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2.08Diterpene BiosynthesisJAKE MacMILLAN and MICHAEL H. BEALEUniversity of Bristol, UK
1[97[0 INTRODUCTION 106
1[97[1 TYPE A CYCLIZATION 108
1[97[1[0 Casbene 1081[97[1[1 Cembrenes 119
1[97[2 TYPE AÐTYPE B CYCLIZATION 119
1[97[2[0 Taxanes 1191[97[2[1 Fusicoccin and Transversial 1111[97[2[2 Miscellany 112
1[97[3 TYPE B CYCLIZATION 112
1[97[3[0 Labdanes and ent!Labdanes 1141[97[3[1 Clerodanes 114
1[97[4 TYPE BÐTYPE A CYCLIZATIONS 115
1[97[4[0 Tricycles from CPP 1151[97[4[1 Tricycles from ent!CPP and syn!CPP 118
1[97[5 TETRACYCLES AND PENTACYCLES 129
1[97[5[0 From ent!CPP by re!Face Cyclization on C!02 1291[97[5[0[0 ent!Kaur!05!ene and ent!kaurenoids 1201[97[5[0[1 Gibberellins 122
1[97[5[1 From ent!CPP by si!Face Cyclization on C!02 1261[97[5[2 From CPP by re!Face Cyclization on C!02 1261[97[5[3 From CPP by si!Face Cyclization on C!02 1271[97[5[4 From ent!CPP by re!Face Cyclization on C!02\ then 8H : C!7 1271[97[5[5 From ent!CPP by si!Face Cyclization on C!02\ then 8H : C!7 1271[97[5[6 From syn!CPP by re!Face Cyclization on C!02\ then 8H : C!7 1271[97[5[7 From syn!CPP by si!Face Cyclization on C!02\ then 8H : C!7 128
1[97[5[7[0 Aphidicolin 1281[97[5[7[1 Scopadulcic acids and thyrsi~orins 128
1[97[5[8 Miscellany 130
1[97[6 SUMMARY AND FUTURE PROSPECTS 130
1[97[7 REFERENCES 130
1[97[0 INTRODUCTION
This chapter concentrates on the cyclic diterpenes[ Their formation can be rationalized byconsidering the di}erent types of cyclization of geranylgeranyl diphosphate "GGPP# that have beenrevealed by studies on the enzymes[ To date\ four di}erent kinds of cyclases "synthases# have beencloned and their functional proteins have been sequenced[ These synthases are]
106
107 Diterpene Biosynthesis
Casbene synthase0Ð2012 catalyzes the formation of "0S\2R#!casbene by ionization of the diphos!phate of GGPP and attack on the resultant allylic 0!carbonium by the terminal double bond"Equation "0##[
OPP HRHS
GGPP Casbene
1
(1)
ent!Copalyl diphospate synthase3\4 catalyzes the formation of ent!copalyl diphosphate "ent!CPP#by proton!induced cyclization "Equation "1##[
H
H
OPPOPP
H
H+
GGPP ent-copalyl diphosphate (ent-CPP)
(2)
Taxadiene synthase5\6 catalyzes the formation of taxa!3\00!diene by a series of cyclization stepsinitiated by ionization of the diphosphate and continued by proton!induced cyclization "Equation"2##[
H
PPO
+
54H H H
H
GGPP
1112
taxa-4,11-diene
H
:B-Enz B+-EnzH
(3)
Abietadiene synthase7\8 catalyzes the formation of abieta!6\02!diene by a series of cyclizationsteps\ initiated by proton!induced cyclization and continued by ionization of the diphosphate"Scheme 0#[
H
H
OPP
H
GGPP
H+
H
H
copalyl diphosphate (CPP)
7
1314
abieta-7,13-diene
OPP
Scheme 1
ent!Kaurene synthase09\00 is not a GGPP cyclase\ but catalyzes the formation of ent!kaur!05!enefrom ent!CPP by diphosphate!induced cyclization[ Thus\ in contrast to abietadiene\ ent!kaur!05!ene is formed from GGPP in two distinct steps catalyzed by two enzymes] ent!CPP synthase and
108Diterpene Biosynthesis
ent!kaurene synthase "Scheme 1#[ However\ in 0886 it was shown that GGPP is converted to ent!kaur!05!ene via ent!CPP in the fungus Phaeosphaeria sp[ L376 by a single enzyme[01 The amino acidsequence\ deduced from the cloned gene\ contains both motifs a and b[
H
H
ent-CPP
H
H
ent-kaur-16-ene
1617
OPP
Scheme 2
Alignment of the amino acid sequences of these synthases reveals two characteristic conservedmotifs\ a and b "Table 0#[ Casbene and ent!kaurene synthases\ in which cyclization is initiated byionization of the diphosphate\ contain only motif a\ which is the suggested binding site for adiphosphateÐdivalent metal complex[ ent!CPP synthase contains only motif b\ nearer the N terminus\and may stabilize the incipient positive charge\ generated by protonation of the 03\04!double bondof GGPP[ Abietadiene synthase catalyzes both types of cyclization and contains both motifs[Taxadiene synthase also contains motif a but motif b is modi_ed and does not align with motif bof the other cyclases^ this modi_ed motif b may be associated with internal deprotonation andreprotonation\ catalyzed by taxadiene synthase "see Section 1[97[2[0#[
Table 0 Conserved regions in geranylgeranyl diphosphate cyclases[*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ
Motif a Motif bCyclase "I\L\V#DDXXD D"I\V#DDTAM Ref[*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐCasbene synthase LIDDTID * 0ent!Kaurene synthase "pumpkin# VVDDFYD * 4
ent!CPP synthase "arabidopsis# * DIDDTAM 00ent!CPP synthase "maize# * DVDDTAM 02
Abietadiene synthase ILDDLYD DIDDTAM 8
Taxadiene synthase LFDDMAD "DSYDD# 5*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ
The individual cyclases are described in more detail later[ On the basis of the general propertiesof the GGPP cyclases and ent!kaurene synthase\ the origins of the diverse structures of the cyclicditerpenes are discussed under two general headings] Type A\ initiation by ionization of the diphos!phate^ and Type B\ initiation by protonation at the 03\04!double bond[
Type A may be followed by Type B\ catalyzed by the same or separate enzymes[ Likewise\ TypeB may be followed by Type A\ catalyzed by the same or separate enzymes[ Further transformationsof the initial cyclization products are catalyzed by oxidases\ some of which have been characterizedand:or cloned[ These oxidases are discussed where appropriate[
1[97[1 TYPE A CYCLIZATION
1[97[1[0 Casbene
Casbene ""1#\ Scheme 2#03\04 is a phytoalexin\ elicited in seedlings of castor bean "Ricinus communisL[# by the fungus Rhizopus stolonifer\05 or oligogalacturonides from cell wall fragments of thisfungus[06 Its formation has been rationalized07 by cyclization of GGPP to a nonclassical carbocationat C!0\ C!03\ and C!04[ However\ the intermediate "0# is shown in Scheme 2 as a classical carboniumion to illustrate that formation of the cyclopropane ring proceeds by suprafacial approach of there\re!face of the 03\04!double bond of GGPP to C!0 and stereospeci_c loss of the pro!S hydrogenat C!0 of GGPP[08 The overall conversion is catalyzed by a single enzyme\ casbene synthase[ Thenative enzyme "Mr\ ca[ 42 999# has been puri_ed "699!fold# from castor bean seeds and shows a
119 Diterpene Biosynthesis
preference for Mg1¦ over Mn1¦[1\2 A near full!length cDNA clone for casbene synthase has beenobtained from castor bean seedlings\0 encoding a 590 amino acid protein with a predicted Mr of57 589[ Although casbene synthase is located in proplastids of germinating seedlings\19 the deducedamino acid sequence contains no clear proplastid targeting signature[ However\ it does containfeatures\ common to mono! and sesquiterpene cyclases of Type A "see Chapter 1[96#\ includingthe DDXXD motif a "Table 0#[ Expression of the gene is increased during elicitation by pecticfragments[10
OPPHS
HR HR
HSH
14HRH15 +
1 3
–HS+
–OPP
GGPP (1) (2) 1S,3R-Casbene
1617
1617 16 17
Scheme 3
1[97[1[1 Cembrenes
The cembrenes "Scheme 3# are 03!membered ring diterpenes that occur in the turpentine gum ofpines and gum from the trichomes of tobacco leaves[ neo!Cembrene "cembrene A\ "3## representsthe simplest product of the Type A cyclization of GGPP and\ in principle\ may be derived fromGGPP via the carbonium ion ""2#\ Scheme 3#[ 0S!Cembrene "6#\11 the parent compound of thecembrene 3!ols "4# and cembrene 3\5!diols "7#\ may\ likewise\ be formed from "2# via "5#[ However\there is no experimental evidence for these steps\ nor for the postulate12 that 0S!cembrene "6# isformed from the "0R\2S#!enantiomer of "0S\2R#!casbene ""1#\ Scheme 2#[ The biosynthesis of thecembrene!diols "7# has been examined in tobacco by two groups[ Using excised calyces\ Crombie etal[12 observed the incorporation of ð1!03CŁgeranylgeraniol and 0R\S!cembrene into the 3a and 3b!cembrenediols "7#[ In contrast\ Guo and Wagner13 found that a puri_ed enzyme preparation fromtrichome glands catalyzed the formation of both cembrene 3a and 3b!ols "4# from ð0!2HŁGGPP[They concluded that the steps from GGPP to "4# were catalyzed by a single\ soluble polypeptide of47 kDa and that the cembrene diols "7# were probably formed from the cembrene 3!ols "4# by acytochrome P349!dependent monooxygenase[ However\ no de_nitive enzymology has beenreported[
1[97[2 TYPE AÐTYPE B CYCLIZATION
1[97[2[0 Taxanes
More than 099 taxanes occur in Taxus species[ The most detailed biosynthetic studies haveconcerned taxol "paclitaxel\ "05#\ Scheme 4#\ a potent anticancer agent from the bark of Paci_c yew"Taxus brevifolia Nutt#[ These studies\ summarized in Scheme 4 and discussed later\ show that the_rst committed step is the cyclization of GGPP to taxa!3\00!diene "02#[ A cDNA clone for taxadienesynthase has been obtained from stems of T[ brevifolia and functionally expressed in Escherichiacoli[5 The expressed protein catalyzes all steps from GGPP to the taxadiene "02#[ The deduced aminoacid sequence of the polypeptide contains a presumptive plastidial targeting sequence and has anMr of 87 292 compared to 68 999 for the mature native enzyme[6 Sequence comparison with otherplant terpene synthases showed similarities\ including the motifs a and b "Table 0#[
Details of the cyclization of GGPP to taxa!3\00!diene "02# have been established by metabolicstudies[ Using a cell!free enzyme preparation from the bark of young saplings of T[ brevifolia\Koepp et al[14 established the cyclization of ð0!2HŁGGPP to taxa!3\00!diene "02# and the furtherconversion of the derived ð2HŁ!diene by stem discs into taxol "05#\ cephalomannine "06#\ and 09!desacetyl baccatin III "07#[ That the _rst intermediate was the 3\00!diene "02# and not the 3"19#\00!
110Diterpene Biosynthesis
OPPb
b+ a
1
HH
H
H
H
5
H
a
GGPP
–OPP
(3)
H
(4)
5
OH1
H2O
H
–H5
4
OH
+
1
4
(5) (6) (7)
H
OH
(8)
1
6
b
Scheme 4
diene "03# was unexpected[ However\ con_rmation of this result\ and further details of the formationof taxa!3\00!diene "02# from GGPP\ were obtained from an elegant series of experiments by Lin etal[15 These authors used a ½599!fold puri_ed enzyme preparation6 from the bark and adheringcambium of T[ brevifolia to obtain the following information[ First\ the conversion of ð19!1H2ŁGGPPto taxa!3\00!diene "02#\ without loss of deuterium\ eliminated the intermediate formation of the3"19#\00!diene "03#[ Second\ the conversion of ð0!1H1\19!1H2ŁGGPP to taxa!3\00!diene "02# withoutloss of deuterium eliminated the intermediate formation of casbene ""1#\ Scheme 2#[ Third\ incor!poration of label from ð09!1HŁGGPP into taxa!3\00!diene "02# showed that intramolecular protontransfer occurred in the cyclization[ These results are incorporated in the mechanism shown inScheme 4[ Thus\ ionization of GGPP promotes bond formation in the substrate from C!0 to C!03\followed by ring closure via re!face attack at C!04 to give cation "8#[ Deprotonation of "8# byremoval of its 00a!H gives 0S!verticillene "09#^ rapid reprotonation at C!6 by the same enzyme baseinitiates transannular cyclization of "00# by re!face attack of C!2 at C!7 to give the taxenyl 3!cation"01# which is deprotonated to "02#[ Thus\ these results establish that taxa!3\00!diene "02# is formedfrom GGPP by a single enzyme\ catalyzing Type A then Type B cyclization[
Following the cyclization of GGPP to taxa!3\00!diene "02#\ the latter is hydroxylated to the 4a!ol "04# by a microsomal enzyme preparation from stems of T[ brevifolia or cultured cells of T[cuspidata[16 The enzyme preparation has the characteristic properties of a cytochrome P349!dependent monooxygenase[ No isotope e}ect was observed for the ð19!1H2Ł!labeled substrate andno mechanistic details of this hydroxylation with allylic rearrangement are known "cf[ the formationof gibberellin A2 from gibberellin A4\ discussed in Section 1[97[5[0[1#[ The intermediacy of the dienol
111 Diterpene Biosynthesis
H
PPOH
H
H H
H
H H
15
H
11
H
10
H
:B-Enz
1
7
H
GGPP
H
H-B+-Enz
R
(9)
7
(10)
+
HO
3
H
8
R1O
5
O
(12)
OH
4
O
4
AcO
20
R2O
5
BzO
(13) taxa-4,11-diene
4
20
H
(11)
H
5
AcO
AcO
(16) taxol, R1=Ac, R2=CO(S)CHOH(S)CHPhNHBz
(17) cephalomannine, R1 = AC,
R2=CO(S)CHOH(S)CHPhNHCOCMeZCHMe
(18) desacetylbaccatin III, R1=R2=H AcO OAc
+
(19) taxuyunnanine C
14
+11
(14) R=H(15) R=OH
1
12 1111
1
Scheme 5
"04# was demonstrated by its occurrence in bark of T[ brevifolia and its conversion into taxol "05#\cephalomannine "06#\ and 09!desacetylbaccatin III "07# by stem discs of T[ brevifolia[16
Although there has been much speculation on the mechanism of formation of the oxetane ring\no metabolic studies have been reported[ Regarding the N!containing side chain\ b!phenylalanine\phenylisoserine\ and the intact N!benzoylphenylisoserine are incorporated into taxol^ phenyl!alanineÐammonia lyase does not seem to be involved since cinnamic acid is not incorporated[17
A nonmevalonate pathway to taxuyunnanine C "08# in cell cultures of T[ chinensis has beenreported18 and is discussed in Chapter 1[92[
1[97[2[1 Fusicoccin and Transversial
Fusiccocin ""13#\ Scheme 5# is the major component of a related family of phytotoxic metabolitesfrom cultures of Fusicoccum amy`dali Del[ No enzymological studies have been reported[ However\its biosynthesis from GGPP "Scheme 5# by Type AÐType B cyclization can be proposed from theresults of labeling experiments[29\20 Label was incorporated from ð2!02CŁMVA into "13# at the C!2\C!6\ C!00\ C!04 in the rings and C!13 in the side chain\ and only four of the expected _ve 2H labelswere incorporated from ð1!03C\3R!2HŁMVA[29 In addition\ it was shown20 that the intensities of the02C!signals of C!6 and C!04 were suppressed\ compared to those of C!2\ C!00 and C!13\ in the NMRspectrum of fusiccocin "13#\ obtained from "3R#!ð2!02C\3!1H1ŁMVA[ These results support pathwaya "Scheme 5#\ from GGPP via "12# to "19#\ then C!5 to C!1 bond formation\ initiated by protonationat the re!face at C!2 of "19#\ to give "10# which is converted to "11# by two consecutive 0\1!hydrideshifts\ from C!1 to C!5 and from C!5 to C!6\ rather than one 0\2!hydride shift from C!1 to C!6[
In contrast\ biosynthesis of the related transversial "16# from Cercospora tranversiana does notinvolve hydride shifts from C!1\ C!09\ or C!03 as shown by NMR spectrometry of "16#\ obtainedfrom ð1\1\1!1H2\0!02C0Ł and ð1\1\1!1H2\1!02C0Łacetate[21 The proposed pathway b\ shown in Scheme5\ includes the suggestion that the stereochemical di}erences between fusicoccin and transversial at
112Diterpene Biosynthesis
H
H
H H
HH
H
H
H
H
PPO
HH
H
HH
H
H
H
HH
H
H
HCHOH
H
H
H
OH
6
7
HO
2
3
O
6
H
+
MeOCH2 H
7
AcO+
HO
a
b
OH
H+
O
H+
33
2
67
8
3
re-face protonation at C-3
a
b
(20)
H
H
H
H
2
a
O(23)OH
(24)
OAc
GGPP
OH
(26) (27)(25)
3
7
11
15
24
(21) (22)
O
Scheme 6
si-face protonation at C-3
C!2 may be the result of the protonation of the intermediates "19# and "14# on the re! and si!faces of C!2\ respectively[ The proposed intermediate\ transversadiene "15#\ is a metabolite of C[transversiana22 but its conversion to "16# has not been reported[
1[97[2[2 Miscellany
The jatrophane\ latherane\ tigliane\ daphnane\ and ingenane skeleta are close structural relativesof casbane and cembrenes[ No biosynthetic studies have been reported but a putative biosyntheticrelationship has been proposed[23 The verrucosanes are also putative products of Type A cyclization\followed by Type B[24
1[97[3 TYPE B CYCLIZATION
As illustrated in Scheme 6\ protonation of the 03\04!double bond of E\E\E!GGPP\ followed bythe attack of C!09 on C!04\ then C!6 on C!00\ gives four possible products\ depending on theconformation of the prochiral substrate[ The chairÐchair conformation "17# gives the 7!carboniumion "18# of copalyl diphosphate "CPP\ "29## with the {{normal|| anti\anti absolute stereochemistry"Type B!0#[ The antipodal chairÐchair conformation "20# of GGPP gives the 7!carbonium ion "21#of ent!copalyl diphosphate "ent!CPP\ "22## with the enantiomeric anti\anti absolute stereochemistry
113 Diterpene Biosynthesis
"Type B!1#[ The chairÐboat conformation "23# gives the 7!carbonium ion "24# of the {{normal|| syn!CPP "25# "Type B!2#[ The chairÐboat conformation "26# gives the 7!carbonium ion "27# of syn!ent!CPP ""28#^ Type B!3#[
H R
H
H
H
H
R
H R
H H
R H
H
R
H
H
H
R
H
15 10
11 7
814
H
R
H
+
Type B-1: chair–chair-"normal"
HH
H
R
Type B-2: chair–chair-"antipodal"
Type B-3: chair–boat-"normal"
Type B-4: chair–boat-"antipodal"
H
R
H+
H+
+
H
H
R
+
+
(28) (30) CPP
(31) (32) (33) ent-CPP
(34) (35) (36) syn-CPP
(37) (38) (39) syn-ent-CPP
(29)
R
H
8
H+
H+
R = OPP
Scheme 7
Each of these cyclization types can be inferred from studies on the diterpenes that are formed byfurther elaboration of the immediate products "see later sections#[ However\ the only cyclase thathas been characterized is ent!CPP "22# synthase "Type B!1#[ The formation of ent!ð03CŁCPP "22#from ð03CŁGGPP was _rst established25 using a soluble enzyme preparation from the mycelia of thegibberellin!producing fungus\ Gibberella fujikuroi[ The enzyme was partially puri_ed from thisfungus25 and from the endosperm of Marah macrocarpus[26 However\ detailed information on ent!CPP synthase comes from the cloning3 of the GA0 locus in Arabidopsis thaliana\ using the GA!responding dwarf mutant ga0 and a genomic subtraction technique[ This gene has been shown4 toencode an ent!CPP synthase with a molecular mass of 75 kDa which is imported into pea chloroplastsand processed to a 65 kDa protein[ The amino acid sequence of ent!CPP synthase contains themotif b "Table 0# and is similar to those of other plant cyclases "for a review\ see ref[ 26#[ The An0gene from Zea mays "maize# contains motif b and probably also encodes an ent!CPP synthase[02
Type B cyclization of Z!isomers of GGPP has not been studied in detail[ However\ the E\Z\E!GGPP was excluded as a precursor of 8\09!syn!diterpenes in cell cultures of Oryza sativa[28
The immediate products of these Type B cyclizations of E\E\E!GGPP are modi_ed by functionalgroup transformations to yield labdanes and their rearrangement products\ the clerodanes[ These5\5!bicyclic products are discussed in the following sections[
114Diterpene Biosynthesis
1[97[3[0 Labdanes and ent!Labdanes
Labdanes\ derived via "18#\ and ent!labdanes\ derived via "21#\ are of widespread occurrence butfew de_nitive biosynthetic studies have been reported[ Indeed\ it has been suggested that they maybe derived from GGPP by a two!step process via monocyclic intermediates on the basis that bothlabdane and retinanes co!occur in di}erent populations of Bellardia trixa`o[39 Another point ofbiosynthetic interest is the reported co!occurrence of labdanes and ent!labdanes[ For example"Scheme 7#\ the enantiomers "39# and "30# occur in the resin of Eperua purporea30 and ent!sclarene"31# and sclarene "32# have been isolated from di}erent specimens of Dacridium intermedium[31 Theco!occurrence of enantiomeric labdanes is discussed by Carman and Du.eld32 but no enzymologicalstudies on this point have been reported[
H
H
H
H
H
H
H
H
CO2H CO2H
H
H
Br
OH
OH
(40) (41)
(42) (43) (44)
Scheme 8
In addition to the previously discussed formation of ent!CPP "22# from GGPP\ the biosynthesisof {{normal|| labdanes has been investigated in tobacco "Nicotania tobaccum L[#[33\34 Cyclization ofGGPP to cis!abienol "34#\ as shown in Scheme 8\ occurs in extracts from trichome glands of theleaves of the cultivar T[I[ 0957[33 The synthase activity was not present in the epidermal or sub!epidermal tissue from which the glands were taken[ It was soluble\ Mg1¦ stimulated\ and una}ectedby conditions that inhibit cytochrome P349 oxygenases[ From these results it was suggested thatthe enzyme!bound intermediate\ CPP 7!carbonium ion "18#\ is hydrated at C!7\ followed by elim!ination of the elements of HOPP as shown in Scheme 8[ It may be that capture of the 7!carboniumion by H1O diverts a Type A cyclization\ resulting in the elimination of 01!H from the 02!cation[ Ina related study\34 labdenediol "35# and sclareol "36# were both formed from GGPP in cell!freeextracts of another cultivar\ 13A\ of tobacco[ The enzyme activities for the formation of eachproduct were not separated after protein puri_cation\ or by thermal inactivation or in productinhibition studies[ It was therefore concluded that labdenediol "35# and sclareol "36# are the directproducts of one synthase\ operating on GGPP[ Although these products appear to require hydrolysisof the diphosphate\ they could also arise from an aborted Type A cyclization whereby the allyliccarbonium ion is captured by H1O^ thus a diphosphatase would not be required[ It remains to bedetermined if the two cultivars\ T[I[ 0957 and 13A\ of tobacco contain two distinct GGPP cyclases[
The occurrence of the 2!bromo!ent!labdane\ aplysin!19 ""33#\ Scheme 7#\ from the mollusc\ Aplysiakurodai\ suggests35 that Type B cyclization can be initiated by Br¦ as well as H¦[
1[97[3[1 Clerodanes
The clerodanes36 comprise a large family\ considered to be derived from rearrangement of the 7!carbonium ions "18# and "21#[ As shown in Scheme 09\ trans!clerodanes can\ in principle\ be theproducts of concerted 0\1!shifts including C!08 to C!4 "sequences a#[ In the case of the cis!clerodanes\a similar series of shifts cannot be concerted^ for a stereoelectronic shift of C!07 to C!4\ sequences
115 Diterpene Biosynthesis
H
H OH
H
H OH
OH
H
H OH
OH
H
H
OPP+
8GGPP
(45)
(46)
(47)
(29)
Scheme 9
b would require a conformational change in ring A[ A consequence would be the prediction thatthe 2b!H of the bicyclic intermediates is lost in the formation of the 2\3!dehydro!trans!clerodanesand that the 2a!H is lost in the formation of the 2\3!dehydro!cis!clerodanes[ However\ this pointhas not been examined experimentally[ Evidence for the proposed rearrangements come from theisotope ratios in a series of furanoclerodanes\ obtained from Tinospora cordifolia\ after administeringð3R!2H\1!03CŁMVA[37 Particularly instructive is the compound ""37# or its enantiomer\ Scheme 09#in which C!07\ derived from the 1!03C of MVA\ was shown to be retained at C!4 from the degradativeevidence that C!08 was unlabeled[ Indirect evidence for the shift of C!07 from C!3 to C!4 is providedby the results of the labeling of heteroscyphic acid A ""38# or its enantiomer\ Scheme 09# in culturedcells of the liverwort Heteroscyphus planus[38 However\ the most interesting result from this studyis the nonequivalent labeling\ indicating that the GGPP precursor is formed from MVA!derivedFPP and non!MVA!derived IPP "see Chapter 1[92#[
1[97[4 TYPE BÐTYPE A CYCLIZATIONS
The initial 5\5!bicyclic products of Type B cyclization of GGPP can undergo Type A cyclizationby two di}erent stereochemical routes[ Following ionization of the diphosphate\ the 7"06#!doublebond can attack the si! or re!face of C!02 and give two series of tri!\ tetra!\ and pentacyclic systemswhich are then further modi_ed by oxidation and:or by rearrangement[ The outcome from each ofthe 5\5!bicyclic products CPP "29#\ ent!CPP "22#\ syn!CPP "25#\ and syn!ent!CPP "28# "see Scheme6# is discussed separately[
1[97[4[0 Tricycles from CPP
The possible pathways to known 5\5\5!tricyclic diterpenes are outlined in Scheme 00[ The re!faceand si!face cyclization of CPP "29# on C!02 gives the 7!carbonium ions "43# and "52#\ respectively[Deprotonation of "43# leads to virescene "40# and sandaracopimaradiene "44#\ and deprotonation
116Diterpene Biosynthesis
H
H
R
H
H
R
HR
H
HR
H
HR
H
HR
H
HR
H HR
H
1819
+
+a
b
+
19 18
+
1819
+
a19
b
18
19
+
1819
(29)
trans-clerodanes(trans-ent-neo-clerodanes)
+
18
+
cis-clerodanes(cis-ent-neo-clerodanes)
ent-trans-clerodanes(trans-neo-clerodanes)
a b
ent-cis-clerodanes(cis-neo-clerodanes)
a b
(32)
* denotes carbon from [2-14C]MVA; # denotes carbon from [2-13C]MVA; thick lines show 13C–13C coupling from[4,5-13C2]MVA
O
O
*
T
OHO
H
OT
T
OC
HO
O
19
H CO2H
*
H
*
18
58
1918
2
3
9
7
6
11
1214 15
#
*
[4R-3H1,2-14C]MVA
[2-13C]- and [4,5-13C2]MVA
(48)
(49)
#
5#
Scheme 10
117 Diterpene Biosynthesis
of "52# leads to pimara!7"03#\04!diene "53# and pimara!7\04!diene "56#[ Metabolic evidence for thesesteps includes the conversion of ð03CŁCPP "29# to the ð03CŁpimaradiene "56# in cell!free extracts ofTricothecium roseum49 and the NMR spectrum of virescenol B "49#\ derived from ð0\1!02CŁacetate inOospora virescens\ shows 02CÐ02C coupling\ consistent with its derivation via GGPP and "43#[40 Inthe step "29# to "52# an overall anti!SN1? displacement of the diphosphate anion has been establishedin the biosynthesis of rosenonolactone "see later# in which the 05Z!H and the 05E!H "see "52## arerespectively derived from the 4!proR and 4!proS!H of MVA[41 Also\ the 0\1!shift "a# of the methylfrom C!02 to C!04 in "44# or "53# to form "48# is stereoselective\ at least for cryptotanshinone "57#"41# and ferruginol "58#[43 The expected 02C!isotopic labeling "see structures# was observed for eachcompound\ derived from ðU!02C5Łglucose in cell cultures of Salvia miltiorrhiza[ This information\together with determination of the absolute stereochemistry at C!04 in "57#\ established that the C!06 methyl group migrates to the si!face of C!04[42 Con_rmation of this result was obtained43 byshowing that it is the migrated methyl group that becomes the proR!methyl in the isopropyl groupin ferruginol "58#[
Enzymological studies provide detailed evidence for the biosynthesis of abieta!6\02!diene "48#and abietic acid "51# by the steps shown by bold arrows in Scheme 00[ Abietadiene synthase isconstitutive in stems of lodgepole pine "Pinus contorta# and both constitutive and wound!induciblein stems of grand _r "Pinus `randis#[44\45 Partial puri_cation8 of the soluble extract from grand _rgave a native protein of 79 kDa that catalyzed the cyclization of ð2H0ŁGGPP to abietadiene ""48#\89)# and sandaracopimaradiene ""44#\ 8)# in the presence of a divalent cation\ preferably Mg1¦[Using cDNA from wound!induced stem and PCR degenerate primers\ based on the amino acidsequences of tryptic digests from the native protein\ three cDNA clones were obtained and func!tionally expressed in E[ coli\7 thereby con_rming that a single protein catalyzed the conversion ofGGPP to abietadiene in a multistep sequence of Type BÐType A cyclizations[ The clone that yieldedthe highest levels of cyclase activity encoded a protein of 88[4 kDa\ including a putative plastidtargeting sequence\ rich in serine and threonine[ The deduced size "77 kDa# of the mature proteinagrees with the molecular mass assigned to the partially puri_ed\ native protein[ The deduced aminoacid sequence also contained both motifs a and b "Table 0#\ consistent with the catalytic propertiesof a Type BÐType A cyclase[ A possible mechanism is shown in the bold arrow sequence of Scheme00 whereby the enzyme!bound intermediate CPP "29# is transformed either by 02re!face cyclizationto "43# and sandaracopimaradiene "44# or by 02si!face cyclization to "52# and the pimaradiene "53#[Rearrangement of "44# or "53# to "47#\ followed by deprotonation\ yields abietadiene "48#[ Theisolation of sandaracopimaradiene "44# as a minor product from GGPP\ using both the puri_ednative protein and the cloned cyclase\ indicates that 02re!face cyclization of "29# may be the preferredroute[
The formation of abietic acid "51# from abietadiene "48# by stepwise oxidation at C!07 has beendemonstrated44 in cell!free extracts from stems of lodgepole pine and grand _r[ The _rst two stepsto the 07!ol "59# and the 07!al "50# are catalyzed by a microsomal fraction with the expectedproperties of cytochrome P349 oxygenases but showing di}erent sensitivities to known inhibitorsof plant P349s[ The third step from the 07!al "50# to the acid "51# is catalyzed by soluble fractions^the activity is not inhibited by CO\ does not require O1\ and uses NAD¦ as a cofactor\ indicating asoluble aldehyde dehydrogenase[
Simple variations of the position of deprotonation of the intermediate "47#\ followed by stepwiseoxidation\ would account for the origin of the common resin acids\ isopimaric acid "41#\ palustricacid "42#\ neo!abietic acid "46#\ and laevopimaric acid "55#\ but no experimental details have beenpublished[
Rearrangement of the tricyclic carbonium ion ""52#\ Scheme 00# accounts for the origins ofrosenonolactone ""60#\ Scheme 01#[ This metabolite of Trichothecium roseum has been shown to beformed from CPP "29# by migration of the C!8 hydrogen in "52#\ derived from the pro!3R hydrogenof MVA\ to C!7 and of the C!09 methyl to C!8[46\47 These two shifts "Scheme 01# would lead to "69#\analogous to the formation of the clerodanes "Scheme 09#[ However\ the exact timing of lactoneformation from "69# is not known[ Carbon!08\ not C!07\ is derived from C!1 of MVA[48 This pointis returned to in discussing the gibberellins and the evidence that Type!B cyclization of E\E\E!GGPP is all!trans[
The biosynthesis of pleuromutillin ""63#\ Scheme 01#\ a metabolite of Pleurotus mutilus\ has beenstudied in detail[59 The various rearrangements\ summarized in Scheme 01\ were proposed from theresults of labeling in "63# from ð0!03CŁacetic acid\ ð1!03CŁMVA\ ð0!03CŁGGPP\ "2R\3R#!ð3!2H0ŁMVA\ð4!2H1ŁMVA\ and "2R\4R#!ð4!2H0ŁMVA[ Since the absolute stereochemistry of "63# is known\50
GGPP appears to be cyclized to the syn!CPP 7!carbonium ion "24# which is then transformed to"61# by a series of nonconcerted 0\1!shifts[ Type A cyclization of "61# to "62#\ followed by a trans!annular hydride shift\ leads to "63#[
118Diterpene Biosynthesis
HOH
H
H
H
HO2CH
H
HO2CH
HH
HH
OPP
HH
HZ
HE
H
H
HO2CH
H
HO2CH
H
RH
H
H
H
H
H
H
H
H
HO2CH
H
HO2CH
H
O
+
H
13
O
O
+
H+
a
+
H+
a
*
HO
*
*
* 15*
H
*
*H
15 **
16 pro--S
17 pro-R
18 1918H
19
OH
(50) (51) (52) (53)
(54) (55) (56) (57)
(30) (58) (59) (60) R=CH2OH(61) R=CHO(62) R=CO2H
(63) (64) (65) (66)
(67) (68) (69)
si
si
Scheme 11
1[97[4[1 Tricycles from ent!CPP and syn!CPP
In cell cultures of Oryza sativa "rice# that have been exposed to either UV light51 or chitin\28 ent!CPP "22# is cyclized to ent!sandaracopimaradiene "66#\ presumably by si!face attack of C!06 at C!02 via ""64#\ Scheme 02#[ In the same system\ syn!CPP "25# is cyclized to 8b!pimara!6\04!diene "67#\
129 Diterpene Biosynthesis
H
H
H
HCO
O
H
HH
H H
OPP
(35)
+ +
H
18
H+
H
+
O OCOCH2OH–OR
H
H
19
OPP
(63) (70) (71)
(72) (73) (74)
Scheme 12
presumably via "65#[ Neither ent!sandaracopimaradiene "66# nor 8b!pimara!6\04!diene "67# areformed in nonelicited cell cultures[ The elicitation of the enzymes for the formation of "66# and "67#is of interest vis!a�!vis the phytoalexins\ such as the oryzalexins "e[g[\ 68#52 and momililactones "e[g[\70#53 which are formed in rice plants that have been exposed to UV light or fungal infection[However\ no biosynthetic studies on the formation of "68# from "66# or "70# from "67# have beenreported[ ent!Sandaracopimaradiene "66# is also formed from GGPP in soluble enzyme preparationsfrom seedlings of castor bean[07
The ent!abieta!6\02!diene ""79# Scheme 02#\ reported to occur in Helichrysum chionosphaerum\54
may be formed from ent!CPP "22# via "66# by a sequence similar to the formation of abieta!6\02!diene ""48#\ Scheme 00#[
There are no reports of the cyclization of ent!syn!CPP "28# to tricyclic diterpenes[ The absolutestereochemistry\ assigned to the ent!8b!pimara!6\04!dienes\ reported to occur in Calceolariaspecies\55 has not been established[
1[97[5 TETRACYCLES AND PENTACYCLES
As illustrated in Scheme 6\ Type B cyclization of GGPP gives four bicyclic products ""29#\ "22#\"25#\ and "28##[ Further cyclization of each of these 5\5!bicycles "71# is shown in general terms inScheme 03[ Type A cyclization by attack of C!06 at the re! or si!face of C!02 gives rise to eightpossible stereoisomers of the tricyclic 7!carbonium ion "72#[ These tricycles can undergo cyclizationfrom C!04 to C!7 ""72# to "73## or from C!04 to C!8 after migration of 8H to C!7 ""72# to "74## togive 05 possible tetracyclic carbonium ions that can undergo further rearrangement to give morethan 64 di}erent tetra! and pentacyclic ring systems[ Some\ but not all\ of these ring systems havebeen shown to occur naturally[ Those that are known\ and for which biosynthetic information isavailable\ are discussed in the following sections[
1[97[5[0 From ent!CPP by re!Face Cyclization on C!02
This group is the most abundant and the most studied[ Scheme 04 shows possible intermediatesfrom the 7!carbonium ion "76# from ent!CPP "22# to the natural ent!kaur!05!ene "75#\ ent!beyerene"77#\ ent!trachylobane "78#\ ent!atisir!05!ene "89#\ and ent!atisir!04!ene "80#[ Early evidence for thesepathways came from the observation that MVA and GGPP were converted to "75#\ "77#\ "78#\ and
120Diterpene Biosynthesis
H HOPP
H
H
OPP
H
H
H
H
H
H
H
H
H
H
(33) ent-CPP
H
H
O
13
O
17
HO
13
HO O
17
(36) syn-CPP
+ +
H
H
(75) (76)
(77) (78)
(79) (80) (81)
si si
Scheme 13
"89# in addition to casbene ""1#\ Scheme 2# and ent!sandaracopimaradiene ""66#\ Scheme 02# bysoluble enzyme preparations from castor been seedlings[03\07 Partial puri_cation of the crude enzymepreparation indicated that separate GGPP cyclases were present for the formation of each of thediterpenes except for ent!kaur!05!ene "75# and ent!trachylobane "78#[ However\ subsequent studieshave been directed to the biosynthesis of ent!kaur!05!ene "75# and its conversion to the gibberellinfamily of plant hormones and these studies are now discussed separately[
1[97[5[0[0 ent!Kaur!05!ene and ent!kaurenoids
The Type A cyclization of ent!CPP "22# to ent!kaur!05!ene ""75#\ Scheme 04# was _rst dem!onstrated in a cell!free system from the fungus\ Gibberella fujikuroi\ and in homogenates from seedsof M[ macrocarpus and seedlings of R[ communis[56 The enzyme concerned\ ent!kaur!05!ene synthase\
121 Diterpene Biosynthesis
H
H
H
H
H
H
8
H
+
H9
+
9 8
9
8
OPP
+
cyclization on re or si-face of C-13
16 groups of tetra-/pentacyclic
skeletons
cyclization of C-15 on re or si–face of C-8
9H to C-8 then cyclization ofC-15 on re or si-face of C-8
deprotonation/rearrangement
deprotonation/rearrangement
15
(82) (83)
(84)
(85)
13
17
Scheme 14
has been partially puri_ed from M[ macrocarpus26 and\ more recently\ puri_ed to near homogeneity"Mr\ 70 999# from Cucurbita maxima[09 The gene encoding the C[ maxima enzyme has been clonedand expressed in E[ coli and the recombinant protein converts ent!CPP to "75#[00 The gene producthas an Mr of 78 999 and the sequence contains a possible plastid!targeting N terminal sequence andmotif a "Table 0#\ characteristic of a Type A cyclase[ Some details of the stereochemistry of thecyclization in M[ macrocarpus were provided in earlier investigations with ð06!E!2H0Ł!ent!CPP whichgave rise to ent!kaur!05!ene "75# labeled with tritium at the C!04 endo!position[ Similarly\ ð0"S#!2H0ŁGGPP gave ent!kaur!05!ene with tritium at the 03b!position[57 Support for the migration of C!01 to C!05 comes from feeding ð2\2?!02C1ŁMVA to G[ fujikuroi\ where retention of 02CÐ02C couplingwas observed at C!05 and C!06 in ent!kaur!05!ene ""75#\ Scheme 04#[58
Oxidative metabolism of ent!kaur!05!ene "75# gives rise to the plethora of naturally occurringent!kaurenoid diterpenes and also the gibberellin plant hormones "see Section 1[97[5[0[1#[ Oxidationat C!08 to the acid is a feature common to many kaurenoids and is also the _rst transformation onthe route to the gibberellins[ Because of this\ oxidative metabolism at rings A and B of ent!kaur!05!ene has been much studied "Scheme 05#[ The oxidation of "75# to the alcohol "81#\ and of thealdehyde "82# to ent!kaur!05!en!08!oic acid "83# is catalyzed by microsomal enzymes with theproperties of P349 monooxygenases[69 The conversion of the 08!alcohol "81# to the 08!aldehyde "82#is also catalyzed by a microsomal activity and involves the loss of the 08!pro!R hydrogen atom[60
Studies in a cell!free system from G[ fujikuroi have shown that the hydroxylation of ent!kaur!05!en!08!oic acid "83# to ent!6a!hydroxykaur!05!en!08!oic acid "85# is also catalyzed by a microsomalP349 monooxygenase activity[61 It is likely\ but not proven\ that the 5\05!dienoic acid "86# can alsobe a product of this enzyme[ The biosynthesis of the kaurenolides "e[g[ 090# has been establishedto proceed from "86#\62\63 probably via the unstable epoxide "88# which undergoes spontaneousintramolecular cyclization to "090# under aqueous conditions[63 Further oxidation of ent!6a!hydroxykaur!05!en!08!oic acid "85# at C!5 gives rise to the ent!5a\6a!diol "87# and the ring!Bcontraction product\ gibberellin A01!aldehyde "099#[ Both "87# and "099# appear to be products ofthe same P349!type of activity in the C[ maxima system\64 and thus can be formed from the same
122Diterpene Biosynthesis
H H H
H
H
H
H
H
H
H
H
H
H
(86) ent-kaur-16-ene
(88) ent-beyerene
(89) ent-trachylobane
(90) ent-atisir-16-ene (91) ent-atisir-15-ene
H
H
+
H
H
a
b
+
+
+
(33) ent-CPP
b
a
–H+
H
hydride shift
–H+ –H+
+
H H
re
13
13
OPP
(87)
12
16
14
15
1617
Scheme 15
C!5 free radical "84# from which a 0\1 radical shift results in the extrusion of C!6 "Scheme 05#[Studies with ent!kaur!05!en!08!oic acid "83#\ stereospeci_cally labeled with deuterium at C!5 andC!6\ have demonstrated that the C!5 and C!6 hydroxylations proceed with retention of con_guration\while the dienoic acid "86#\ and hence the kaurenolides "090#\ are formed with stereoselective lossof the ent!6a!hydrogen and nonstereoselective loss of the ent!5a and 5b hydrogen[65 This study alsorevealed that ring contraction to "099# proceeds with stereoselective loss of the ent!5a!hydrogen\complementing earlier results with ð5\5!2H1\03CŁ!ent!kaur!05!en!08!oic acid "83#[66
1[97[5[0[1 Gibberellins
The gibberellins "GAs# are a family of over 099 tetracyclic diterpenoids biosynthetically derivedfrom gibberellin A01!aldehyde ""099#\ Scheme 05#[ They are produced by several fungi\ the mostnotable being G[ fujikuroi\ from which the GAs were _rst isolated[ They also occur in higher plants\where they have an important role as hormones involved in many aspects of normal growth anddevelopment[ As a result of their biological signi_cance there is much literature on the biosynthesis
123 Diterpene Biosynthesis
H
H
HOCH2H OHC H
HO2C H
H
HO2C H
H
O
HO2C
H
HO2C H
H
OH
OHH
H
HO2C
H
H
HO2CC
H
OO
6
H
7
OH
OH
HβHαHαH
HO2C
H
HβHα
·
Hα
gibberellins
H
Hβ6 7
6
HO2C
H
Hα
7
H
OH6
Hα
H6αC O
H7αH
7
C O
H7αH6α
·
(86) (92) (93) (94)
(95) (96) (97)
(98) (99)
(100) (101)
19
Scheme 16
of GAs\ comprehensively reviewed in several recent articles[67Ð79676879 Here discussion will becon_ned to the basic pathway from "099# to biologically active GAs\ with the emphasis on infor!mation arising out of the recent cloning of some of the enzymes involved[ The pathways from GA01!aldehyde "099# are shown in Scheme 06[ Several parallel pathways exist as a result of the di}erenttiming of hydroxylation steps[ The pathways in higher plants "shown in Scheme 06 by bold arrows#di}ers from that in G[ fujikuroi "plain arrows# and they are discussed _rst[ Oxidation of "099# at C!6 to the acid\ with or without hydroxylation at C!02\ gives rise to two pathways\ via GA01 "091# andGA42 "092#[ The sequence of subsequent events involves progressive oxidation and eventual loss ofC!19 with the respective formation of the g!lactones\ GA8 "002# or GA19 "003#\ the parents of thebiologically active GAs[ Stepwise oxidation at C!19 proceeds via the alcohols GA04 "094# or GA33
"095# and the aldehydes GA13 "097# or GA08 "098#[ The stereochemistry of the oxidation of the 19!alcohols to the 19!als is known from deuterium!labeling studies to involve loss of the 19!proRhydrogen atom[70 The next step involves loss of C!19 from the aldehydes "097# or "098# with theformation of the lactones\ GA8 "002# and GA19 "003#\ respectively[ The biologically inactive car!boxylic acids\ GA14 "000# or GA06 "001#\ are also metabolites of the aldehydes\ but are not precursorsof the lactones[
124Diterpene Biosynthesis
H
H
HO2CH
H
HO2C CO2H
R
CHO
H
H
HO2C
HOH2C
CO2H
R
H
H
O
CO2H
CO
OH
O
H
H
HO2C
OHC
CO2H
R
H
H
O
CO2H
CO
19
H
H
HO2C
HO2C
1
3
CO2H
R
20
7
13
(103) GA53, R=OH
H
H
O
(106) GA44, R=OH
(100)
CO2H
(102) GA12, R=H
(107) GA4
(104) GA14
CO
(105) GA15, R=H
R
HO
(122) GA3
(115) ∆-2 GA9, R=H
H
H
O
(109) GA19, R=OH
(114) GA20, R=OH
CO2H(121) GA6(119) GA4, R=H
(113) GA9, R=H
CO
(108) GA24, R=H
(120) GA1, R=OH
R
HO
(112) GA17, R=OH
(111) GA25, R=H
HO
(118) GA8, R=OH
H
H
HO2C CO2HHO
H
H
O
CO2HHO
CO
H
H
O
CO2HHO
CO
OH
H
H
O
CO2H
CO
R
(110) GA7
H
H
O
CO2HHO
CO
R
(117) GA34, R=H
(116) GA5, R=OH
Scheme 17
125 Diterpene Biosynthesis
The enzyme "GA!19!oxidase# involved in the oxidation of C!19 in C[ maxima seeds has beenpuri_ed71 and cloned[72 Related GA!19!oxidases have also been cloned from Arabidopsis thaliana\73
Spinacea oleracea\74 Pisum sativum\75 and Marah macrocarpus[76 These enzymes are members of the1!oxoglutarate!dependent dioxygenase family[ They are soluble enzymes requiring Fe1¦\ ascorbate\and oxygen as well as 1!oxoglutarate\ which acts as cosubstrate with the GA[ The deduced sequencesof these GA!19!oxidases have 41Ð70) identity and contain highly conserved regions[ Two motifs"e[g[\ H132XD and H187R in the C[ maxima enzyme# may be associated with the metal binding site^two histidine residues and an aspartic acid residue have also been identi_ed as metal binding sitesin the related dioxygenase\ isopenicillin!N!synthase\ by X!ray crystallographic studies77 and site!directed mutagenesis[78 There is also an NYYPXCXXP motif that may identify the 1!oxoglutaratebinding site and a LPWKET motif that may be associated with binding of the GA substrate[ Inmany plant species there are multiple enzymes indicating tissue speci_c regulation of GA biosyn!thesis[ However\ it is now evident from heterologous expressing studies in E[ coli that each of theknown GA!19!oxidases catalyze all the steps for the conversion of the C!19 methyl in "091# and"092# through to the g!lactones "002# and "003# and:or to the carboxylic acids "000# and "001#[72\73
The mechanism of oxidative loss of C!19 in the GAs is unusual[ In other systems\ removal ofmethyl groups usually occurs via oxidation to the carboxylic acid followed by decarboxylation\often involving activation by b!carbonyl functions "cf[ loss of 3!methyl groups in cholesterolbiosynthesis# or via oxidation to the aldehyde\ then double bond formation from elimination ofvicinal functions "cf[ aromatase in estrogen biosynthesis#[ In GA biosynthesis\ involvement of the0\2!trans diaxially disposed carboxylic acid function at C!08 appears to lead to a novel mechanism\the details of which are still unknown[ Studies with speci_cally!labeled mevalonate and G[ fujikuroiindicate that adjacent hydrogen atoms at C!0\ 4\ and 8 are retained[89\80 In the same system\ 07O!labeling has shown that both oxygen atoms of the g!lactone arise from the 08!carboxylic acid[81
Results from experiments using cell!free systems of P[ sativum indicate that carbon!19 is releasedultimately as CO1 in the overall conversion of GA01 "091# to g!lactones[82 If a 19!oxidase were theonly enzyme involved\ this result would require two cycles of 1!oxyglutarate turnover\ and thus anintermediate\ between the aldehyde "097# and the g!lactone "002#[ The nature of this intermediate\which could be enzyme!bound\ is not yet known[ The recent availability of recombinant enzymeshould give further insight into this mechanism[
In higher plants the next step in the biosynthesis of bioactive GAs is hydroxylation at C!2b togive GA3 "008# or GA0 "019#[ This step is also catalyzed by 1!oxoglutarate!dependent dioxygenases[Such enzymes have been cloned from A[ thaliana83 and from P[ sativum84 and their function hasbeen con_rmed by heterologous expression in E[ coli[84\85 Both recombinant enzymes convert "002#to "008# and\ less e.ciently\ "003# to "019#^ the fusion protein from A[ thaliana can also oxidize GA4
"005# to the epoxide\ GA5 "010#[85 Previous experiments with partially puri_ed 2b!hydroxylase fromP[ vul`aris embryos and GA19 "003# as substrate had shown that the 1\2!ole_n\ GA4 "005# is also aproduct along with the hydroxy compound "019#[86 The multifunctionality of a 2b!hydroxylase inZea mays has also been suggested from metabolic studies with the dwarf!0 mutant in which theconversion of GA19 "003# to GA0 "019# and of GA4 "005#\ and of GA4 "005# to GA2 "011#\ isblocked[87 Deactivation of the biologically active GA3 "008# and GA0 "019# in plants is accomplishedby further hydroxylation at C!1b\ by 1!oxoglutarate!dependent dioxygenases\88 to give the bio!logically inactive GA23 "006# and GA7 "007#[
The biosynthesis of the majority of the 099 or so gibberellins found in plants follows thebasic linear pathway described above[ Structural variation is achieved by hydroxylation at variouspositions at di}erent stages in the pathway[ Common positions for hydroxylation additional tothose in Scheme 06 are at C!0\ 00\ 01\ and 04[ The result is the occurrence of parallel pathways\often in the same plant\ with cross!over points\ creating metabolic grids of intermediates formedprobably by a small number of enzymes acting on a number of structurally related substrates[ Oneunusual feature of GA biosynthesis in plants is the formation of GA2 "gibberellic acid\ "011## andGA6 "009#[ These GAs are formed\ respectively\ by an unusual ene!hydroxylation of the 1\2!ole_ns\GA4 "005# and 1\2!dehydroGA8 "004#\ initiated by abstraction of the 0b!hydrogen demonstratedusing substrates speci_cally labeled with deuterium[099 The pathway to GA2 and GA6 in plants isdi}erent from that in the fungus\ G[ fujikuroi[ In the fungal pathway\ shown in Scheme 06 by normalarrows\ 2b!hydroxylation occurs early\ at the GA01!aldehyde stage\ then 6!oxidation gives GA03
"093#[ Oxidation at C!19 then occurs\ presumably by the same sequence that occurs in plants\ togive GA3 "096#\ a compound common to both plant and fungal pathways[ In the fungus\ metabolismto GA2 then occurs by 0\1!dehydrogenation to give GA6 "009#\ a process in which the 0a and 1a!Hatoms are eliminated\099 followed by hydroxylation at C!02[ Little is known about the enzymologyof fungal GA biosynthesis[ Preparation of cell!free systems that take precursors beyond GA03 has
126Diterpene Biosynthesis
not been achieved\ and at the present time it is not clear if the enzymes concerned are 1!oxoglutarate!dependent dioxygenases as in plants[
Although the majority of GAs are derived from ent!kaurenoid precursors by variations in thebasic pathways shown in Scheme 06\ the recent discoveries of ring C:D variants in higher plants090\091
and ferns092Ð094092093094 "Scheme 07# indicates that other tetracyclic and pentacyclic hydrocarbonsmay give rise to gibberellin!like structures[ Indeed\ the fungal enzymes do show a remarkable degreeof nonspeci_city and convert a range of {{unnatural|| ent!kaurenonids to {{unnatural|| fungal GAs^this topic and the fungal biosynthesis of GAs is comprehensively reviewed by Bearder[095 However\as shown in Scheme 07 by dashed arrows\ formation of the ring C:D structures present in 8\00dehydro!GAs "e[g[\ "013##\ 01\04 cyclo!GAs "e[g[\ "014#\ "015##\ and antheridic acid "016#\ can berationalized as rearrangements of the 8!radical "012# from GA8 "002#[ An alternative route to "014#could also be from the C!04 radical of "002#[ The only pertinent experimental information comesfrom the observed metabolism of "014# to "015# and "015# to "016# in Anemia phyllitidis cultures[094
H
CO2H
O
CO
CO2H
O
CO
H
H
CO2H
O
CO
CO2H
O
CO
H
H
CO2H
O
CO
CO2H
O
CO
CO2H
O
COOH
b
CO2H
O
CO
a
˙9
HO
bd
˙ce
e
ca d
(113) (123)
(124) (125) (126) (127)
HO
Scheme 18
The all!trans cyclization of E\E\E!GGPP to ent!CPP has been established by] "i# the exclusivelabeling48 of C!07\ and not C!08\ in GA2 from ð1!03CŁMVA "see also rosenolactone "60#\ Scheme01#^ "ii# retention82 of the 3!proR!hydrogen from MVA at C!2 and C!8^ and "iii# the demonstration096
that the 3!proR!hydrogen from MVA at C!2 in ent!kaur!05!ene is lost by hydroxylation withretention of con_guration[ This is one of the few documented cases of the generally accepted all!trans!cyclization of GGPP[
1[97[5[1 From ent!CPP by si!Face Cyclization on C!02
Cyclization of the 02!epimer of the 7!carbonium ion ""76#\ Scheme 04#\ derived from ent!CPP"22# by si!face attack on C!02\ would give ring C:D enantiomers of the structures shown in Scheme04[ None are known[
1[97[5[2 From CPP by re!Face Cyclization on C!02
Cyclization of the 7!carbonium ion ""43#\ Scheme 00#\ derived from CPP "29# by re!face attackon C!02\ gives the carbon skeletons corresponding to those in Scheme 04 but with the {{normal||absolute stereochemistry at C!4\ !8\ and !09[ Only phyllocladene ""017#\ Scheme 08# is known andno biosynthetic studies have been reported[
127 Diterpene Biosynthesis
H
H
H
H
H
H
H
H
H
H
H
H
H
+ +
H
H
(30) CPP
H
H
+
+
re-faceat C-13
+
+H
(128) phyllocladene
(129) kaur-16-ene(63)
(54)
si-faceat C-13
13
13
Scheme 19
1[97[5[3 From CPP by si!Face Cyclization on C!02
Cyclization of the 7!carbonium ion ""52#\ Scheme 08#\ derived from CPP "29# by si!face attack onC!02\ gives the enantiomers of the structures shown in Scheme 04[ Of these enantiomers only kaur!05!ene ""018#\ Scheme 08# is known but no biosynthetic studies have been reported[
1[97[5[4 From ent!CPP by re!Face Cyclization on C!02\ then 8H:C!7
Cyclization of the 7!carbonium ion ""029#\ Scheme 19#\ derived from ent!CPP "22# by re!faceattack on C!02\ then 8H:C!7 in "76#\ provides a possible origin for helifulvanic acid "020#\ isolatedfrom Helichrysum chionosphaerum[53 This plant also contains ent!atisir!05!ene ""89#\ Scheme 04#\ent!kaur!05!ene ""75#\ Scheme 04#\ and ent!abieta!6\02!diene "the enantiomer of "48#\ Scheme 00#\indicating the presence of at least two di}erent GGPP cyclases in H[ chionosphaerum[ The 00a!hydroxy derivative "021# of helifulvanic acid "020# and ent!kaur!05!en!08!oic acid ""83#\ Scheme 05#also co!occur in H[ fulvum\097 indicating that cyclization of the unrearranged ion "76# and therearranged ion "029# takes place in the same plant[
Other products that may arise from the ion "029# have not been reported[
1[97[5[5 From ent!CPP by si!Face Cyclization on C!02\ then 8H:C!7
The postulated cyclization "Scheme 19# of the 7!carbonium ion "022#\ derived from ent!CPP "22#by si!face attack on C!02\ then 8H:C!7 in "64#\ gives the ent!7a!stemarenes\ e[g[\ the villanovanes"023#\ which have been isolated from Villanova titicaenis[098 No experimental evidence for thispathway is available[
1[97[5[6 From syn!CPP by re!Face Cyclization on C!02\ then 8H:C!7
Cyclization of the 7!carbonium ion "025#\ derived from syn!CPP "25# by re!face attack on C!02to "024#\ then 8H:C!7\ is shown in Scheme 10[ Pathway a from "026# via "027# is the probablepathway to the known stemodanes\ stemodin "028# and stemodinone "039#[ Pathway b\ from "026#via "030#\ gives stemar!05!ene "031# which is formed from syn!CPP "25# in rice suspension culturesthat have been treated with chitin[28 Stemar!05!ene "031# is the presumed precursor of the phyto!alexin\ oryzalexin S "032#\ found in UV!irradiated rice leaves[009 It is also the probable precursor ofstemarin "033#\ occurring in Stemodia maritima L[\000 and of the stemodanes that occur in S[chiensis[001
128Diterpene Biosynthesis
H
H
H
H
H
H
HO2C H
H
H
H
H
H
H
H
R2CH2H
H
CH2R3
OH
R1
+
+
+
(33) ent-CPP
(133)
H
(134) villanovanes
+H
(131) R=H, helifulvanic acid(132) R=OH
H
13
(87)
+H
13
(75)
re
R
si
(130)
+
+
Scheme 20
1[97[5[7 From syn!CPP by si!Face Cyclization on C!02\ then 8H:C!7
Cyclization of the 7!carbonium ion "65#\ derived from syn!CPP "25# by si!face attack on C!02\then 8H:C!7\ is shown in Scheme 10[ The sequence via "037#\ "038#\ and "049# leads to scopadulin"034# and aphidicolin "036#[ The sequence via "040# gives the related scopadulcic acids "e[g[\ 041#and the thyrsi~orins "042#[
1[97[5[7[0 Aphidicolin
Aphidicolin "036# is the major metabolite of the fungus Cephalosporium aphidicola[ Its originfrom syn!CPP "25# has not been directly established but can be inferred from the established 7bH!stereochemistry002 and the generation of 1HÐ02C coupling at C!7 in the 1H!NMR spectrum ofaphidicolin\ biosynthesized from ð3!1H1\02CŁMVA[003 These _ndings implicate the intermediate "037#\derived from "65# by a 8b!H:C!7 shift[ The 05!OH is derived from H1O in the _rst step from the05!carbonium ion "049#[004 Subsequent hydroxylation of "035#\ in the main pathway to aphidicolin"036#\ appears to proceed in the order\ C!07\ C!2\ C!06\ on the basis of the observed metabolism ofintermediates[005 However\ the addition of cytochrome P!349 inhibitors indicate that C!2 hydroxy!lation may be the last step[006
1[97[5[7[1 Scopadulcic acids and thyrsi~orins
The biosynthesis of scopadulcic acid B "041# from the herb Scoparia dulcis L[ has not been studiedbut a speculative pathway has been suggested007 from the aphidicolin intermediate "049# via "040#
139 Diterpene Biosynthesis
H
H H
H
H
H
H
H
H
H
RCH2H
H
R
H
H
R
OH
H
H H
H
H
H
H
H
H
H
OH
HOCH2H
H
OH
HO
H
9
H
+
HO2CH
+
H
+
O
OCOPhH
H
8
OH
OR2
+
OR1
+
(36) synCPP
re-face at C-13
+
98 +
++ +
CH2OHH
H
OH
a
si-face at C-13
a
b
b
c
c
(142) R=H, stemar-16-ene(143) R=OH, oryzalexin S
(147) aphidicolin
HO2CH
H
OH
(152) scopadulcic acid B
(144) stemarin
OCOPh
(153) thyrsiflorins
(139) R=H, α-OH, stemodin(140) R=O, stemodinone
CH2OH
(145) scopadulin
3
18
1617
(135) (136) (137) (138)
(141)
(146)
(76) (148) (149) (150) (151)
Scheme 21
130Diterpene Biosynthesis
as shown in Scheme 10[ The co!occurrence of "034# and "041# in S[ dulcis lends credence to theircommon origin[008 The thrysi~orins "042# from the herb Calceolaria thyrsi~ora019 have the samepresumptive origin[
1[97[5[8 Miscellany
Cyclization of syn!ent!CCP "28# by re! or si!face attack on C!02\ then 8H:C!7\ leads to theenantiomers of the compounds shown in Scheme 10 but none are known[ ent!Stemarenes have beenreported to occur in Calceolaria species although the evidence for their absolute stereochemistry isnot de_nitive[010
No tetra! or pentacyclic diterpenes are known from the cyclization of CPP by re! or si!face of C!06 on C!02\ then 8H:C!7^ from the cyclization of syn!CPP by re! or si!face on C!02^ or from thecyclization of syn!ent!CPP by re! or si!face on C!02[
1[97[6 SUMMARY AND FUTURE PROSPECTS
It has been shown that the diverse structures of the cyclic diterpenes arise by di}erent modes ofcyclization of GGPP\ then by oxidation of the cyclization products[ Details of these processes areemerging through the puri_cation and cloning of the enzymes involved[
Four types of GGPP cyclases have been cloned and the function of their gene products has beendetermined[ Thus\ four types of cyclization of GGPP have been established\ depending on theinitiation of cyclization "protonation or diphosphate ionization#[ Furthermore\ each of the fourclasses of cyclases are multifunctional\ catalyzing a series of steps on the enzyme surface[ Since theseenzymes can now be overexpressed in vitro\ detailed study of the mechanisms of cyclization can beanticipated[ Rapid advances in the characterization of other cyclases can also be anticipated usingconserved sequences of the known genes and PCR[ Questions of particular interest to be addressedare the existence of enantiomeric products of the cyclization of GGPP and the diastereotopiccyclization of the 5\5!bicyclic products from the proton!initiated cyclization of GGPP[
The oxidative metabolism of the products of cyclization of GGPP has only been studied indetail for a few groups of diterpenes[ Details information on the soluble 1!oxoglutarate!dependentdioxygenases is emerging from studies on gibberellin biosynthesis[ Characterization of the micro!somal P349 oxidases\ however\ has been less tractable[
The progress that has been made sets the scene for the genetic engineering of diterpene biosynthesisas a means of studying the regulation of pathways and ~ux of precursors into them\ and also tomanipulate diterpene synthesis to give desirable properties[ Uses may range from the production ofrare medicinal terpenes in amenable plants or microorganisms to the manipulation of plant devel!opment by sense or antisense expression of gibberellin biosynthesis genes\ a goal that has alreadybeen achieved in Arabidopsis\ or to the manipulation of the plant defense response via terpenoidphytoalexins and antifeedant compounds[
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