Stereochemistry of the oxidation of gibberellin 20-alcohols, GA15 and GA44, to 20-aldehydes by gibberellin 20-oxidases

Article abstract of DOI:10.1039/a606158c

(20R)- and (20S)-[20-²H₁]-gibberellins A₁₅ and A₄₄ have been used to determine the stereochemistry of the conversion to the 20-aldehyde catalysed by gibberellin 20-oxidases.

Full text of DOI:10.1039/a606158c

View Article Online / Journal Homepage / Table of Contents for this issue
Stereochemistry of the oxidation of gibberellin 20-alcohols, GA15 and GA44, to
20-aldehydes by gibberellin 20-oxidases
Jane L. Ward,a Graham J. Jackson,a Michael H. Beale,*a Paul Gaskin,a Peter Hedden,a Lewis N. Mander,a
Andrew L. Phillips,a Hideharu Seto,b Manuel Talon,c Christine L. Willis,d Theresa M. Wilsonc and
Jan A. D. Zeevaartc
a IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, UK
BS18 9AF
b Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia
c MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
d School of Chemistry, University of Bristol, Cantock’s Close, Bristol, UK BS8 1TS
(20R)- and (20S)-[20-2H1]-gibberellins A15 and A44 have
been used to determine the stereochemistry of the conversion
to the 20-aldehyde catalysed by gibberellin 20-oxidases.
The syntheses of (20R)- and (20S)-[20-2H]-GA15 are sum-
marised in Scheme 2. The route relies on the use of bulky tert-
butyldiphenylsilyl (TBDPS) protecting groups to distinguish
between the alcohol functions in the triol 1, allowing manipula-
tion of C-20, the most sterically hindered of the three
hydroxymethyl groups. Triol 1 and its [2H6]-isotopomer 1a
were prepared by LiAlH4 (LiAlD4) reduction of GA25 trimethyl
ester, itself prepared by deoxygenation of GA13 trimethyl
ester.
The stepwise oxidation and subsequent removal of carbon-20
from C20-precursors is one of the most important sequences of
reactions in the biosynthesis of biologically active C19 gib-
berellin (GA) plant hormones (Scheme 1). This conversion is
unusual in that carbon-20 is lost at the aldehyde oxidation level
in a reaction which also involves concerted formation of the
19,10-lactone function, a characteristic structural feature of the
C19-GAs, such as GA9 and GA20. The 20-carboxylic acid is also
a biosynthetic product, but is not an intermediate in C19-GA
formation. Genes encoding GA 20-oxidases have been cloned
from several plants.1–5 These enzymes are 2-oxoglutarate-
dependant dioxygenases, an important class of soluble, FeII-
containing oxygenases .6 Functional expression of GA 20-oxi-
dase clones in E. coli has provided the means for a detailed
study of the mechanism. Although studies in cell-free systems
of spinach7 indicated that more than one enzyme may be
involved in the conversion of 20-methyl compounds via the
20-alcohol and 20-aldehyde to the C19-lactone, all GA 20-oxi-
dases so far cloned are able to convert substrates with 20-methyl
functions through to C19-lactones and/or 20-carboxylic acids.
Here we report studies on the stereochemistry of the
conversion of the 20-alcohol to the 20-aldehyde. Results are
compared in cell-free systems from spinach with those obtained
using recombinant enzyme from high-level expression of the
Arabidopsis thaliana GA 20-oxidase clone At23532 in E. coli
using the pET9d vector;† this enzyme converts GA12 to GA9 in
high yield.
Treatment of 1 with TBDPSCl and imidazole gave the
7,19-bis-TBDPS derivative 2 in high yield. Swern oxidation of
2 gave the 20-aldehyde 3. Reduction of the aldehyde with
NaBD4 was stereoselective (see later) and returned the
[20-2H1]-alcohol 4, which was protected as the acetate, before
removal of the TBDPS groups with Bu4NF to give 7,19-diol 5.
Oxidation to the 7,19-dicarboxylic acid 6 followed by hydroly-
sis yielded (20R)-[20-2H]GA15 (d1-65%, d0-35%), in the
lactone form 7. The corresponding (20S) isomer 8 (d1-99%, d0-
1%), was prepared in an analogous way from 1a, reducing the
20-deuterio-aldehyde 3a with NaBH4 as shown in Scheme 2.
1
The configurations of 7 and 8 were assigned by H NMR
spectroscopy. In fully protio species, the 20-hydrogens resonate
at d 4.12 (d, J 12.5 Hz) and 4.48 (dd, J 12.5 and 2.5 Hz). The
downfield signal was assigned to the 20-proR proton by virtue
of the long range W-coupling (2.5 Hz) to 1b-H, as confirmed by
a difference NOE experiment, which revealed a clear NOE from
6-H to the signal at d 4.48 and the absence of any enhancement
of the upfield doublet at d 4.12, assigned to the 20-proS
hydrogen. Both 7 and 8 were labelled stereospecifically.
The corresponding [20-2H1] isotopomers of the 13-hydroxy-
lated gibberellin, GA44 9 and 12, were synthesised as shown in
R
R
R
R
20
Me
13
HOH2C
OHC
O
CO
Me
CO2H
CO2H
CO2H
CO2H
Me
CO2H
Me
CO2H
Me
O
CO2H
19
R = H GA12
R = OH GA53
R = H GA9
R = OH GA20
R
R
R
OH
O
HO2C
CO
Me
CO
CO2H
CO2H
CO2H
Me
CO2H
Me
R = H GA15
R = OH GA44
R = H GA24
R = OH GA19
R = H GA25
R = OH GA17
Scheme 1 Reactions catalysed by GA-20-oxidases
Chem. Commun., 1997
13

View Article Online
Table 1 Deuterium incorporations in enzymic products
Substrate d1 :d0(%)
Enzyme Product d1 :d0(%)a
20
MeO2C
HOY2C
iii (a Y = H)
1
6
iv (b Y = 2H)
X
7 CO2Me
CY2OH
7 (20R) Open lactone 65:35
8 (20S) Open lactone 99:1
9 (20R) Open lactone 95:5
12 (20S) Open lactone 61:39
At2353
At2353
At2353
At2353
Spinach GA19
Spinach GA19
GA24
GA24
GA19
GA19
0:100
97:3
0:100
50:50
85:15
13:87
Me
CO2Me
Me
CY2OH
19
1
Me3GA13 X = OH
Me3GA25 X = H
i, ii
v
9 (20R) Lactone
12 (20S) Lactone
95:5
61:39
AcO
Y
Z
C
R1
a GA24 and GA19 were identified by GC–MS comparison (KRI and spectra),
as Me or Me, Me3Si derivatives, with authetic samples. Due to weak
molecular ions isotope enrichments were calculated on M+ 2 32 (m/z 342)
vii (Y = H, Z = 2H), ix
viii (Y = 2H, Z = H), ix
R2
CY2OSiPh2But
CY2OSiPh2But
2 R1 = CY2OH
3 R1 = CYO
for GA24 and M+ 2 28 (m/z 434) for GA19
.
Me
R2
Me
4 R2 = CY2OSiPh2But
5 R2 = CY2OH
6 R2 = CO2H
cell-free systems from C. maxima endosperm9 and pea
cotyledons,10 whereas homogenates of spinach leaves oxidise
the closed-lactone form of GA44.7 Although the product of
metabolism of GA15 open lactone was mainly GA9, under short
incubation times§ the intermediate 20-aldehyde GA24 was
readily detectable by GC–MS.
x
vi
xi
xii
Z
Y
1a–6a, 7 Y = H, Z = 2H
1b–6b, 8 Y = 2H, Z = H
O
Incubation§ of (20R)- and (20S)-[20-2H1]-GA15 7 and 8, and
GA44 9 and 12 in the open-lactone form‡ with recombinant
enzyme and GC–MS analysis of the 20-aldehyde (GA24 and
GA19, respectively) products, revealed that, for both GA15 and
GA44 substrate, there is stereospecific loss of the 20-proR
hydrogen (Table 1). In contrast, incubation of 9 and 12, in the
closed-lactone form, with the spinach cell-free system7 and
analysis of the 20-aldehyde (GA19) produced showed that, in
this case, the 20-proS hydrogen is lost (Table 1). Thus, this cell-
free system contains an enzyme that is capable of oxidising C-
20, presumably in the lactone form, and it does so with the
opposite stereochemistry to that observed with the recombinant
A. thaliana protein, which only accepts the open-lactone form.
In the lactone form, the 20-proR hydrogen is fixed in a severely
sterically hindered position under ring B and, thus, it is not
surprising that the lactone 20-oxidase, present in spinach,
removes the 20-proS hydrogen. However, in the enzymes that
accept only the ‘open-lactone’ form, the substrate presumably is
held such that the 20-proR hydrogen is more exposed.
CO
CO2H
Me
7 (20R) and 8 (20S)
Scheme 2 Synthesis of (20R)- and (20S)-[20-2H1]-GA15. Reagents: i, KH,
CS2, MeI; ii, Bu3SnH; iii, LiAlH4; iv, LiAl2H4; v, ButPh2SiCl, imidazole;
vi, (COCl)2, Me2SO, Et3N; vii, NaB2H4; viii, NaBH4; ix, Ac2O, pyridine; x,
Bu4N+F2; xi, Jones oxidn; xii, NaOH, aq. MeOH.
2H
OMOM
OMOM
2H
13
iii
CO
CO
Me
CO2Me
CO2Me
Me
10
See ref. 8
iv, v
OH
OMOM
OHC
O2HC
Footnotes
CO2Me
CO2Me
† Full details of the functional expression and characterisation of GA
20-oxidases will be reported elsewhere.
‡ The lactone-opened form of 20-hydroxymethyl GAs was produced by
treatment with 2 mol dm23 NaOH.
§ Recombinant enzyme incubations were carried out in a total volume of
100 ml of TrisCl buffer (1 mol dm23, pH 7.5) containing 70 ml enzyme
preparation [45% pure enzyme (estimated from SDS gel), 2.5 mg protein
ml21]. GA substrate (5 mg), 2-oxoglutarate (8 mmol dm23), ascorbic acid (8
mmol dm23), dithiothreitol (8 mmol dm23), FeSO4(1 mmol dm23), catalase
(2 mg ml21) and bovine serum albumin (4 mg ml21).
Me
O
CO2Me
Me
O
CO2Me
11
vi, vii, ii
i, ii
OH
OH
2H
H
H
2H
C
C
CO
Me
CO
Me
CO2H
CO2H
9 (20R)
12 (20S)
References
Scheme 3 Synthesis of (20R)- and (20S)-[20-2H1]-GA44 via GA19
.
1 T. Lange, P. Hedden and J. E. Graebe, Proc. Natl. Acad. Sci. USA, 1994,
91, 8552.
2 A. L. Phillips, D. A. Ward, S. Uknes, N. E. J. Appleford, T. Lange,
A. K. Huttly, P. Gaskin, J. E.Graebe and P. Hedden, Plant Physiol.,
1995, 108, 1049.
3 Y-L. Xu, L. Li, K. Wu, A. J. M. Peeters, D. A. Gage and
J. A. D. Zeevaart, Proc. Natl. Acad. Sci. USA, 1995, 92, 6640.
4 K. Wu, D. A. Gage and J. A. D. Zeevaart, Plant Physiol. 1996, 110,
547.
5 D. N. Martin, W. M. Proebsting, T. D. Parks, W. G. Dougherty,
T. Lange, M. J. Lewis, P. Gaskin and P. Hedden, Planta, 1996, 200,
159.
Reagents: i, NaB2H4; ii, NaOH, aq. MeOH; iii, 2H2O, K2CO3; iv, KH, DMF,
THF, O2; v, CH2N2; vi, Dowex 50W(H+); vii, NaBH4.
Scheme 3. (20R)-[20-2H1]-GA44 9 (d1-95%, d0-5%) was
produced by NaBD4 reduction of GA19 dimethyl ester followed
by hydrolysis of the esters (2 mol dm23 aq. NaOH–MeOH, 1:1,
reflux, 2 h). For the synthesis of (20S)-[20-2H1]-GA44, the
starting point was ketone 10, an intermediate in the synthesis8 of
GA19. Deuterium exchange at C-20, oxidative cleavage8 and
methylation gave [20-2H1]-GA19 dimethyl ester, 13-methoxy-
methyl ether 11. Deprotection at C-13 and then reduction of the
aldehyde followed by demethylation gave the required (20S)-
[20-2H1]-GA44 12 (d1-61%, d0-39%). The stereochemistry at
C-20 was assigned by 1H NMR spectroscopy as above.
6 A. G. Prescott and P. John, Ann. Rev. Plant Physiol. Plant Mol. Biol.,
1996, 47, 245.
7 S. J. Gilmour, J. A. D. Zeevaart, L. Schwenen and J. E. Graebe, Plant
Physiol., 1986, 82, 190.
Incubation of unlabelled GA15, as the lactone or as the
20-hydroxy-19-carboxylic acid (‘open lactone’),‡ with recom-
binant At2353 20-oxidase and analysis of the products by GC–
MS revealed that only the open lactone was accepted as a
substrate by the enzyme. A similar result has been obtained in
8 R. D. Dawe, L. N. Mander and J. V. Turner, Tetrahedron Lett., 1985, 26,
363.
9 P. Hedden and J. E. Graebe, J. Plant Growth Regul., 1982, 1, 105.
10 Y. Kamiya, Y. Takahashi and J. E. Graebe, Planta, 1986, 169, 524.
Received, 6th September 1996; Com. 6/06158C
14
Chem. Commun., 1997