4.5. Fase de Implementación
4.5.1. Configuración de los equipos de Radio para la comunicación del PC con
3,18.1 [
17
Q-Formyl]formylmethyl(pyridine)cobaloxlme (79) An IR spectrum of (79) is shown in Fig*6
. The two additional isotopically substituted carbonyl bands had absorptions at 1640 cm- 1
(C»17
0) and 1630 cm- 1
(C«18
0). The IR spectrum showed that the relative intensities of180
»170
i180
carbonyl absorptions were in the ratio of 2 t 3 t 5. This compound was prepared by the hydrolysis of (EtOlgCHCHgCofdmgH^py with 1 mol equiv. of 31 atom-% [17
0]water. The relative intensities of the IR carbonyl absorptions suggested that most of the isotopic label from the 31 atom-% [170
]water was incorporated into the cobaloxime (79). When this cobaloxime was purified through a silica-gel column, its IR carbonyl absorptions showed180
t170
t180
in the ratio of2
t 1 t 2. The decrease in the intensities of the isotopic O O absorptions were a result of the acidicity of the silica-gel, which caused isotopic exchange between the labile aldehyde functional group and water absorbed on the silica-gel.-123
3.18
An interesting feature of this labelled cobaloxime is that the carbonyl stretching values were at much lower frequencies compared to typical organic aldehydes. For example, the carbonyl absorption of acetaldehyde appeared at 1730 cm
-1
and that of an a ,p-unsaturated or aromatic aldehyde in the region 1710-1685 cm-1 . The weakening of the carbonyl bands in17
the O-enriched cobaloxime had been observed by Dolphin et al [47] on the unlabelled compound
(79). They suggested that this phenomenon arose from an inductive effect due to the interaction of the more electropositive Co d - orbital« with the p-carbonyl group. We observed similar effects in the 1H NMR and 170 NMR spectra of this compound. The characteristic formyl proton of pentanal appeared at 9.66 ppm, whereas that of the labelled cobaloxime was shifted upfield to 9.10 ppm, J ■ 3.5 Hz. Ibis upfield shift was also observed for the
170
resonance which appeared at 544 ppm (relative to water) compared to[
17
0]pentanal at 583 ppm.2 [
17
0]Pentanal and [17
0-formyl]formyl(pyridine)cobaloxlme The observed pseudo-first order rate constants for the hydration of the title compounds are shown in Table6
. The rate constants were obtained by plotting In1
(intensity of170
[water] signal) againsttime (t).
17
A mechanism for the hydration of [* 0]pentanal and the title cobaloxime, thought to go, via a
gem-diol, is shown in Scheae 84. An interesting observation is that the kinetics of hydration of the cobaloxime (79) in aqueous dioxan, showed a half-life Tjya» 4 h at 50 °C, which is extremely slow relative to aromatic aldehydes or even ketonic compounds. In accord with Eq. 2, the rate of hydration of a similar amount of (79), but in dioxan containing phosphate buffer (pH
6
.8
) at 50 °C, has a half life of 2 h. The lack of reactivity of the formyl group in 0CHCH8
Co(dmgH)8 py was evident when attempts to reduce the compound to HOCH8
CH8
Co(dmgH)8 py with NaBH4
and H A I H4
failed, although conversion can be achieved with diborane (50). This lack of reactivity may be attributed to the occurrence of a significant sigma to n-hyperconjugation as in complex (80), thus deactivating the carbonyl groups towards nucleophilic addition by the water molecule. Brown[51] observed that the pKa of H00CCH
8
Co(dmgH)8 py was 7.14 and that of H08
CCH8
CH8
Co(dmgH)8 py was 5.70. The weak acidity of the former complex was a consequence of a - n hyperconjugation. o - n Hyperconconjugation was described [52] as the stabilization of a neighbouring cationic centre by the delocalisation of a a bond. The length or angles around such a o bond are not changed as the transition state is approached. Such delocalisation without changing the reactant geometry is also known as 'vertical stabilisation'. This effect is in contrast to that of the bridged-ion theory of neighbouring group participation.-125- ♦
*0
*oH h*o h II h2o ( h RCH2
C-H — ► RCHoC-OH — — RCH2
C>0H | + I H H RCH2
C-0 + h2
o* HR ■ Pr or -Co(dmgH)
2
py, * ■ Oxygen-17 Scheme 84(
8 0)
3.18.3 [
17
0Ac] -2-Acetoxyethyl(pyrldlne)cobaloxlme (76) The rate constants £or the hydrolysis of cobaloxime (76) are shown in Table6
. Pig. 7 shows a 170 NMR spectrum taken during the hydrolysis of cobaloxime (76) at 50 °C in aqueous dioxan. The intensity of the I170
g]acetate signal increased with time, whilst that of the water and the dioxan remained constant throughout the experiment; no other signals17
were detected. The appearance of 0 in the acetate ion indicated that the hydrolysis involved breakage of the alkyl-oxygen bond. From Ingold's classification of the mechanism of ester hydrolysis, the neutral hydrolysis (76) is a BAL1 type, a relatively uncommon reaction, the one example known being CF
3
CO.OC(Me)g (Section 3.6.2). The hydrolysis of(76) in aq. dioxan at 50°C has a half-life of 3 h. N .B., the neutral hydrolysis of ethyl acatate is exceptionally slow (the estimated half-life at 25°C is
75 years), [cf 22]. The by-product from the hydrolysis of (76) was HOCHgCHgCofdmgH^py' which was shown by 0 NMR spectroscopy to be free of 0 signal. When (
170
]H0
CH2
-CH2
-Co(dmgH)gpy was held at 5 0 % in aqueous dioxan containing1
mol equiv. of acetic acid, for 24 h, on removal of the solvent and redissolving the cobaloxime in CDCI3
the sample showed an0
signal comparable to an untreated sample in CDClg pulsed similarly for 1 h.A mechanism which is consistent with these results (Scheme 8 5 ), proposes a n-ethylene intermediate of cobaloxime(III), which undergoes rapid capture of H
2
O to give H0
C H2
C H2
Co(dmgH)8 py or loses ethene to give HOCo(dmgH)a p y • The rates of these reactions are such that the relative amount of the former compared to the latter is in the ratio of 3 t v 1 (from *H NMR spectroscopy). Ionisation C H 2CH *OC-Me 5 3 5 I Internal (Co) return Intimate ion pair cotd.127 CH 2=C Co c h
2
*°\-VC-Me
o ' dissociation Solvent separated ion pair n-complex H20 I k H 20 c h2
c h,o h , 0 * ----> I2
2+ Me<(- k (Co)0
1
+ (Co)&
----> h2
c«c h2
♦ + * ■ Oxygen-17 Schsms 85Tabla
6
. Rate constants for the hydration and hydrolysis of170
-enriched compounds l170
]PentanalHydration of (
17
0]pentanal in 0.1 M phosphate buffer, pH6
.8
.0 - 2
k (5 °C) - (1.6 i 0.1) x 10 s
Half-life (Ti/a ) ■ 43 seconds or u 1 min. [
17
0-Formyl)Formylmethyl(pyridine)cobaloximeHydration of cobaloxime (
79
) in dioxan t water (3 »2
) k (40 °C) - (1.6 t 0.4) » 10"6
s_1
k (50 °C) - (4.5 t 0.4) x l0'B s"1
0
-4 .1 k (60 °C) > (1.6 t 0.2) x 10 s T - 4 h (50 °C)1/2
N.B. Hydration of (79) in dioxan : phosphate (pH
6
.8
) gave «- k (50 °C) « (1.0 + 0.1) x 10-5
s-t T ■ 2 h1/2
[17
0Ac]-2-Acetoxylethyl(pyridine)cobaloximeHydrolysis of the title cobaloxime in dioxan : water (4 : 2).
A duplicate experiment was recorded, k (50 °C) - (5.5 + 0.1) x 10"
5
s_1
k (50 °C) - (
6.6
± 0.4) x I0_5
s“1
T
1/2
178
p
p
Chapter 4 4. INTRODUCTION
A cobaloxime model for the diol-dehydrase catalysed reaction, for example, HOCH
2
[18
OlH-CH-(CH2
)gCo(dmgH)2 py required a 1,2 (18
OH)-migration, terminating at [18
0]pentanal (cf Section 1.6.3). Because the [180
)pentanal from the cobaloxime is released into an aqueous acetic buffer, rapid reversible hydration may occur resulting in the loss of the 180 label. However, the isotopic composition of [180
]pentanal can be ascertained provided the labile compound is rapidly trapped (e.g. by conversion to an alcohol). This Chapter begins with a discussion of the properties of the reducing enzymes such as yeast and liver alcohol dehydrogenases and their possible applications as trapping agents. This is followed up by a brief description of a synthetic route to a C-418
0-enriched alkylcobaloxime. Finally, in the Experimental Sections, there are presented results which indicated that the hydration of 23 atom-« (I80
]benzaldehyde was relatively slow compared to a 25 atom-« (*8
O]BuCH0 in aqueous-organic solvent. These results implied that an organic solvent may be used as trapping agent, and hence an alternative cobaloxime model is proposed in the Discussion Section. 4.1 ALCOHOL DEHYDROGENASES [1 a-b)These are zinc metalloensymes of broad specificity, oxidising a wide range of aliphatic and aromatic alcohols to their corresponding aldehydes and ketones using NADH
-130 and NAD* as coenzymes. The two most studied enzymes are those from yeast and horse liver. At pH 7, the equilibria of the redox reactions lie heavily in favour of carbonyl reduction. Scheme
86
shows a mechanism for the oxidation of alcohols. The basic mechanism is essentially electrophilic catalysis mediated by the zinc atom at the24-
active site. Polarization of the alcohólate anion by Zn acting as a Lewis acid facilitates the subsequent hydrogen transfer step. This process involves only one side of the nicotinamide ring of NADH.
Scheme