;Hill,H.A.O.;Pratt, J. M.;Vanston,N. J.; Williams, R. J. P. J. Chem. Soc. 1965,6485. (38) Reenstra, W. W.; Jencks, W. P.J. Am. Chem. Soc. 1919,101,3780. (39) Randall, W. C.; Alberty, R. A. Biochemistry 1966,5, 3189. (40) (a) Marques, H.M.;Bradley, J. C.; Campbell, L. A. J. Chem. Soc., Dalton Trans. 1992,2019. (b) Marques, H. M.; Breet, E.L. J.; Rinsloo, F. F. J. Chem. Soc., Dalron Trans. 1991,2941. (c) Marques, H. M. J. Chem. Soc., Dalton Trans. 1991, 304. (d) Marques, H. M.;Mgan, T.J.; Marsh, J. H.; Munro, 0.Q.Inorg. Chim. Acra 1989, 166,249. (e) Bradley, J. C.; Marques, H. M.;Brown, K.L.; Brooks, H. B. Manuscript submitted to J . Chem. Soc., Dalron Trans.

Organocobalt Corrinoids

J. Am. Chem. Soc.. Vol. 115, No. 15, 1993 6695

h

recorded as a function of time in 6.5 M aqueous H2SO4, in which solution it is ca. 90% base-off at equilibrium.36 The reaction being spectrally interrogated is thus the base-on to base-off transition of H2OCbl (eq l), in which the first step, formation

A

of the base-off species, is rate limiting. This step is the reverse of the final reaction in Scheme I1 in which the base-off HzOCbl product, formed by rapid air oxidation of the direct homolysis product, base-off cob(II)alamin, is converted to the base-on species. In this H2S04 solution, slow spectral changes were observed which were similar to, but in the opposite direction of, the slow spectral change following the aerobic decomposition of a-NpCbl in neutral aqueous solution. In addition,when a-NpCbl was thermolyzed anaerobically in the presence of HTEMPO at D 25 OC, strictly first-order kinetics were observed and there was no slow secondary spectral change. This is expected since, in anaerobic solution, cob(I1)alamin persists, and its base-off to baseon conversion (pK-n = 3. 10)19is expected to be extremely fast. Since base-on and base-off HzOCbl's have different 3lP NMR E chemical shifts (0.14 and -0.42 ppm, respectively),36 the baseon/base-off transition of HzOCbl can also be monitored by 31P NMR. Thus, 31P NMR studies of seven known base-off alkylcobalt corrinoids in D20, including a-RCbl's (R = Ado, 1 ' ~ ~ ' I ' ~ ' I I I ' I ~ I ' CH3CH2, CH3, CF3, CF~CHZ), a- and @-CF3CH2-(trimethyl0.5 0.0 -0.5 -1. 0 PPM benzimidazolyl)cobamides$l and (CN)zCbl, showed that all such Figure 5. "PNMR spectra of a-NpCbl(l.5 mM) in -0.2 M N b O A c base-off species have chemical shifts (6 = -0.47 0.16 ppm) at 25 OC: (A) 7 min; (B) 27 min; (C) 72 min; (D) 207 mh, (E) 1068 similar to that of base-off HzOCbl and other protonated, basemin. Theuflield-most peabarcduetoa-NpCbl (A) andbasaoff H20Cbl off @-RCbl'sthat have been studied (6 = -0.45 f 0.03 ~ p m ) . ~ ~(C, D),as all base-off cobamidcs studied to date have 3lP chemical shifts Consistent with these values, a-NpCbl has a 31P chemical shift of-0.46+0.16ppm. Thepeakat0.07ppm(A)isduetobase-onH~OCbl, at -0.48 ppm (at - 5 "C). The chemical shift of base-on and the peak at -0.03 ppm is due to NH3Cb1, unavoidabiy formed from H20Cb1, on the other hand, was found to vary from 0.07 to 0.14 HzOCbl in the NH,OAc containing solvent (see text). ppm, depending on the concentration (1.5-20 mM). In order to obtain 31Pspectra of freshly purified a-NpCbl, crude a-NpCbl 11.2 min; vide infra) was -81% complete (27 min, Figure 5B), was purified by HPLC using ammonium acetate as the solute in a large resonance43 persisted at -0.48 ppm that must be attributed the aqueous solvent (see Experimental Section) instead of the to baseoff HZOCbl, i.e., the air oxidationproduct of the immediate usual ammonium phosphate buffer to avoid the Occurrence of a product of C& homolysis, base-off cob(I1)alamin. This large inorganic phosphate resonance in the 31Pspectra. In the resonance disappeared only slowly, with a half-time of 385 min, same ammonium acetate containing solution, the 3lP chemical with the concomitant growth of the resonance at -0.03 ppm, due shift of HzOCbl was found to be -0.03 ppm, presumably due to to &NHpCbl (formed from the final HzOCbl product in 0.2 M formation of @-NH&bl. At 25 OC, the NMR spectrum of NH40Ac). This half-time agrees well with a value of 362 min a fresh sample of a-NpCbl (1.4 mM, in ca. 0.2 M NHdOAc), obtained from kinetic analysis of the slow UV-visible spectral obtained as soon as possible after make up (ca. 7 min), showed change during aerobicthermolysisof a-NpCbl (vide infra). Taken threepeaksattributable to a-NpCbl (6 = -0.48 ppm),unavoidable together with the fact that the slow secondary spectral changes contamination with HzOCbl(6 = 0.07 ppm), and the @-NH&bl were absent when a-AdoCbl was decomposed anaerobically with (6 = -0.03 ppm) formed from thecontaminating H20Cbl (Figure HTEMPO (since ligand substitution in cob(I1)alamin must be 5A).42 When the thermolysis of the starting a-NpCbl (t1/2 = fast), these experiments satisfactorily demonstrate that the reaction observed in aqueous solutions of a-NpCbl is adequately (41) Brown, K.L.; Wu,G. 2.Organometallics 1993, 12, 496. described by Scheme 11. (42) In the first obtainable 3IP spectrum of a-NpCbl (Figure SA), the two peaks at 0.07 and -0.03 ppm are attributed to contaminating HzOCbl and Although the absorbance change due to the slow formation of @-NH,Cbl. The peak at 0.07 ppm is due to HZOCbl, 88 confirmed by the base-on species was noticeable at the end of the thermolysis obscrvationofanauthenticsampleofH2OCblinDlOat thesamcmcentration. The second peak at -0.03ppm was shown to be base-on @-NH3Cblby control of a-NpCbl, its effect on the analysis of the kinetics is minimal experimentsin which samples of HZOCbl( 1.5 mM) were dissolved in N)40Ac since the base-onlbase-off reaction was at least 30-fold slower and NaOAc solutions(both 0.2 M). In NaOAc, H2OCbI has a chemical shift than that of the primary thermolysis reaction, and the spectral of0.07ppm (Le., thesameshift it hasinDzO), whileinN)40Ac,thechemical change associated with formation of base-on/base-off reaction shift was -0.03 ppm. Evidently, the exchange between NH3 and H20 in the inert cobaltcomplexwasslowenoughforH~0Cbltoappearinthefmtspcct" was much smaller than that of the thermolysis. Furthermore, that was recorded at 7 min after making up the sample. The base-on HZOCbl the kinetic data for thermolysis of a-NpCbl were collected at peak at 0.07 ppm disappeared in the second spectrum (Figure 5B), taken at 35 1.6 nm, near the isosbesticpoint of the slower reaction. At this 27 min, bccause all H20Cbl had been converted to NH3Cbl. The samplewas

i n A

*

prepared from a lyophilized solution of a-NpCbl by HPLC separation. A preparationofthescalenecessaryfor~~PNMRnormallywouldtakcamini" of 3 4 h. Apparently, about 25%of the a-NpCbl had decomposed by the time the sample was ready, in spite of the care taken to keep the purified solution cold at all times.

(43) a-NpCbl and base-off HzOCbl (the intermediate of the faster thermolysis reaction) have the same 3IP chemical shift at 4 . 4 8 ppm. This is as expected as all basesff @-RCbl'sand a-RCbl's have asentially the same 31Pchemical shift.

'

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Zou and Brown

wavelength,the overall spectral change for the secondary reaction was at least 10 times smaller than that for the thermolysisreaction, further minimizing the influence of the secondary reaction. Consequently, the kinetic data for a-NpCbl thermolysis were successfully fitted to an equation consisting of an exponential term for the primary thermolysis reaction and a linear term for the slower secondary reaction as shown in eq 2, restricting the

A = Cl(1 - exp(-k,t))

+ C2t + C3

data set to about 9 half-times of the faster kinetic phase. The observed rate constants thus obtained for a-NpCbl thermolysis are collected in Table I. An Eyring plot from these data is shown in Figure 4, and the activation parameters from the Eyring plot are given in Table 11. The rate constants are smaller at all temperatures than those for a-NpCbi+. A rate constant for anaerobic thermolysis of a-NpCbl in the presence of HTEMPO is also given in Table I for comparison. In order to permit evaluation of the rate constant for the slow secondary spectral change and comparison to the results of the 3lP NMR experiment discussed above, the UV-visible spectral changes for thermolysis of a-NpCbl were also followed to completion at 358 nm, 25 OC, for both phases of the reaction. In this case, a double exponential equation was used to obtain rate constants for both phases of the reaction as shown in eq 3, where

A = Cl(l - exp(-kit)) + C2(1- exp(-k2t))

+ C3

(3)

kl is the rate constant for the faster thermolysis reaction and kz is for the slower base-on reaction. The rate constant, kl, thus obtained from eq 3 agreed well with those for the primary thermolysis reaction obtained by fitting truncated data sets to eq 2, and the value of kz agreed well with that obtained from the 31P NMR experiment as discussed above. Figure 6 s (available as supplemental material) shows such a set of kineticdata for the thermolysisof a-NpCbl that was followed to completion for both the thermolysis and the slow base-on reaction at 358 nm. These data were fitted, respectively, to a double exponential equation (eq 3), to a single exponential equation for the entire data set, and to eq 2 for the first 110 min. These comparisons clearly show that eq 2 can give accurate rate constant for the thermolysis reaction, and the effect of the secondary reaction on the thermolysis is minimal.

Discussion We have reported previou~ly,~ as have other groups,zp~8c that reductive alkylation of factor B or H2OCbl with neopentyl halides produces only a single alkylcobalt corrinoid, p-NpCbi+ or O-NpCbl, respectively. The current study shows, however, that substantial amounts of a-NpCbi+ and a-NpCbl can be produced by reductive alkylation with NpBr below ambient temperature. Apparently, the failure to observe the formation of a diastereomers in earlier studies is due to the extreme lability of the a-neopentylcobalt corrinoids. Normal workup procedures at r c " temperature would have clearly lead to the destruction of these elusive complexes since they decompose rapidly (Table I). When we started this project, our initial plan was to attempt the preparation of a-NpCbi+by anaerobicphotolysis of 8-NpCbi+, a method that has been successfullyused to prepare a-alkylcobalt corrinoids that were only minor products or were unobtainable by reductive alkylation.20 As expected, anaerobic photolysis of @-NpCbi+produced a-NpCbi+ in 10-15% yield, by HPLC. It quickly became clear to us, however, that the newly formed a-NpCbi+ was quite unstable at room temperature and that workup and HPLC procedures needed to be carried out at -0 OC to prevent massive decomposition. This finding prompted us to reexamine the reductive alkylation reactions of factor B and H20Cblwith NpBr. Indeed, when the reaction and purification

steps were carried out at lower temperatures (-0 "C), both a-NpCbP and a-NpCbl were positively identifiedinand separated from reaction mixtures resulting from the reductive alkylation of factor B and HZOCbl, respectively, with NpBr. In fact, more a diastereomer was produced than /?diastereomer in both cases under appropriate conditions (see Experimental Section). Reductive alkylation has been consequently used throughout this study for the preparation of a-neopentylcobaltcorrinoidsbecause of the high yields and overall convenience of the preparations. However, due to the lability of the a-neopentylcobalt corrinoids, purification immediately before use was necessary in order to provide samples of sufficient purity (>95%). The high yields of a-neopentylcobalt corrinoids by reductive alkylation and the recent synthesisof a-adenosylcobaltcorrinoids's seem to suggest that the stereochemicaloutcome of the reaction, i.e., the ratio of a-and 8-alkylcobalt corrinoids, does not depend on the size of the bulkiness of the organic ligand involved. Understanding the factors that control the stereochemistry of reductive alkylation reactions remains an important objective that is under study in our laboratory. Unlike 8-AdoCbl and &Ad0Chi,13~in which Co-C bond heterolysis competes with homolysis due to the presence of the 8 oxygen atom in the organic ligand,the mechanism of thermolysis of 8-NpCbl and B-NpCbi+ has been firmly established to be strictly Co-C bond homolysis?.& Co-C bond fission by heterolysis is not likely to compete with the homolysis because of the lack of a /3 heteroatom in the neopentyl ligand. In addition, the lack of any /3 hydrogens in this ligand also precludes any possible 8 elimination reactions. The activation enthalpies (AH*) and entropies (AS*)for the thermolysis of base-on 8-NpCbl and &NpCbi+ in water have been reported,h.7.8cas well as values in other Thus, the thermolysis of 8-neopentylcobalt corrinoids is well studied and documented, although there are some discrepancies in the reported activation parameters (Table 11). Thermolysesof a-NpCbl and a-NpCbi+ are expected to follow the same mechanism, Le., COX bond homolysis. However, there is no prior study in the literature on the mechanism of an a Co-C bond cleavage. Evidence to support the homolytic mechanism in the Co-C bond cleavage in the a-neopentylcobalt corrinoids was thus sought by anaerobic thermolysis of a-NpCbl (and a-NpCbi+) in the presence of a nitroxide free radical trap, HTEMPO. After completion of the thermolysis at 25 OC (- 120 min, >10 half times), the only product detectable by GC/MS in the pentane extract of the reaction mixture was NpHTEMPO as identified by comparison of its mass spectrum to that of independently synthesized authentic NpHTEMPO. This indicates that the neopentyl ligand formed a free radical during the thermolysis. By GC quantitation, the yield of NpHTEMPO was found to be 92 6% for a-NpCbl and 70 3% for a-NpCbi+. In addition, the corrinoid after HTEMPO-trapped anaerobic thermolysis was spectrally identical to cob(I1)alamin (or cob(1I)inamide) with an absorbance maximum at 470 nm,44and the yields of the cobalt(I1) corrinoids were found to be 102 2% and 78 f 1%for a-NpCbl and a-NpCbi+, respectively. The reduction of the yields of Np-HTEMPO and cob(I1)inamide for a-NpCbi+ below 100% is attributed to unavoidable aerobic decomposition of the a-NpCbi+ during purification, as described above. These results established that the mechanism of thermolysis of a-NpCbl and a-NpCbi+, like their 8 diastereomers, is homolytic cleavage of the Co-C bond (Scheme I). Evidence to support a homolytic mechanism was also obtained by product analyses of aerobic thermolysis of a-neopentylcobalt corrinoids. Alkyl radicals rapidly react with oxygen4sva to form

*

*

(44) Beaven, G. H.; Johnson, E. A. Nature 1955, 176, 1264. (45) Mailard, B.; Ingold, K. U.; Scaiano, J. C. J. Am. Chem. Soc. 1983, 105.5095. . .., .....

(46) Marchaj,A.; Kelley, D. G.; Bakac, A.; Espenson,J. H. J. Phys. Chcm.

1991, 95,4440.

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Organocobalt Corrinoids

differential cage effects in the two isomers.48 However, it seems unlikely to us that much of this large observed difference in reactivity can be attributed to cage effects given the similarity of the caged pair which must be generated from a-and &NpCbi+. RCH,' 0, RCH,OO' '/,RCHO '/,RCH,OH It has been suggested,7~~~ that in base-on 8-RCbl's with bulky l / 2 0 , (4) organic ligands,thermal homolysis is driven by steric interactions of the bulky organic ligand with the upward projecting a, c, and g acetamide side chains. This steric effect is expressed as an conversion to its Schiff s base with aniline (i.e., N-(2,2-dimethelevated entropy of activation for Co-C bond homolysis since in ylpropylidene)benzenamine),the anticipated aldehyde product the ground state, the steric interactions between the side chains from the neopentyl radical was detected among the aerobic and the organic ligand reduce the freedom of rotational motion thermolysis reaction products of both a-NpCbi+ and a-NpCbl about the side chain C-C bonds, while in the transition state this by GC and GC/MS. Careful GC quantitation of the yields of motional restriction is significantly relieved by the incipient the Schiff s base from pivalaldehyde, using a standard curve separation of the corrinoid and organic ligand fragments. The constructed with the authentic, independentlysynthesizedSchiff s fact that the base-off speciesof such BRCbl's (and the analogous base, showed that the aldehyde was produced in slightly greater 8-RCbi's) are 103-fold more stable than the base-on &RCbl's than 50% yield from both a-NpCbi+and a-NpCbl. These results has been attributed to a downward folding of the corrin ring in are consistentwith the persistenceof a COX homolysis mechanism these species (originally suggested by Schrauzer and Grate,), for the aerobic thermolysis and validate Scheme I for these which reduces the steric interference with side-chain motion in decompositions. the ground state, hence reducing the entropic driving force for Different radical scavengers have been used in studies of reaction.7 This release of ground-state steric interactions is seen carbon-cobalt bond homolysis reactions.6~8~J~ Some of the most in the differential activation parameters for base-on 6-NpCbl widely used compounds are nitroxide derivatives, such as and the base-off species ( A M * = -2 f 2 kcal mol-', but A M * TEMP0.38*c*dJ3C An alternative is to use 02 as a trap. Oxygen = 10.2 f 4.9 eu).7 If this interpretation of the reaction energetics in air-saturated water has been shown to be an efficient scavenger of organocobalt corrinoid Co-C bond homolysis is correct, one for a wide variety of alkyl free radicals with second-order rate would anticipate that a-NpCbi+ would substantially more labile constants in excess of 1 X lo9 M-1 s-1.45.46 For thermolysis of than the /3 diastereomer (as is the case) since the downward a-neopentylcobalt corrinoidsin this study, oxygen-saturatedwater projecting corrin ring side chains (the b, d, e propionamides and was used because of the labilities of the compounds being studied the f side-chain secondary amide) are both more numerous and and the difficulties involved in achieving strictly anaerobic longer than the upward projecting side chains. However, in this condition without some degree of prior decomposition. Furrelatively simple picture of Co-C bond activation, the increase thermore, control experiments showed that thermolysis of a-neoin reactivity of the a diastereomer would be expected to be found pentylcobalt corrinoids in 02-saturated water and in anaerobic mostly in the entropy of a c t i ~ a t i o n .The ~ ~ results for a-NpCbi+ solution containing excess HTEMPO (10 mM) had essentially show that just the opposite is the case; there is a loss of entropic the same rates for both a-NpCbl and a-NpCbi+ thermolysis driving force relative to the fl diastereomer, and the enthalpy of (Table I). activation is lowered by 8.3 kcal mol-'. If our picture of the There have been some discrepanciesin the literature regarding energetics of Co-C bond homolysis is substantially correct, then the values of AH* and AS* for /?-NpCbi+ and 8-NpCbl these results suggest that, as is the case with the 0-RCbi relative thermolyses in aqueous solution (Table 11).h37.8c These discrepto the base-on 0-RCbl, there is a flexing of the corrin ring, this ancies are most likely due to varying methods employed for time in the upward direction, which tends to relieve the steric temperature measurements by different researchers as well as restriction of side-chain motion in the ground state. If this is the other technical difficulties associated with such measurement^.^' case, the upward corrin flex in a-NpCbi+ must be more extreme In the case of 0-NpCbl, additional disagreement occurs because than the downward corrin flex in &NpCbi+ since a large reduction different methods have been used to evaluate the amount of basein A S is observed rather than the expected increase due to the on species present at each temperature and hence to correct the observed rate constants for the base-on/base-off equilibri~m.k,~,~C fact that the downward projecting side chains are more numerous and larger. Such an accentuated upward flex could conceivably In the following analysis, the values from our previous report7are lead to a sufficientlydistorted ground-statecoordination geometry used since these data were obtained under the same conditions toalter (weaken) theorbitaloverlapin theCOX bondand produce as those for the a-neopentylcobalt corrinoid thermolyses in this the observed lowering of AH*.It should be pointed out that the study. capability of the corrin to undergo such an upward flex (for which The enthalpy and entropy of activation for the thermolysis of there is no prior evidence) could be extremely important in the a-NpCbi+ were found to be 21.4 f 0.4 kcal mol-' and 0.9 1.3 "activation" of AdoCbl for Co-C bond homolysis by AdoCbleu, respectively, from the Eyring plot (Table 11). This enthalpy requiring enzymes. The possibility that the a and 6 diastereomers of activation is about 8 kcal smaller than that for &NpCbi+ of NpCbi+ adopt two very different corrin ring conformations is homolysis under identical reaction conditions. In addition, consistent with recent NMR evidence that suggeststhat the corrin although the entropy of activation of a-NpCbi+ remains positive, ring conformations of 0-AdoCbi and a-AdoCbi are significantly it is reduced by some 10 eu from that of &NpCbi+. With different.18.52 This possibility is currently being further studied. extrapolation of the data of a-NpCbi+ to 35 OC, the lowest Another possible contributor to thevery low AS*for a-NpCbi+ temperature for which data are available for b - N ~ C b i + , ~ is the very length of the downward projecting b, d, e, and f side a-NpCbi+ was found to be 5.3 X 103-fold more reactive than the B diastereomer. This results from 8.3 kcal of activation by the (48)Koenig, T.W.;Hay, B. P.; Finke, R. G. Polyhedron 1988, 7, 1499. (49)Brown, K.L.;Brooks, H. B.; Behnke, D.; Jacobsen, D. W. J. Biol. reduction is AH*,countered by 3.0 kcal of stabilization due to Chem. 1991. 266. 6737. the reduction of LW*. At 5 OC, after extrapolating the data for (50) In the ground state, the @-RCbl'sare favored over the a-RCbl's by @-NpCbi+,the ratio of the rate constants for a-NpCbi+ and about 17:l or about 1.7 kca1.I' Binding constants for exogenous ligands to RCbi's are 10% to 90% higher for @-RCbi'sthan the a diastereomers (Le., &NpCbi+ was 2.3 X lo4. Some of this difference in the observed @coordinationis favored by 0.05 to0.38kcal)."1 However, thecurrent results rate constants for trapped homolysis of the carbon+obalt bonds represent the first example of kinetic comparison of a and @ diastereomers of the neopentylcobinamide diastereomers may be due to and so it is not apparent how different AH* for Co-C homolysis is likely to alkylperoxy radicals that subsequentlydecompose to an equimolar mixture of the alkyl-derived aldehyde and alcohol (eq 4).7J1 After

+

-

-

+

+

-

*

be. (47)Private communication with Professor R. G. Finke of the University of Oregon.

(51) Brown, K. L.;Satyanarayana, S. Inorg. Chim. Acra 1992,201, 113. (52)Brown, K.L.;Zou, X. J . Am. Chem. Soc. 1993,115, 1478.

6698 J. Am. Chem. SOC.,Vol. 115, No. 15, 1993

Zou and Brown

a-NpCbl to 35 OC, the lowest temperature at which data for base-off @-NpCblwere obtained’) in favor of a-NpCbl. At 5 OC, the ratio of the rate constants is 1.1 X 104. The differences in enthalpies and entropies ( A M * = 7.5 kcal mol-1 and PAS*= 8.4 eu) are almost the same as the differencesbetween the NpCbi+ pair. Again, both AH*and A S have lower values. Presumably, similar factors responsible for the effects in the diastereomeric NpCbi’s are involved here as well. In conclusion, this study shows that a-NpCbi+ and a-NpCbl can be prepared by reductive alkylation of factor B and HzOCbl with NpBr or by anaerobic photolysis from their corresponding @ diastereomers. Like the @ diastereomers, thermolysis of a-NpCbi+ and a-NpCbl leads to Co-C bond homolysis, but the rates of homolysis of a-NpCbi+ and a-NpCbl are more than 103-104 times faster than those of the corresponding @ diastereomers. The differences in the rates of homolysis are due to a reduction in enthalpiesof activation in the a diastereomers despite a decrease in the (positive) entropies of activation as compared to @-neopentylcobaltcorrinoids. It is suggested that the a-alkylcobalt corrinoidsmay adopt comn ligand conformationsinvolving an upward corrin ligand flex as opposed to 8-NpCbi+, which presumably displays a downward corrin flex.

chains. Thus, if the length of the breaking Co-C bond in the homolytic transition state is similar in the a and @ diastereomers, the resulting geometry may free the upward projecting side chains completely or nearly completely from motional restrictions by the departing Np ligand in the @ transition state but not in the a transition state since the side chains involved are longer. Thus, any relief of steric restrictions on side-chain mobility may be much smaller in the a transition state than in the @ transition state leading to a small AS*for the a diastereomer. The relative importance of this effect and the presumed upward corrin flex remain to be elucidated. The enthalpy and entropy of activation for a-NpCbl thermolysis are statistically indistinguishablefrom those for a-NpCbi+. This suggests that the presence of the uncoordinated benzimidazole nucleotide in a-NpCbl has little or no steric consequences for the homolysis reaction, perhaps because it is partially (or mostly) *tucked-in” (Le., the benzimidazole B3 nitrogen is hydrogen bonded to the g side-chain amide),S’ as is the case for other a-RCbl’s.54 The nearly identical enthalpies and entropies for a-NpCbi+ and a-NpCbl seem to support, retrospectively, the conclusion that Co-C bond homolysis and subsequent formation of the base-on species of HzOCbl in a-NpCbl thermolysis could be treated, for practical purposes, as two separate reactions (Scheme 11). Comparisoncould also be made between the rates of thermolysis of a-NpCbl and @-NpCbl. However, such a comparison is not apt, sincecoordinationof the axial nucleotidein the @diastereomer has been shown to cause a 10z-103-fold rate enhancement in base-on over base-off @-RCbl’~.Z*3b*~ A more meaningful comparison would be that between a-NpCbl and base-off @-NpCbl (Table 11). The ratio of rate constants between the latter two complexes is 3.4 X 103at 35 OC (after extrapolating the data for

Acknowledgment. This research was supported by the National Science Foundation (Grant No. CHE 89-96104), the NSF EPSCoR program (Grant EHR 91-08767), the State of Mississippi, and Mississippi State University. The authors are grateful to Professor Earl G. Alley of this department for assistant with GC/MS analysis and to Professor R. G. Finke and M. D. Waddington for communication of their results prior to publication.

(53) (a) Brown, K. L.; Brooks, H. B.; Gupta, B. D.; Victor, M.; Marques, H. M.; Scooby,D. C.; Goux, W. J.; Timkovich, R. Inorg. Chem. 1991, 30, 3430. Ib~Brown.K.L.:Brooks.H.B.:Zou.X.:Victor.M.:Rav.A.:Timkovich. R. Ino& Chem.’ 1996, 29, 4841. (c) Brown, K. L.;Peclt’Siler, S . Inorgi Chem. 198%. 27,3548. (54) Brown, K. L.; Evans, D. R. Inorg. Chim. Acta 1992,197, 101.

Supplementary Material Available: Figure 6S, showing the treatment of the kinetic data for aerobic thermolysis of a-NpCbl, and text giving a brief description of the methodology (2 pages). Ordering information is given on any current masthead page.