Eur. J. Biochem. 136, 303-311 (1983) 0 FEBS (1983)

Stereochemistry of the rearrangement of 2-aminoethanol by ethanolamine ammonia-lyase David GANI, 0. Caryl WALLIS, and Douglas W. YOUNG School of Chemistry and Molecular Sciences, University of Sussex, Falmer, Brighton

(Received April 26/June 6, 1983) - EJB 830425

1. (1 R)-2-Amin0[l-~H ,]ethanol and (1 S,2RS)-2-amino[l ,2-2H2]ethanols have been synthesised by decarboxyl- ation of (2S,3R)-[3-2Hl]serine and (2S,3S)-[2,3-2H2]serine respectively.

2. The stereochemical integrity of these labelled 2-aminoethanols has been ascertained from the 'H-NMR spectra of their N,O-dicamphanoyl derivatives.

3. This assay has also been used to confirm that samples of (2%)- and (2S)-2-amin0[2-~H~]ethanols prepared from (2R)- and (2S)-[2-'H l]glycines are stereochemically pure.

4. Ethanolamine ammonia-lyase rearranges ( I R)-Zamino[ 1 -'H ,]ethanol to acetaldehyde at approximately the same rate as it rearranges unlabelled 2-aminoethanol, whilst (1 S,2RS)-2-amino[l ,2-2H2]ethanol is rearranged at the same rate as the (l,l-2H2)-labelled substrate. The isotope effect is approximately kH/k2H = 8.

5. The 2H-NMR spectra of the 3,5-dinitrobenzoates of the ethanol produced by reduction in situ of the acetaldehyde formed in the rearrangements show that the 1-2H, label migrates in (IS,2RS)-2-amino- [1,2-2H2]ethanol and 2-amin0[l,l-~H~]ethanol but not in (lR)-2-amin0[1-~H~]ethanol.

6. The above results indicate that the adenosylcobalamin-dependent ethanolamine ammonia-lyase catalyses the rearrangement of 2-aminoethanol with migration of the 1 -pro-S-hydrogen atom.

A large number of rearrangements catalysed by coenzyme- B, (AdoCbl) may be summarised briefly by the reaction (1)$(2) (Scheme 1). Studies of the mechanism of these rearrangements [l ,2] have proved stimulating and fruitful and have been aided by determination of the stereochemical course of the reactions [3, 41. The results of these stereochemi- cal studies have shown that the reactions may take a variety of courses, rearrangement being accompanied by retention and inversion and by racemisation [3,4]. Ethanolamine ammonia-lyase is an AdoCbl-requiring enzyme which normally catalyses the rearrangement of 2-aminoethanol (3) to the carbinolamine (4). This then deaminates to yield acetaldehyde (5 ) (Scheme 1). The enzyme can also use both stereoisomers of 2-aminopropanol as substrates and Diziol eta]. [5] recently made the fascinating discovery that, while the enzyme catalyses the rearrangement of (2S)-2-aminopropanol with retention of stereochemistry, the (2R)-isomer is rearranged with inversion of stereochemistry. Thus the same enzyme catalyses the rearrangement of two related substrates, the two reactions having entirely different stereochemical courses.

The stereochemical course of the rearrangement of the natural substrate for ethanolamine ammonia-lyase, 2-amino-

This paper is dedicated to the memory of Professor A. W. Johnson, deceased 5th December, 1982.

Abbreviations. AdoCbl, adenosylcobalarnin [see Eur. J . Biochem. 45, 7-12 (1974)]; NMR, nuclear magnetic resonance (s, singlet; d, doublet; t, triplet; br, broad) ; sh, shoulder ; Me&, tetramethylsi- lane ; TLC, thin-layer chromatography.

Enzymes. Ethanolamine ammonia-lyase (EC 4.3.1.7) ; alanine amhotransferase or glutamic-pyruvic transaminase (EC 2.6.1.2) ; alcohol dehydrogenase (EC 1.1.1.1).

(3)

Scheme 1

ethanol (3), is of interest in view of a report [6] that the centre C-2, which receives the migrating hydrogen, became racemised in the process. The steric course of this rearrange- ment at the migrating centre C-1 was, however, unknown, although work using nonchirally labelled substrates had suggested [7] that the enzyme was capable of distinguishing the two hydrogen atoms at C-1. This paper reports our findings on the steric course of the rearrangement at the migrating centre C-1 . Some of these results have already been communicated in preliminary form [8].

EXPERIMENTAL PROCEDURES

Analytical and spectroscopic measurements

Melting points were determined on a Kofler hot-stage apparatus. Infrared spectra were recorded on Perkin-Elmer 257,457 and 477 instruments. 'H-NMR spectra were recorded on Perkin-Elmer R12 (60 MHz) and R32 (90 MHz)

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instruments. High-resolution ’H-NMR (360 MHz) and ’H- NMR (55.28 MHz) spectra were obtained by Dr I. H. Sadler and his colleagues (University of Edinburgh) using a Bruker WH360 instrument. Specific rotations were recorded using a Perkin-Elmer PE241 polarimeter and a 1-dm path-length cell. Optical spectroscopic measurements were made using a Cary 14 spectrophotometer. Mass spectra were obtained using a Kratos MS25 instrument. Microanalyses were performed by Mrs G. Olney.

Materials Ethanolamine ammonia-lyase was prepared and assayed

as outlined below. [2,2-’H2]Glycine was prepared by a modification of the synthesis of Darapsky and Hillers [9] in which cyanoacetazide was exchanged with Na02H/2H20 and the Curtius rearrangement was conducted in CH3CH20’H. (2S,3R)-[3-’H1]Serine and (2S,3S)-[2,3-2Hz]serine were prepared by the method of Gani and Young [lo]. (-)Camphanoyl chloride was prepared from (-)camphanic acid supplied by both the Aldrich Chemical Co. Ltd (Gillingham, Dorset, UK) and Fluka (Fluorochem Ltd, Glossop, Derbyshire, UK) using the method of Gerlach [I 11. N-Phthaloyl-2-aminoethanol [12] and N-acetyl-2-aminoetha- no1 [I31 were prepared by methods devised by Wenker. Samples of glutamic-pyruvic transaminase were obtained both from Sigma London (Poole, Dorset, UK) and Boehringer-Mannheim (Lewes, Sussex, UK). Yeast alcohol dehydrogenase, NADH, pyridoxal5’-phosphate, L-serine, 3 3 - dinitrobenzoyl chloride and 35% ’HC1 were obtained from Sigma London. AdoCbl was obtained both from Sigma London and Glaxo Laboratories Ltd (Stoke Poges, Bucks, UK) and was purified and its concentration determined as described previously [14,15]. All solutions containing AdoCbl were kept dark and dispensed in a dim light. 2-Aminoethanol, Dowex 50W X8 (US mesh 20-50, H’ form) and glycine were obtained from BDH Ltd (Poole, Dorset, UK). 99.8% ’H20, LiAlH4 and thionyl chloride were supplied by the Aldrich Chemical Co. Ltd (Gillingham, Dorset, UK) and para-methoxyacetophenone by Koch Light Laboratories Ltd (Colnbrook, Bucks, UK). Merck GFz54 silica (type 60, Art 7730) was used for preparative TLC.

Synthesis of labelled 2-aminoethanols

(1R)-2-Arnino[l-’H,]ethanol and (IS,2RS)-2-amino- [ 1 ,2-’H,]ethanols were prepared by decarboxylation of the corresponding labelled samples of serine ; 2-amino[l,l -’H2]- ethanol by reduction of methyl or ethyl glycinate with LiA1’H4 ; and 2-amino[2,2-’H2]ethanol, (29-2-amino- [2-2H ‘]ethanol and (2R)-2-amin0[2-~H~]ethanol by reduction of the labelled methyl glycinates with LiAlH4 as follows.

2-Aminoethanol (3) from L-serine (6). L-Serine (60 mg, 0.57 mmol) and para-methoxyacetophenone (120 mg, 0.8 mmol) were ground together in a mortar and the mixture was heated from 160 to 190°C in a stream of nitrogen. The effluent was passed through aqueous barium hydroxide which became cloudy when the temperature of the mixture reached 190°C. After about an hour, no further C 0 2 was evolved and the reaction was cooled and heated to reflux with 12 M hydrochloric acid ( 5 ml) for 2 h. Water ( 5 ml) was added and the solution was washed well with chloroform ( 5 x 20 ml). The aqueous solution was boiled with animal charcoal

(x 50 mg) for several minutes, filtered and lyophilised to yield a solid (52 mg, 93%), mp 76-78°C (lit. [16] mp 75-77°C). (Found C, 24.0%; H, 8.7%; N, 14.2%; C2H8C1N0 requires C, 24.6% ; H, 8.2% ; N, 14.4%.) 6 (10% 2HC1-2Hz0) 2.91 ppm (2H, t , J = 6Hz, CH’N), and 3.60 ppm (2H, t, J = 6 Hz, CH20).

( I R)-2-Amin0[l-~H~]ethanol hydrochloride (3, I-HR = ’H). This was prepared from (2S,3R)-[3-’Hl]serine (6, HB = ’H) as described above. The product, mp 73 - 76 “C, had similar spectra to the undeuteriated compound except that the ‘H-NMR spectrum showed 6 (10% ’HCI-’H20) 2.94 ppm (2H, d, J = 7 Hz, CHzNH3f) and 3.60 ppm (IH, br t, J = 7 Hz, CH-OH).

( I S,2 RS)-2-Amino[l,2-2Hz]ethanol hydrochloride (3, I - Hs=’H, 50% of one 2-H=’H). This was prepared from (2S,3S)-[2,3-2H2]serine (6, HA = ’H) as described above. The product, mp 72-75°C had similar spectra to the unlabelled material except that the ‘H-NMR spectrum showed 6 (10% 2HC1-2Hz0) 2.97 ppm ( lH, br m, CHNH:) and 3.66 ppm (IH, br m, CHOH).

(2R)-[2-’H1]Glycine (11, HR = ’H). Glycine (50 mg, 0.667 mmol) was dissolved in 2Hz0 (5 ml) and the solution was freeze-dried. The residue was dissolved in ’H20 (1 ml) with K2HP04 (15 mg, 0.086 mmol) which had similarly been exchanged in ’H20. The pH was adjusted to 7.1 with 35% 2HC1 in ’H20 and pyridoxal 5’-phosphate (3 mg) and glutamic-pyruvic transaminase (90 units) were added. The solution was left in the dark at 37 “C for 90 h and the enzyme was denatured by boiling for several minutes. The mixture was centrifuged and the supernatant solution was lyophilised to yield (2R)-[2-’H l]glycine and buffer (69 mg). The glycine was characterised as its camphanic acid derivative below.

using [2,2-2H2]glycine and H 2 0 in the method above. It was characterised as its camphanic acid derivative below.

N-Cumphanoylgfycine (12). Glycine (1 50 mg, 2 mmol) was dissolved in 1.2 M aqueous sodium hydroxide (2 ml) and this solution was shaken with a solution of (-)camphanoyl chloride (435 mg, 2.01 mmol) in toluene (2 ml) at room temperature for 2 h. The aqueous phase was washed with chloroform (2 x 10 ml), acidified to pH FZ 2 with 6 M aqueous HCl and extracted with chloroform (3 x 10 ml). The extracts were dried (Na2S04) and the solvent was removed in vacuo to yield an oil which crystallised on scratching with water,mp67-7O0C(lit. [17]mp73.5-75”C),m/z255(Mt), 6 (C2HC13) for H-2 of glycine 4.14ppm (2H, ABX,

N-Camphanoyl-(2 S)-(2-’Hl]glycine (12, HR= ’H).This was prepared from (2R)-[2-ZH,]glycine (11, HR = ’H) using the method described above for the unlabelled com- pound. Spectra were similar to those of the unlabelled compound but the mass spectral parent ion was one mass number higher, mjz 256. The ‘H-NMR spectrum (C2HC13) showed mainly one proton 6 = 4.16ppm (d, J = 5.1 Hz) for the glycine moiety although there was evidently unlabelled compound present. The ’H-NMR spectrum (CHClJ had one absorption at 6 = 3.99 pprn (s) as shown in Fig. 2 b. Thus although incorporation was incomplete, all deuterium had been incorporated stereospecifically.

N-Camphanoyl-(2 S)-[2-2Hl]glycine (12, Hs = 2H) .This was prepared from (2S)-[2-’H1)glycine (11, Hs = ’H) using the method described above for the unlabelled compound. Spectra were similar to those of the unlabelled compound but the mass spectral parent ion was one mass number higher, mlz256. The ‘H-NMR spectrum (C’HCl3) had only one

(2S)-(2-2H1]Glycine (11, Hs = ‘H). This was prepared

J A B = 18.5H2, JAx = Jsx = 5.2 Hz).

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proton for the glycine moiety, 6 = 4.05 pprn ( lH, d, J = 5.1 Hz) and the 'H-NMR spectrum (CHCl,, Fig. 2c) had one absorption at 6 = 4.16 ppm (s).

N-Carnphan0yl-[2,2-~H~]glycine (12, Hu = Hs = 2H). This was prepared from [2,2-2H2]glycine (11, HR = Hs = 'H) using the method described above for the unlabelled compound. The parent ion, m/z 257, was two mass numbers higher than in the unlabelled compound, there was no absorption corresponding to the glycine moiety in the 'H-NMR spectrum (C2HC13) and the 'H-NMR spectrum (CHC13, Fig. 2 a) had singlets at 6 = 3.98 ppm and 4.12 ppm.

Merhyl glycinate hydrochloride. Thionyl chloride (2 ml, 27.9 mmol) was added dropwise to dry methanol at - 15 "C over 5 min with stirring. Glycine ( 2 g, 26.7 mmol) was added and, after stirring for a further 5 min at - 15 "C, the solution was heated to reflux for 10 min. The reaction was cooled to 0°C and dry diethyl ether was added to precipitate the product, which was filtered and washed with cold dry diethyl ether (3 x 15 ml), (2.65 g, SO%), mp 170- 172 "C (lit. [IS] mp 175 "C); 6 ('H20, external Me,Si) 3.84 ppm (3H, s, OCH3) and 3.99 ppm (2H, s, CH2).

Methyl [2,2-'H2]g1ycinate hydrochloride. This was prepared from [2,2-2H2]glycine in the same way as the unlabelled compound above; mp 174- 176 "C (dec); 6 (2H20, external Me,Si) 3.62 ppm (s, OCH3).

Methyl ( 2 R)-[2-'Hl]glycinate hydrochloride. This was prepared from (2R)-[2-'Hl]glycine in the same way as the unlabelled compound above ; mp 174- 175 "C (dec) ; 6 ('H20, external Me4Si) 3.76ppm (3H, s, OCH3) and 3.90 ppm (lH, s, CH).

Methyl ( 2 S)-[2-'H1]glycinate hydrochloride. This was prepared from (2S)-[2-'Hl]glycine in the same way as the unlabelled compound above ; mp 169 - 172 "C (dec) ; 6 (2H20, external Me,Si) 3.74 ppm(3H, s, OCH3) and 3.88 ppm (lH, s, CH).

2-Aminoethanol ( 3 ) from methyl glycinate hydrochloride. Methyl glycinate hydrochloride (4.52 g, 36 mmol) was added slowly over 5 min to a stirred suspension of LiAlH4 (2 g, 52 mmol) in dry tetrahydrofuran (50 ml). The reaction was heated to reflux for 2 h and cooled. A saturated aqueous solution of sodium sulphate was added carefully until efferves- cence ceased. The solids were separated and extracted with tetrahydrofuran (300 ml) in a soxhlet for 18 h. Removal of the solvent in vacuo gave a viscous oil which distilled at 66-75 "C and 10 mm Hg (1.68 g, 76%). Spectra were ident- ical with those of an authentic sample.

2-Arnin0[l,l-~H~]ethanol (3 , I-HR = I-Hs = ' H ) . This was prepared in 70% yield by substituting LiA12H4 for LiAlH, in the above method, 6 (C2HC13, 'H20) 2.54 pprn (s, CHIN).

2-Amin0[2,2-~H~]ethanol hydrochloride (3 , 2-Hu = 2-Hs

hydrochloride using the reduction conditions described above for preparation of the unlabelled compound. After addition of the aqueous sodium sulphate, the precipitate was extracted thoroughly with tetrahydrofuran/methanol (9 : 1) and the extracts were acidified to pH = 2 with 12 M aqueous hydro- chloric acid. Removal of the solvent gave the product in 55% yield, 6 ('H'O) 3.63 ppm (br d, J = 1.5 Hz).

(2 R)-2-Arnin0[2-~H~]ethanol hydrochloride (3 , 2-& = ' H ) . This was prepared from methyl (2R)-[2-2Hl]glycinate hydrochloride using the method described above for the (2,2-ZH2)-labell~d compound; 6 ('H20) 2.96 pprn (lH, br t, J = 7Hz, CHNH,) and 3.63 ppm (2H, d, J = 7Hz,

= 2 H ) . This was prepared from methyl [2,2-'H2]glycinate

CH20H).

( 2 S)-2-Amin0[2-~H~]ethanol hydrochloride (3 , 2-HS = 2H). This was prepared from methyl (2S)-[2-2Hl]gly- cinate hydrochloride using the method described above for the (2,2-2y2)-labelled compound, S ('H20) 3.02 ppm (lH, br m, CHNH3) and 3.65 ppm (2H, br m, CH20H).

Assay for the stereochemical purity of labelled 2-aminoethanols

After synthesis of N-acetyl-0-camphanoyl-2-amino- ethanol and N-phthaloyl-U-camphanoyl-2-aminoethanol, it was found that when N,O-dicamphanoyl-2-aminoethanol was prepared, the 360-MHz 'H-NMR spectrum of this derivative allowed the stereochemical purity of the samples of chirally labelled 2-aminoethanols to be assessed.

N - Aceryl- 0 - camphanoyl- 2 - aminoethanol. N-Acetyl-2- aminoethanol(ll0 mg, 1 mmol) and (-)camphanoyl chloride (195 mg, 0.9 mmol) were stirred in dry diethyl ether (3 ml) in an atmosphere of nitrogen. The mixture was cooled in an ice/salt bath and dry pyridine (0.1 ml, 1.3 mmol) in dry diethyl ether (2 ml) was added slowly. After addition, the mixture was stirred at room temperature for 2 h and the ethereal solution was decanted from the precipitated pyridinium hydrochloride. Diethyl ether (10 ml) was added and the solution was washed with 0.1 M aqueous hydro- chloric acid and water and dried (Na2S04). The solvent was removed in vacuo to yield a gum which crystallised from ethyl acetate/light petrol (bp 60 - 80 "C) as needles (1 55 mg, 61 YO), mp 108-llO"c, [a]D-12.5" (c 2.154, CHC13). (Found c , 59.4%; H, 7.2%; N, 5.0%; C,,H,,NO, requires C, 59.4%; H, 7.4%; N, 4.95%.) m/z 283 (M') ; v,,, (KBr) 3258 cm-' (NH), 1777 cm-' (lactone), 1724 cm-' (ester) and 1638 cm-' (amide); 6 (C2HC13) 0.92 ppm, 1.07 ppm and 1.09 ppm (3 x 3H, s, C-Me), 1.5-2.7 ppm (7H, m), 3.57 ppm (2H, m; becomes t, J = 6 Hz on addition of 'H2O, CH2N), 4.32 ppm (2H, t, J = 6 H z , CH20) and 6.4ppm (lH, br, s, NH, exchanges in 2Hz0). Addition of tris(dipivaloy1methanato) europium caused the CHzO and CH2N protons to broaden and move downfield, but not to become further resolved.

N-Phthaloyl-0-camphanoyl-2-aminoethanol. N-Phthaloyl- 2-aminoethanol (145 mg, 0.76 mmol) was stirred in dry diethyl ether (5 ml) in an ice bath and dry pyridine (3 ml) and (-)camphanoyl chloride (I 65 mg, 0.76 mmol) were added. The mixture was stirred at room temperature for 90 min and was then allowed to stand at room temperature for a further 30 h. Ice was added followed by 1 M aqueous potassium hydroxide to bring the pH to about 8. The solution was extracted with chloroform (3 x 50 ml) and the extracts were washed with water (2 x 10 ml). The solvent was removed in vacuo to yield a solid which was dried azeotropically with chloroform/ethanol (10: 1, 3 x 5 ml). The residue was recrystallised from chloroform/light petroleum (bp 40-60 "C) to yield 183 mg (65%), mp 124- 125 "C. A sample was further purified by preparative TLC (silica; CHC13/ light petroleum, 5 : 1) and further recrystallisation from chloroform/light petroleum (bp 60-80 "C), mp 124- 125 "C,

N, 3.8%; C20HzlN06 requires C, 64.7%; H, 5.7%; N, 3.8%.) m/z 371 (M' ) ; v,,, (nujol) 1785 cm-' (lactone), 1730 cm'-' (sh, ester), 1710 cm-' (imide); 6 (C2HC13) 0.897 ppm, 0.976 ppm and 1.063 ppm (3 x 3H, s, C-Me); 1.65 ppm (fH, m), 1.86 ppm (1H, m), 2.02 ppm (lH, m) and 2.36 ppm (lH, m) (all camphanoyl C-Hs), 4.02 pprn (2H, m, CH2N), 4.48 ppm (2H, t, J = 5 Hz, CH20), 7.72 ppm and 7.85 ppm (4H, 2 x m, aromatics). Addition of tris(6,6,

[ a ] ~ -22.7' (C 13.21, CHC13). (Found C 64.7% ; H, 5.8% ;

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7,7,8,8,8-heptafluoro-2,2-dimethyloctane-3,5-dionato) euro- pium caused resolution of the triplet at 6 = 4.48 ppm.

N,O-Dicamphanoyl-2-aminoethanol (10). 2-Aminoetha- no1 hydrochloride (18 mg, 0.183 mmol) was dissolved in dry pyridine (0.8 ml). (-)Camphanoyl chloride (200 mg, 0.923 mmol) was added, followed by dry diethyl ether (2 ml). The solution was stirred at room temperature for 2 days, water (2ml) was added and the solvents were removed in vucuo. The residue was dissolved in chloroform (5 ml) and washed successively with 1 M aqueous hydrochloric acid (1 ml), saturated aqueous sodium bicarbonate (2 x 2 ml) and water (1 ml). The solution was dried (Na2S04), filtered through a small pad of chromatographic silica and the solvent was removed to yield a solid (28 mg, 36%), mp 121 "C, [a];' -26.6" (c 1.453, CHC13). (Found C, 62.5%; H, 8.0%; N, 3.4%; CZ2N3'NO7 requires C, 62.7%; H, 7.4%; N, 3.3%.) mjz 421.2102 (C22H31N07 requires 421.2100); vmax (nujol) 3390 cm-' (NH), 1790 cm-' (lactone), 1755 cm-' (br, ester) and 1678 cm-' (amide). The 'H-NMR spectrum of the 2-aminoethanol moiety is shown in Fig. 1 a.

N,O-Dicamphanoyl-2-amino[ 1 ,I -'HJethanol (10, I-HR = I -H, = ' H ) . This was prepared from 2-amino[l,l- 2Hz]ethanol using the method described above for the unlabelled compound. The mass spectral parent, m/z 423, showed about 100% dideuteriation and the 'H-NMR spectrum of the 2-aminoethanol moiety is shown in Fig. 1 b.

N,O-Dicamphanoyl-2-amin0[2,2-~H~]ethanol (10, 2-H, = 2-Hs = 'H). This was prepared from 2-amino[2,2- 2H2]ethanol using the method described above for the unlabelled compound. The mass spectral parent ion region is outlined in Table 2 and the 'H-NMR spectrum of the 2- aminoethanol moiety is shown in Fig. 1 c.

N,O-Dicamphanoyl- ( I R)-d-amino[ 1-'Hl]ethanol (10, 1-HR = ' H ) . This was prepared from (lR)-2-amin0[l-~H~]- ethanol using the method described above for the unlabelled compound. The mass spectral parent ion, m/z 422, showed about 92% 'HI and the 'H-NMR spectrum of the 2- aminoethanol moiety is shown in Fig. 1 d.

N,O-Dicamphanoyl- (1 S,2 RS) -2-aminoll ,2-'Hzjethanol. This was prepared from (1 S,2RS)-Zamino[ 1 ,2-'H2]ethanol using the method described above for the unlabelled com- pound. The mass spectral parent region, mjz 423,422 indicated about 58% 'H2, 42% 'HI and the 'H-NMR spectrum of the 2-aminoethanol moiety is shown in Fig. 1 e.

N,O-Dicamphanoyl- (2 R)-2-amino[2-'Hl]ethanol (10, 2- Hp, = ' H ) . This was prepared from (2R)-2-amino[2-'H1]- ethanol using the method described above for the unlabelled compound. The mass spectral parent, m/z 422, 421 indicated incorporation of about 62% 2H1 and the 'H-NMR spectrum of the 2-aminoethanol moiety is shown in Fig. 1 f.

N,O-Dicamphanoyl-(2 S)-2-amino[2-'H1]ethanot (10, 2-Hs = ' H i . This was prepared from (2S)-2-amin0[2-~H~]- ethanol using the method described above for the unlabelled compound. The mass spectral parent region mlz 423, 422 indicated about 14% 'H2, 86% 'H1 and the 'H-NMR spectrum of the 2-aminoethanol moiety is shown in Fig. 1 g.

Ethanolamine ammonia-lyase

This was purified from cultures of Clostridium sp. and bound cobamides were removed as previously described [19]. 2-Aminoethanol was removed before use by dialysis against 10 mM potassium phosphate buffer, pH 7.4 and the same buffer was present in all enzyme incubations. Enzyme activity

was measured using the spectrophotometric method of Kaplan and Stadtman [20] with 10 p M AdoCbl in each assay. Enzyme concentration was determined using the method of Lowry [21] and by measuring absorbance at 280 nm to obtain protein concentrations. The results were corrected using factors from dry weight determinations [22]. The concentra- tion of active sites was calculated using a molecular weight of 520 000 [23], six active sites per molecule [22] and correcting for 'blocked' active sites by multiplying by the specific activity of the preparation and dividing by the specific activity of the most active preparation obtained (84 U/mg). All enzyme concentrations reported are active-site concentrations.

Kinetics of the AdoCbl-catalysed rearrangement

The substrates were dissolved in distilled water and the pH of the solutions was adjusted to 7.4 with HCl or KOH. White precipitates which formed with (1 R)-2-amino- [ l-'Hl]ethanol and (1 S,2RS)-2-amino[l ,2-'H2]ethanol were removed by centrifugation. Potassium phosphate buffer, pH 7.4 was added to the solutions to give a final concentration of 10 mM. Further white precipitates were removed by centrifugation. The final concentration of substrate solutions was checked using a spectrophotometric assay similar to that described by Kaplan and Stadtman [20] for the assay of enzyme activity, acetaldehyde production being coupled to oxidation of NADH in the presence of yeast alcohol dehydrogenase. The complete assay system (1 ml) contained potassium phosphate buffer pH 7.4, 50 mM ; NADH, 0.1 mM; yeast alcohol dehydrogenase, 10 U ml- ' ; ethanol- amine ammonia-lyase, 60 nM ; substrate 2-aminoethanol, 30 - 70 p M ; and AdoCbl, 10 p M. The reaction was started by addition of AdoCbl and the absorbance at 340 nm was measured until no further change took place. The initial concentration of substrate was calculated from the total decrease in absorbance at 340 nm, corrected for the change caused by addition of AdoCbl, using &340 = 6.2 x lo3 M- ' cm-' for NADH. Concentrations of the product acetaldehyde formed in the enzyme reactions were determined in the same way from measurements of the decrease in absorbance at 340 nm in the presence of excess NADH and yeast alcohol dehydrogenase.

Time courses for the deamination of 2-aminoethanol and its deuterated isomers were obtained using the above spectrophotometric assay. Initial concentrations in the reac- tion mixture (1 ml) were potassium phosphate buffer pH 7.4, 10 mM ; NADH, 0.1 mM ; yeast alcohol dehydrogenase, IOU ml-'; substrate, 17-6Op M and AdoCbl, l o p M. Ethanolamine ammonia-lyase was used in 5.3 nM or 22 nM concentrations in the reactions with 2-aminoethanol and (1R)-2-amino[l-2Hl]ethan~l and in 32 nM or 110 nM concen- trations in the reactions with (lS,2RS)-2-amin0[1,2-~H~]etha- no1 and 2-amino[l,l-'Hz]ethanol. The reactions were started by addition of AdoCbl, the temperature was 24°C and the path length in the cuvette was 1 cm. Measurements of absorbance commenced shortly after mixing (normally 10 - 20 s) and the substrate concentration ([So]) at this time (to) was calculated from the total absorbance change during the subsequent reaction. The low K, with these substrates necessitated the use of the integrated form of the Michaelis- Menten equation described by Hollaway et al. [24] to determine values for apparent K, and for k,,,. An example of data plotted from one time course with each substrate is shown in Fig. 3. Average values of these parameters obtained

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by analysis of eight time courses for each substrate are shown in Table 1.

Isolation of the 3,s-dinitrobenzoates (13) from the coupled lyase/dehydrogenase reactions

Three reactions were conducted using potassium phosphate buffer, pH 7.4, 10 mM; yeast alcohol dehydrogenase, 0.36 mg ml-’ (300 U mg-l) and other com- ponents as follows: (a) 2-amino[l,l-’Hz]ethanol, 6.8 mM ; NADH lyophilised to remove ethanol, 10 mM ; and ethanol- amine ammonia-lyase, 2.5 p M in a total volume of 25 ml ; (b) (1S,2RS)-2-amino[l,2-’H2]ethanol, 1.4 mM ; NADH lyophilised to remove ethanol, 2 mM ; and ethanolamine ammonia-lyase, 3.1 p M in a total volume of 20 ml; and (c) (1 R)-2-amino[l-’H1]ethanol, 6.2 mM ; NADH lyophilised to remove ethanol, 10 mM ; ethanolamine ammonia-lyase, 0.5 p M in a total volume of 25 ml. The reaction mixtures were warmed to 24 “C, and the reaction was started by adding AdoCbl to a final concentration of 24 p M. Samples taken at intervals to monitor the absorbance at 340 nm showed the reactions to be complete within 5.5 min in each case. The reactions were terminated after 8.5 min for (a) and (c) above and after 10 min for (b) above by addition of 10% HCl to a final concentration of 0.1 M. Dowex 50WX8 ( 5 ml, wet) was added and after several minutes was removed by centrifuga- tion. Solid NaOH (4g) was added slowly with cooling followed by solid sodium acetate (1 8). 3,5-Dinitrobenzoyl chloride (2 g) in benzene (20 ml) was added and the mixture was shaken vigorously for 2 min. Water (50 ml) was added and the mixture was extracted with diethyl ether (2 x 50 ml). The extracts were dried and the solvent was removed in vacuo to yield a pale yellow solid which was purified by preparative TLC (silica gel, CHC13). The fraction of RF 0.6 proved to be the desired ester (13).

The sample from 2-amino[l ,l-2Hz]ethanol had ions, m/z 242 and 240, showing that about 55% of the product was derived from the enzymic reaction. The unlabelled material was derived from ethanol in the NADH used. The ’H-NMR spectrum is shown in Fig. 4a. The sample from (1S,2RS)- 2-amino[1,2-’H2]ethanol had ions, m/z 242, 241 and 240, indicating that about 52% of the product was derived from the enzymic reaction and this had about 50% ’H2,45% ’HI. The ’H-NMR spectrum is shown in Fig. 4c. The sample from (1R)-Zamino[l-’Hl]ethan~l had ions, m/z 241 and 240, showing that about 52% of the product was derived from the enzymic reaction. The ’H-NMR spectrum is shown in Fig. 4 b.

2-Arnino[2,2-’H2]ethanol (8 mg) was also treated as described above to yield a 3,5-dinitrobenzoate, 2H-NMR (CHC13 ref. to C2HC13 at 7.25 ppm) 6 = 1,45 ppm (s, Me). The mass spectral data are shown in Table 2.

RESULTS AND DISCUSSION To assess the absolute stereochemistry at C-1 in the

rearrangement of 2-aminoethanol by the AdoCbl-mediated enzyme ethanolamine ammonia-lyase, it was first necessary to synthesise samples of 2-aminoethanol(3) stereospecifically labelled at C-1 with deuterium or tritium. The availability of NMR techniques to assess the position and stereochemistry of the label in the substrates and to follow the fate of the label in the enzymic rearrangement made deuterium the obvious choice. Since we had synthesised (2S,3R)-[3-2Hl]-

Me-Me Me-Me Me-Me

(9)

Scheme 2

serine (6, HB = ’H) and (2S.3S)-[2,3-2Hz]serine (6, HA = ’H) [lo], conversion of these to samples of labelled 2- aminoethanol by decarboxylation seemed an attractive and straightforward method of obtaining these compounds.

L-Serine (6) was therefore heated with para-methoxy- acetophenone at 160 - 190 “C in an atmosphere of nitrogen to effect decarboxylation. The resultant product was hydrolysed by heating to reflux with 12 M hydrochloric acid to yield 2-aminoethanol hydrochloride in 93% yield. When (2S,3R)-[3-’Hl]serine and (2S,3S)-[2,3-’Hz]serine were treated in this way, samples of 2-aminoethanol were obtained which were evidently deuteriated. Since there was a real possibility that the centre C-3 of serine (C-1 of 2-aminoetha- nol) might have been racemised in the reaction, it was necessary to develop an assay for the stereochemical purity of these compounds.

The enantiotopic hydrogens HA and HB in some alcohols (7) and amines (8) have been differentiated by converting them to esters [25] or amides [17] of (-)(1 S,4R)-w-camphanic acid (9). These now diastereotopic hydrogens HA and HB can have quite different chemical shifts when the ‘H-NMR spectrum is run in the presence of a lanthanide shift reagent. We therefore prepared the N-acetyl-0-camphanoyl and N-phthaloyl-0-camphanoyl derivatives of 2-aminoethanol. The ‘H-NMR spectrum of the N-acetyl compound did not show the expected resolution of the 1-pro-S and 1-pro-R hydrogens of the 2-aminoethanol moiety, even in the presence of shift reagents. These hydrogens were resolved in the ‘H- NMR spectrum of the N-phthaloyl compound in the presence of tris(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyloctane-3,5-dio- nato) europium but spectral broadening was a problem. When, N,O-dicamphanoyl-2-aminoethanol (10) was pre- pared, however, all four stereoheterotopic hydrogens 1-Hs, 1-HR, 2-Hs and 2-HR appeared at different chemical shift values in the 360-MHz ‘H-NMR spectrum (Fig. 1 a). This spectrum was complex but when 2-amino[l ,l-ZHz]ethanol and 2-amino[2,2-’H2]ethanol were prepared by reduction of ethyl or methyl glycinate hydrochloride with LiA12H4 and by reduction of methyl [2,2-’Hz]glycinate hydrochloride with LiA1H4 respectively, then the ‘H-NMR spectra of the N,O- dicamphanic acid derivatives (Fig. 1 b and 1 c) showed the protons on C-1 and C-2 of the 2-aminoethanol moiety as separate AB systems. There was coupling between the protons H-2 and the amido NH.

The N,O-dicamphanoyl-2-aminoethanols (10) derived from the samples of 2-aminoethanol prepared by decarboxyl- ation of (2S,3R)-[3-’Hl]serine (6, HB = ’H) and (2S,3S)-

308

/ I .-

1.4 1.2 1.0 3.8 3.6 6(ppml

Fig. 1. 360-MHz ‘H-NMR spectra (C’HC13) of N,O-dicamphanoyl- 2-aminoethanols (10). (a) Unlabelled ; (b) (l,l-’H2)-labelled ; (c) (2,2- ’HZ)-labelled ; (d) (1 R)-(1-’HI)-labelled ; (e) (1 S,2RS)-(1 ,2-’H2)- labelled ; (f) (2R)-(2-’H l)-labelled ; (8) (2-S)-(2-’H I)-labelled. The spectrum (a) has been computer line-narrowed, and the spectrum (e) has had the spectrum of a small amount of ethyl camphanoate removed by computer subtraction

[2,3-’H2]serine (6, HA = ’H) had ‘H-NMR spectra (Fig. 1 d and l e respectively) which showed that the labelling was stereospecific. The decarboxylation of serine had, therefore, not been accompanied by racemisation of the centre C-3 (C-1 of 2-aminoethanol). The product of decarboxylation of (2S,3R)-[3-’Hl]serine (6, HB = ’H) must therefore be (1R)- 2-amino[l-’Hl]ethano1(3, 1-HR = ’H) and the product from decarboxylation of (2S,3S)-[2,3-’H2]serine (6, HA = ’H) must be (1S,2RS)-2-amino[l,2-’Hz]ethanol (3, 1-Hs = ’H, 50% of one 2H). The mass spectra of the N,O-dicamphanoyl- 2-aminoethanols indicated that whilst deuterium had been retained in the (1 R)-(I -’H ,)-labelled compound, about 50% of one deuterium had been lost in the decarboxylation leading to the (lS,2RS)-(l ,2-’H,)-labelled compound. The ‘H-NMR spectrum (Fig. 1 e) indicated that this loss was entirely at C- 2 and that the deuterium in the 1-pro-S position was intact.

Since all four stereoheterotopic hydrogens of 2-amino- ethanol could be distinguished by our assay, it seemed appropriate to check the optical purity in the synthesis of C-2 chirally labelled 2-aminoethanols used by Rttey et al. [6] in the work which deduced that the centre C-2 of 2-aminoethanol was racemised in the rearrangement catalysed by ethanolamine ammonia-lyase. The 2-pro-R-specific [26] L-alanine aminotransferase (glutamic-pyruvic transaminase) was used to prepare (2R)-[2-’Hl]glycine (1 1, HR.’= ’H) from glycine and ’HzO and (2S)-[2-’H1]glycine (1 1, HS = ’H) from [2,2-’Hz]glycine and HzO. In view of the warning

I I L.5 1.0 3.5

6(ppml Fig. 2. 55-MHz ’ H - N M R spectra (CHC13) of N-camphanoylglycines (12). (a) Unlabelled ; (b) (2R)-(2-2H1)-labelled ; (c) (259-(2-’H,)- labelled

Me. ,Me

(13)

sounded by Armarego et al. [17] on the stereochemical purity of [2-’H l]glycines prepared by enzymatic methods, we converted these compounds to their N-camphanoyl amides (12). The ‘H-NMR spectra and, more importantly, the ’H- NMR spectra (Fig. 2) of these compounds indicated that they were stereochemically pure. The labelled samples of glycine were separately converted to esters which were reduced with LiA1H4 to yield (2R)-2-amino[2-’H1]ethanol (3, 2-H, = ’H) and (2S)-2-amino[2-’H1]ethanol (3, 2-Hs = ’H). The ‘H- NMR spectra of the N,O-dicamphanic acid derivatives (10) of these compounds (Fig. 1 f and 1 g) showed them to be stereochemically pure.

Having prepared samples of 2-aminoethanol stereospeci- fically deuteriated in all four C-H bonds, we were now in a position to address the question of the stereochemistry of the AdoCbl-catalysed rearrangement of this the normal substrate with ethanolamine ammonia-lyase. 2-Aminoethanol (3), 2-amino[l,l-’H2]ethanol (3, ~ - H R = 1-Hs = ’H), (1R)- 2-amino[l-’H1]ethano1 (3, ~ - H R = ’H), and (lS,2RS)-2- amin0[1,2-~H~]ethanol (3, l-Hs= ’H, 50% of one 2-H = ’H) were therefore incubated separately at 24 “C with ethanol-

309

0 0 50 100 50 100

t ~ l o g l ~ ~ ~ S o l ~ " S , - ~ P , 1 ) ) Is1

Fig. 3. Plots of data from a time course for the reaction of ethanolamine ammonia-lyase with 2-aminoethanols. (a) Unlabelled (0) ; (l,l-'Hz)- labelled (0 ) ; (c) (lR)-(l-ZH1)-labelled (0); (d) (1S,2RS)-(1,2-ZHz)- labelled (H). Ethanolamine ammonia lyase concentration was 5.3 nM for (a) and (c) and 32 nM for (b) and (d). Initial substrate concentra- tion was 30 p M in each case. t =Time after measurements commenced; [P,] = product molar concentration at time t ; [So] = substrate molar concentration at to

Table 1. Kinetic parameters for the rearrangement of 2-aminoethanols Results are the average of eight determinations for each substrate. Enzyme concentrations were 5.3 nM or 22 nM for a and b and 32 nM or 110 nM for c and d. Initial substrate concentrations varied over 17-60 p M

Substrate kc,, Apparent kc&)/ K n kcat

s- PM a. 2-Aminoethanol (3) 122 & 3 3.5 & 0.6 1 b. (1R)-2- 124 10 5.4 f 1.7 0.98

C. (1S,2RS)-2- 14.7 & 1.7 3.6 & 1.1 8.3

d. 2-Amino[1,1-2Hz]ethanol 15.8 1.4 3.6 1.3 7.7

Amino[l J H ,]ethanol

Amino[ 1 ,2-'H2]ethano1

amine ammonia-lyase in the presence of an excess of yeast alcohol dehydrogenase and NADH. The reactions were started by addition of AdoCbl and the rates were followed by measuring the change in absorbance at 340 nm due to the disappearance of NADH in the coupled reduction of the acetaldehyde ( 5 ) formed in the lyase reaction. One set of results is shown graphically in Fig. 3 and the kinetic parameters averaged from eight sets are summarised in Table 1 . Although the reactions used different concentrations of enzyme, Vfor the rearrangement has been shown [20] (and unpublished results) to be directly proportional to enzyme concentration between 5.3 nM and 22 nM for 2-amino- ethanol or 5.3 nM and 110 nM for 2-amin0[l,l-~H~]ethanol. The results show that the rate of the reaction with (1R)-2- amin~[l-~H~]ethanol (3, 1-HR = 'H) is comparable to that with the unlabelled substrate, whilst the rate of the reaction with (lS,2RS)-2-amin0[1,2-~H~]ethanol (3, I-Hs = 'H, 50% of one 2-H='H) is similar to that with the (1,1-'H2)- labelled derivative. The deuterium isotope effect, kH/kZH = 8, compares well with the value of 7.4 found by Weisblat

I , I

L.5 L.0 3.0 2.0 6lppml

Fig. 4. 55-MHz 'H-NMR spectra (CHC13) of the ethyl 3,5- dinitrobenzoates (13) derived ultimately from the rearrangement catalysed by ethanolamine ammonia-lyase of ( a ) 2-amino/l,l- 2Hz]ethanoZ; (b) (IR)-2-amin0[l-~H~]ethanol; and [c) (I S,2 RS)-2-amino[l ,2-'HZ]ethanol. The large reversed signal at about S = 3.2 ppm is the quadrature image of residual CZHCI,

and Babior [27]. Since only the compounds which contain deuterium in the I S position show this isotope effect, it must be the 1-pro-S hydrogen which migrates in the rearrangement of 2-aminoethanol by ethanolamine ammonia-lyase.

These findings were confirmed when the samples of ethanol produced in the coupled lyase/dehydrogenase reactions were isolated as their 3,Sdinitrobenzoates (1 3). Since the lyase and the AdoCbl were present in only catalytic amounts, dilution of the label by the 5'-hydrogens of the coenzyme was not anticipated. The commercial NADH used in these experiments was lyophilised to remove the ethanol originally present. This was not totally effective, however, so that the product ethanol was always contaminated with unlabelled material from the NADH. The 'H-NMR spectra (Fig. 4) of the 3,5-dinitrobenzoates (13) were therefore more reliable than the corresponding 'H-NMR spectra in assessing the course of the reaction. The 'H-NMR spectra of the 3,5-dinitrobenzoates (1 3) derived from the reactions of 2-amino[l ,l-2H2]ethanol (Fig. 4 a) and of (lS,2RS)-2-amino- [I ,2-2H2]ethanol (Fig. 4 c) showed evidence that deuterium had migrated to the methyl group (HA in 13). The spectrum of the 3,Sdinitrobenzoate (13) from the reaction of (1R)-2- amin~[l-~H~]ethanol (Fig. 4 b) showed that the deuterium had not migrated and remained on the methylene group (HB in 13). The rearrangement had therefore occurred with migration of the I -pro4 hydrogen.

There was a small additional deuterium signal in the spectrum (Fig. 4c) of the 3,5-dinitrobenzoate (13) derived from (IS,2RS)-2-amino[l ,2-2H2]ethanol. This occurred at the chemical shift for the methylene group (HB in 13). Since, when 2-amin0[2,2-~H,]ethanol (3, 2-HR = 2-Hs = 'H) was used in the coupled lyase/dehydrogenase reaction, all of the label remained on the methyl group (HA in 13), the most likely explanation for this unexpected signal was that it was due to a small amount of racemisation in the synthesis of (1 S,2RS)-2-amino[l ,2-'H2]ethanol. The 'H-NMR spectrum of the N.0-dicamphanoyl derivative of this compound (Fig. 1 e) indicated that if any (1RS)-labelled material were

310

Table 2. Mass spectra (electron impact) of derivatives of substrate and product in the coupled lyaseldehydrogenase reaction using 2- arnin0[2,2-~H~]ethanoI The substrate derivative was N,O-dicamphanoyl-2-amino[2,2- 2H2]ethanol (10) ; the product derivative was ['Hlethanol 33- dinitrobenzoafe (13)

Derivative Mass Relative Corrected Norma- of ion intensity for P + 1 lised for

'H2 = 100

Substrate (10) 421 (Hz) 422 (2H1) 423 ('HZ) 424 (2H3)

Product (13) 240 (H2)= 241 ('HI) 242 ('H2) 243 ('H3)

2.45 2.45 19.95 19.3 100 95.2 23.86 0 100 100 28.5 17.1 72.2 70.3 9.3 1.3

2.6 20.3 100

0 142.2 24.3 100 1.8

a Due to ethanol in the original NADH

Scheme 4

(20) Scheme 5

racemisation, without implying that the enzymic process itself is non-stereospecific. The reversibility of step (3) in Scheme 4 was supported by the fact that, when we examined the mass spectrum of the 3,5dinitrobenzoate (13) obtained from the coupled lyase/dehydrogenase reaction on 2-amino- [2,2-'H2]ethanol (Table 2), there seemed to be more monodeuteriated material than in the original 2-amino[2,2- 2Hz]ethanol and some trideuteriated material also seemed to be present. Small differences such as these found in mass spectra must, however, be treated with extreme caution, in view of isotope effects in mass spectral fragmentation. An attempt was made to obtain more convincing results by scaling up the amounts of enzyme and AdoCbl used in the

OH /

H \ C-CHO Hs\H

present then the amount must be very small. The isotope effect in the AdoCbl-catalysed rearrangement, however, would enhance the amount of deuterium at C-1 after the reaction so that a detectable 'H-NMR signal might be seen at the chemical shift of HA in the spectrum of the 3,5-dinitrobenzoate

Our results show that it is the 1-pro-S hydrogen which migrates in the rearrangement of 2-aminoethanol(3) catalysed by ethanolamine ammonia-lyase. RCtey et al. [6] have shown that the centre to which this hydrogen migrates, C-2, is racemised in the process. These results might be explained by considering the mechanism outlined in Scheme 4. Although it has been shown that step 1 where the C-1 hydrogen is removed is irreversible when 2-aminoethanol is substrate [28], when acetaldehyde was incubated with the enzyme in the presence of NH,f ions, tritium was transferred from C-5' of the coenzyme, AdoCbl (14), to the a-carbon of acetaldehyde [28]. This implies that steps (3) and (4) in Scheme4 are reversible and so the intermediate compound (19) allows for

(1 3).

:MoH H H H NH2

reaction with 2-amin0[2,2-~H~]ethanol. Although control experiments indicated that we could assess deuterium in AdoCbl by FAB (fast-atom-bombardment) mass spectrometry, we were unable to obtain sufficient quantities from the enzymic experiment to verify the results.

The (IS) stereospecificity of the lyase for 2-aminoethanol is in keeping with the finding of Diziol et al. [5] that the hydrogen 1-Hs migrates when both (2S)-2-aminopropanol (20) and (2R)-2-aminopropanol (21) are used as substrates. The migrating hydrogen becomes 2-Hs of the resultant propionaldehyde (22) with both substrates so that the stereochemistry of this centre is retained when (2S)-Zamino- propanol (20) is substrate whilst it is inverted when (2R)-2- aminopropanol (21) is substrate. There are differences in mechanism between the rearrangements of 2-aminoethanol and 2-aminopropanol since the whole reaction is irreversible for the former substrate [28] but reversible for the latter substrate [29]. The result of Diziol et al. implies that when 2-aminopropanol is substrate, the intermediate corresponding

311

to compound (18) of Scheme 4 can exist long enough for rotation about the C-I -C-2 bond to occur and that hydrogen addition is specific to but one face of this intermediate. This

2-aminoethanol.

11. Gerlach, H. (1968) Helv. Chim. Acta, 51, 1587-1593. 12. Wenker, H. (1937) J . Am. Chem. SOC. 59,422. 13. Wenker, H. (1935) J . Am. Chem. sot. 57, 1079-1080.

R. & White, H. A. (1975) FEBS Lett. 53, 193-198. explanation would also Serve for the racemisation at c-2 of 14. Joblin. K. N.. Johnson, A. W., Lappert, M. F., Hollaway, M.

15. Hollaway, M. R., White, H. A., Joblin, K. N., Johnson, A. W.,

We thank Dr I. H. Sadler (Edinburgh University) for 'H-NMR and 'H-NMR spectra ; Prof. H. R. Morris, Imperial College London for FAB mass spectra; and the Science and Engineering Research Council (U.K.) for financial assistance.

REFERENCES 1. Babior, B. M. & Krouwer, J. S. (1979) CRC Crit. Rev. Biochem.

2. Zagalak, B. (1982) Nuturwissenschaften, 69, 63 -74. 3. RCtey, J. & Robinson, J. A. (1982) Stereospecificity in Organic

ChemistryandEnzymology, pp 185-207 and 310-311, Verlag Chemie, Weinheim.

4. Retey, J. (1982) in Stereochemistry (Tamm, Ch., ed.) pp. 249 - 282, Elsevier Biomedical Press, Amsterdam.

5 . Diziol, P., Haas, H., Retey, J., Graves, S. W. & Babior, B. M. (1980) Eur. J . Biochem. 106, 21 1-224.

6. RCtey, J., Suckling, C. J., Arigoni, D. & Babior, B. M. (1974) J . Biol. Chem. 249, 6359 - 6360.

7. Babior, B. M. (1969) J . Biol. Chem. 244, 449-456. 8. Gani, D., Wallis, 0. C. & Young, D. W. (1982) J . Chem. Soc.,

9. Darapsky, A. & Hillers, D. (191 5) J . Prukt. Chem. 92, 297 - 341.

6, 35 - 102.

Chem. Commun., 898 - 899.

IOa.Gani, D. & Young, D. W. (1982) J . Chem. SOC., Chem.

IOb.Gani, D. & Young, D. W. (1983) J . Chem. SOC. Perkin Trans. Commun. 867 - 869.

1, in the press.

Lappert, M. F. & Wallis, 0. C. (1978) Eur. J. Biochem. 82,

16. Merck Index (1968) 8th edn, Merck and Co. Rahway, NJ, p.

17. Armarego, W. L. F., Milloy, B. A. & Pendergast, W. (1976) J .

18. Aldrich Chemical Co. (1983-4) Catalogue of Fine Chemicals

19. Joblin, K. N., Johnson, A. W., Lappert, M. F. &Wallis, 0. C.

20. Kaplan, B. H. & Stadtman, E. R. (1968) J . Biol. Chem. 243,

21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

22. Hollaway, M. R., Johnson, A. W., Lappert, M. F. & Wallis, 0.

23. Kaplan, B. H. & Stadtman, E. R. (1968) J . Biol. Chem. 243,

24. Hollaway, M. R., Antonini, E. & Brunori, M. (1969) FEBS Lett.

25. Gerlach, H. & Zagalak, B. (1973) J. Chem. SOC., Chem. Commun.

26a. Besmer, P. & Arigoni, D. (1968) Chimiu (Aarau) 22, 494. 26 b. Besmer, P. (1970) Dissertation 4435, Eidgenossische Technische

27. Weisblat, D. A. & Babior, B. M. (1971) J . Biol. Chem. 246,

28. Carty, T. J., Babior, B. M. & Abeles, R. H. (1971) J. Biol. Chem.

29. Carty, T. J., Babior, B. M. & Abeles, R. H. (1974) J. Biol.

143 - 154.

426.

G e m . Soe., Perkin Trans. I , 2229-2237.

( U K ) , p. 616.

(1976) Biochim. Biophys. Acta, 452, 262-270.

1787- 1793.

(1951) J . Biol. Chem. 193, 265-275.

C . (1980) Eur. J . Biochem. 111, 177-188.

1794-1803.

4, 299 - 306.

274-275.

Hochschule, Zurich.

6064 - 6071.

246, 6313-6317.

Chem. 249, 1683 - 1688.

D. Gani and D. W. Young*, School of Chemistry and Molecular Sciences, University of Sussex, Falmer, Brighton, Sussex, Great Britain BN1 9QJ 0. C. Wallis, School of Biological Sciences, University of Sussex, Falmer, Brighton, Sussex, Great Britain BNI 9QG

* To whom all correspondence should be addressed