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Transcript
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
NPTEL Phase – II (Syllabus Template)
Course Title: Bio-Organic Chemistry of Natural Enediyne
Anticancer Antibiotics
Module II:
Synthesis/Biosynthesis of Enediynes Class of Natural Products: Classifications of Natural
Enediynes-Calicheamicins/ Esperamicins class of enediynes (Class I), The Dynemicins class of
enediynes (Class II), and The Chromoprotein class of enediynes (Class III); Mechanism of DNA
Cleavage by Each Class; Chemical Synthesis of a Few Members of Enediynes Natural Products;
Biosynthesis of a Few Members of Natural Enediynes.
2.1. Classifications of Natural Enediynes
2.1.1. Introduction
Enediynes are a class of bacterial natural products characterized by either nine- and tenmembered rings containing two triple bonds separated by a double bond. In the mid to late
1980s, it became clear that an emerging series of naturally occurring antitumor antibiotics such
as calicheamicin, esperamicin, dynemicin, kedarcidin chromophore and C-1027 chromophore
possessing the enediyne core and showed biological activity through the generation of active
biradical specis via Bergman cyclization. In addition to that neocarzinostatin (NCS)
chromophore which does not contain the classical conjugated enediyne system also demonstrated
very similar DNA cleavage mechanism via the generation biradical species through the MyersSaito cyclization. The enediynes are in their native form biologically inactive but undergo
cycloaromatization reactions after being activated by a triggering reaction and produces the
active biradical species. Therefore, the enediyne group in those compounds is often called a
warhead. For example, the strain imposed by the double bond in calicheamicin or by the epoxide
in dynemicin imparts stability to the system. Cycloaromatization of these natural products then
give rise to cytotoxic diyl radicals which are capable of inducing DNA strand scission at low
concentration by abstracting –H atom from the sugar phosphate backbone of DNA. Several of
the naturally occurring enediynes have entered clinical trials against cancer and in Japan
neocarzinostatin is used clinically.
The biological profile of the calicheamicin and esperamicins are:
(a)
(b)
(c)
(d)
subpicogram potency against Gram positive bacteria,
activity in the biochemical induction assay at very low concentrations,
high potency against a number of animal tumor models and,
induction of double-stranded DNA cleavage with minimal concurrent single strand
breakage.
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.1.2. Classifications of Natural Enediynes
During the 1980s enediynes as a new class of natural products have been introduced with the
structural elucidation of neocarzinostatin and calicheamicin. Since this time, thirteen enediynes
have been structurally confirmed, which includes two probable enediynes isolated as inactive
degradation products (Table 1; Figure 1).
Table 1: Enediyne Natural Products
Name
Auromomycin
Largomycin
Actinoxanthin
Sporamycin
Neocarzinostatin
C-1027
Maduropeptin
Kedarcidin
N1999A2
Sporolides A and B
Cyanosporasides A and B
Esperamicin
Calicheamicin
Dynemycin
Namenamicin
Shishijimicin
Uncialamycin
Producer
Nine-Membered Enediynes
Streptomyces macromomyceticus
Streptomyces pluricolorescens
Actinomyces globisporus
Streptosporangium pseudovulgare
Streptomyces carzinostaticus
Streptomyces globisporus
Actinomadura madurea
Actinomycete L585-6
Streptomyces sp. AJ9493
Salinispora tropica
Salinispora pacifica
Ten-Membered Enediynes
Actinomadura verrucosospora
Micromonospora echinospora ssp. calichensis
Micromonospora chersina
Polysyncraton lithostrotum
Didemnum proliferum
Unknown
Year
1968 [3]
1970 [4]
1976 [5]
1978 [6]
1985 [1]
1991 [7]
1994 [8]
1997 [9]
1998 [10]
2005 [11]
2006 [12]
1985 [13]
1987 [2]
1990 [14]
1996 [15]
2003 [16]
2005 [17]
These natural antitumor antibiotics are classified under three classes:
(a) The Calicheamicins and Esperamicins.
(b) The Dynemicins
(c) The Chromophore types; Kedarcidin chromophore, C-1027 and Neocarzinostatin.
Even though these natural antitumor antibiotics possess phenomenal cytotoxicity against
tumor cells they are too toxic and indiscriminant for use as drugs, hence efforts have been made
to synthesize various derivatives of these compounds. A notable example is gemtuzumab
ozogamicin (Mylotarg), which is a derivative of calicheamicin conjugated to a humanized antiCD33 antibody; the drug is indicated for the treatment of acute myeloid leukemia (AML).
However this has been withdrawn because of its strong cytotoxicity.
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Figure 1. Structures of Enediyne class of natural products.
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.2. Mechanism of DNA Cleavage
2.2.1. Calicheamicins and Esperamicins
The Calicheamicins (also known as the LL-E 33288 antibiotics) produced from
Micromonospora echinospora spp. Calichensis, a bacterium was discovered by May. D. Lee et
al., in 1987. It is the most important member of the enediyne class of natural products, and
possesses potent cytotoxicity against murine tumor cells.
Esperamicin A1 is also another member of the enediyne family of antibiotics exhibiting
activity against marine tumor models in the 100ng/kg range. The families of Esperamicins were
isolated from the bacterial Actinomadura verrucosospora and their structure elucidation was
reported in 1987-89. The antitumor antibiotic drugs, calicheamicin, dynemicin, and esperamicin,
all possessed bicyclo-[7,3,1]-enediyne substructure and become active p-benzyne biradical
intermediates due to Bergman cyclizations. Precisely the reactive intermediate is proposed to be
a 1, 4-dehydrobenzene derivative which is suggested to arise thermally from (Z)-enediyne in a
cyclic version of the Bergman reaction.
The mechanistic studies have revealed that at a minimum, three common features are
essential to the show the potent DNA cleavage activity by these antibiotics:
(a) non-destructive high-affinity binding to DNA,
(b) a chemical trigging mechanism leading to a high–energy intermediate
(c) rapid formation of biradical specis at physiological temperatures which is mainly
responsible for DNA strand scission.
Natural enediyne Calicheamicins and Esperamicins family
SSSMe
O
SSSMe
O
NHCO2Me
Me
H
HO
O
O
OH
Me
O
O
O
OH
O
N
H
O
Me
S
HO
MeO
Me
OMe
I
OMe
O
Calicheamicin  1I
O
Me
O
HO
MeO
OH
HO
O
O
O
NH
O
OH
Me
O
MeO
MeO
EtHN
H
O
O
NHCO2Me
O
N
H
OH
OMe
OMe
NH
O
Me
Me
Esperamicin A1
Figure 2. Structures of Calicheamicin  1I and Esperamicin A1.
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Page 4 of 89
O
Me
SMe
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
The esperamicins and the calicheamicins both share similar structures and their structures
possess three distinct domains: (a) an oligosaccharide chain, (b) a trisulphide moiety, and (c) an
enediyne core. Each of these domains has a specific function in DNA cleavage.
(a) The oligosaccharide chain recognises and targets selected base pair sequences in the
minor groove of DNA. Thus, the molecule binds selectively to the minor groove through
hydrophobic, electrostatic interactions and hydrogen bonding of the sugar side chain with
DNA. The natural enediynes are actually stable until they are bonded to DNA and then
become activated.
(b) After binding to minor groove, the trisulphide then serves as a molecular trigger which
upon reductive activation produces thiolate. The thiolate then performs an intramolecular
Michael addition onto the proximally positioned enone moiety to unlock the enediyne
warhead. This leads a change in the geometry of the molecule from a trigonal bridgehead
to a tetragonal centre. Thus, ―cd‖ distance between the two triple bonds is reduced. The
decrease has been calculated to be from 3.35 to 3.16 Å distance which is close enough for
spontaneous Bergman cyclization according to Nicolaou‘s theory.
(c) Bergman cycloaromatization of the enediyne structural motif generates a p-benzyne
diradical which abstracts hydrogen from DNA backbone. The reaction of the DNA
backbone radicals with molecular oxygen results in double strand cleavage which
ultimately lead to permanent damage of the genetic material.
The enediyne systems in both the calicheamicin and esperamicin could easily be triggered to
aromatize via a free-radical intermediate by cleavage at the methyl trisulfide moiety. This
aromatization process is responsible for the remarkable DNA damaging effects of the
calcheamicin and the esperamicins.
Mechanism of DNA Cleavage by Calicheamicins
Trisulfide reduction
initiates the activation
Intercalates
into DNA
Calicheamicins
Responsible for
DNA strand scission
Scheme 1. Mechanism of DNA cleavage by Calicheamicin.
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.2.2. Dynemicins
Dynemicin A (DNY-A) is the first known member of the family dynemicin A 1.187. It was
isolated from Micromonospora chersina M956-1109 strain and the recent member
deoxydynemicin A 1.187b was obtained from Micromoonspora globosa MG331-HF6.
Dynemicin contain a bicyclo[7.3.1]enediyne substructure which may be related biosynthetically
to the cores of calicheamicin and esperamicin. The dynemicins has a striking hybrid structure
that contains (a) the cyclic enediyne, (b) an anthraquinone chromophore. Unlike the other
members of this class, it exhibits antibacterial and antitumor activity with low toxicity. As a
result of their intriguing and unique structural characteristics, various strategies have been
developed to provide a synthetic route towards the natural and the non-natural dynemicin and its
analogues.
Natural enediyne Dynemicins family
OH O
H
HN
Me
CO2H
O
O H
N
PhO
O
OMe
H
R
O
OH
R = -OH; Dynemicin A
Dynemicin Model
R = H;
Deoxydynemicin A
Figure 3. Structures of natural Dynemicin A and its model compound.
Nicolaou et al., reported the synthesis of the dynemicin model compound (Figure 3) to
demonstrate the mechanism of cyclization reactions of dynemicin A. In this model the critical
distance (cd) was found to be 3.59 Å (popularly known as cd distance), a value that agrees with
the X-ray crystallographic analysis of dynemicin A (3.54 Å).
The mechanism of the cyclization reaction is as below (Scheme 2):
(a) Protonation of the epoxide group initiates the formation of diol
(b) Spontaneous Bergman cyclization to form benzenoid biradical.
(c) Rapid trapping of the biradical by the hydrogen donor present to give the cyclized
product.
This cyclization is analogous to those observed for dynemicin A. The pharmacological
activity of this model compound is believed to be related to dynemicin A‘s ability to cleave DNA
following its intercalation into DNA with its anthraquinone which in actual fact is typical of
most enediyne cyclization reactions. It is the benzenoid biradical that is actually responsible for
the cleavage of the DNA molecule as illustrated in the scheme below, (Scheme 3).
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Scheme 2. Mechanism of the cyclization reaction of a dynemicin model compound.
Mechanism of Biological Action of Dynemicin A
OH O
H
HN
Me
CO2H
O
OMe
Dynemicin
OH O
OH O
Me
H
HN
HO
CO2H Path A
OH
H
OH OH HN
Me
CO2H
Path B
O
OH O
H
HN
HO
OMe
Me
CO2H
H+
OMe
OMe
OH OH OH
OH OH OH
OH OH OH H2O or Nu
Proton Transfer
Nucleophilic attack
Me
H
OH OH HN
HO
OH O
CO2H
OH O
OH OH OH
OH OH OH
Me
CO2H
OH
OH OH OH
OMe
OMe
Cycloaromatization
CO2H
DNA
OMe
OH
DNA
OH
Me
H
OH OH HN
HO
Cycloaromatization
H
OH OH HN
HO
CO2H
H
OMe
OH
Me
H
HN
HO
O2
OH O
Me
H
HN
HO
DNA Diradical
OH O
CO2H
H
OMe
O2
DNA Double
Strand Cleavage
OH O
H
HN
HO
Me
CO2H
OH
OH O
OH O
OH O
OH
DNA Double
Strand Cleavage
OMe
OH
Scheme 3. Mechanism of biological action of dynemicin A.
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CO2H
H
DNA
Diradical
O2
Me
H
HN
HO
Page 7 of 89
OH
OMe
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.2.3. The Chromoprotein Types Enediyne Class
2.2.3.1. Neocarzinostatin Chromophore (NCS)
NCS is the first enediyne antibiotic that was first isolated from a culture of Streptomyces
carzinostaticus var. F-41 in 1965. Its potent antibacterial and antitumor activities derived from
the inhibition of DNA synthesis and DNA degradation in cells. It is composed of a very unstable
chromophore and a carrier apoprotein. The neocarzinostatin core is slightly different from the
basic enediyne structure. It contains the bicyclo[7.3.0]dodecenediyne and shows its biological
activity through the involvement of the allene eneyne system. Meyer-Saito cyclization (MSC) is
believed as the key step in the mechanism of action of the antitumor agent neocarzinostatin
chromophore through which it produced a 3, 7-dehydroindene derivative as shown below
(Scheme 4). The generated biradical via MSC is responsible to abstract H-atom from DNA
backbone leading to the damage of DNA.
RSH
OMe
O
O
C
MeO
O
O
O
O
OH
O
Path A
MeHN
HO
HO Me
Path B
O
Neocarzinostatin
Chromophore
O
O
O
Ar
O
O SR
O
OH
Sugar O
O
O
C C
OH
C
C
O
O SR
Ar
Sugar O
O
O
O
O
RS
O
OH
RS
Ar
OH
Ar
O
O
O
O
O
Sugar O
Triggering pathways for the
neocarzinostatin chromophore
DNA
H- abst ruction
Sugar O
O
Ar
RS
H2O
DNA diradical
(Cleavage)
O
O
H
O
O
O
RS
O
OH
O
Sugar O
OH
Ar
H
O
HO
O
Sugar O
Scheme 4. Mechanism of DNA cleavage by Neocarzinostatin (zinostatin).
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
The new antibiotic N1999A2 isolated from the broth filtrate of Streptomyces sp. AJ9493,
possesses a novel 9-membered ring enediyne chromophore similar to neocarzinostatin. But
N1999A2 is not chromoprotein. The most interesting feature is that the stable N1999A2 exists as
enediyne chromophore alone. The antibiotic N1999A2, the structure of which is given below
(Figure 4.), revealed more random DNA cutting profile than neocarzinostatin chromophore.
OMe
Cl
HO
O
HO
HO
O
O
OH
O
MeHN
HO
HO Me
O
N1999A2
Figure 4. Structure of enediyne antibiotic N1999A2.
2.2.3.2. C-1027 Chromophore
C-1027 is one of the most potent antitumor antibiotic chromoproteins. It composed of an 11kDa apoprotein and a highly reactive chromophore. The C-1027 chromophore is in equilibrium
with its active biradical form in the apoprotein and unlike NCS does not need nucleophiles or
radicals for its activation. The p-benzyne biradical thus generated exerts its potent biological
activity by abstracting hydrogen atoms from the sugar portion of double stranded DNA, which
ultimately leads to oxidative cleavage of DNA (Scheme 5).
Mechanism of Biological Action of C-1027 Chromophore
MeO
O
C-1027 Chromophore
O
O
N
H
MeO
MeO
O
O
O
O
N
O
H H- abstruction
O
DNA
O
N
OH
OH
O
N
O
OH
O
Cl
OH
O
O
OH
OH
O
O
Cl
O
O
OH
O
O
DNA diradical
(Cleavage)
Cl
Scheme 5. Cycloaromatization process of C-1027 chromophore.
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O
O
N
O
O
Page 9 of 89
O
O
N
H
O
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.2.3.3. Kedarcidin Chromophore
Kedarcidin is a new chromoprotein antitumor antibiotic that was isolated from the
fermentation broth of a novel actinomycete strain. It consists of an apoprotein and a cytotoxic,
highly labile, non protein chromophore. The apoprotein is water soluble while the chromophore
is solvent-extractable, cytotoxic and highly unstable. As with NCS, the antitumor activity of
kedarcidin is due primarily to the chromophore. The enediyne core is activated by chemical
reduction (e.g. sodium borohydride) followed by spontaneous cyclization to a biradical
intermediate and DNA cleavage (Scheme 6).
Me
Me
R
O
O
MeO
Cl
OMe H
N
NH
R
OH
O
O
o
Nucleophilic
H
Attack
O
RO
O
O
Me
HO Me
OH
O
O
Kedarcidin Chromophore
Me
OH Cycloaromatization
RO
Me NMe2
NMe2
OH
O
OH
O
Me
HO Me
Nu
OH
O
Nu
Nu
O
O
O
O
Me
Me
N
Me
Me
HO Me
HO
OH
O
DNA
DNA Single Strand Cleavage
O2
DNA Radical
R
O
Proposed mechanism of action of the kedarcidin chromophore
OH
O
Nu
Me
NMe2
RO
OH
O
Me
HO Me
OH
O
Scheme 6. Mechanism of Biological action of Kedarcidin chromophore.
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.3.
Chemical Synthesis of a Few Members of Enediynes Natural Products
2.3.1. Introduction
Figure 5. History and basic strategies of total synthesis of enediyne natural products.
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.3.2. Total Synthesis of Neocarzinostatin
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.3.3. Total Synthesis of Calicheamicin
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.3.4. Total Synthesis of Dynemicin A
Retrosynthetic Analysis for the synthesis of Dynemicin A
Carboxylation
Me
N
CO2TIPS
(Myers)
OH O
H
HN
O
Me
CO2H
O
OMe
OMe
40
(Schreiber)
H
H
HN
Me
CO2H
O
OMe
Dynemicin A
OH O
O
H
OH
Intramolecular
Nucleophilic Addtion
(Danishefsky)
42
OH
Transannular
Diels-Alder
Tandem Stille
reaction
Yamaguchi
alkynylation
Me
N
CO2MOM
O
OMe
H
Yamaguchi
alkynylation
Me
AllocN
Amidation
OH
43
OTBS
Yamaguchi
alkynylation
OMe
OMe
O
Suzuki reaction
Stille reaction
41
O
Epoxidation
Me
Me
N
HN
44
45
OTBS
OH
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O
Diels-Alder
reaction
O
H
OH
O
Yamaguchi
macrolactonization
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NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Myers's Synthesis of Dynamicin A
Me
Me
O
Me
O
O
1) Pd(PPh3)4, Na2CO3
[49], 1,4-dioxane, 1000 C
2) 4-ClC6H4OH, 1800 C
Me
3 steps
MeO
Me
O
EtO
Me
46
MeO
O
TfO
OMe
OMe
NHBOC
50
1) KHMDS
CeCl3, THF
2) p-TsOH
acetone
H
Me
3 steps
AllocN
TBS
Me
OMe
OMe
O
OH
AllocN
OH
OTBS
Me
AllocN
O
O
OMe
OMe
O
OTBS
MgBr
52
OMe
TBS
1) [52], EtMgBr
THF, AllylOCOCl
2) mCPBA, DCM
PH 7 buffer
N
N
B(OH)2
49
48
47
OMenMe
OMe
OMenMe
OH
OH
OTBS
54
53
55
OTBS
OTMS
57
Me
N
9 steps
O
OTMS
CO2TIPS
OTMS OTMS
O
Me
CO2H
MnO2, 3HF·Et3N
O
H
OSMT H O
OTMS
56
Dynemicin A
OMe
O
OMe
O
H
H
N
58
Myers, A. G.; Tom, N. J.; Fraley, M. E.; Cohen, S. B.; Madar, D. J. J. Am. Chem. Soc. 1997, 119, 6072-6094
Danishefsky's total synthetic route to Dynemicin A
Me
H
OH
Me
O
CHO
4 steps
O
ZnCl2,
CHO DCM, 25 °C
H
60%
OMe
OMe
59
OMe
60
Me
CHO
AllocN
O
Ph
OH
OH
OTBS
OTBS
62
63
Me
O
Ph
O
Ph
12 steps
Teoc
Teoc
Me3Sn
SnMe3
N
Pd(PPh3)4, DMF, 75°C
O
OH
OH
O
OH
OH
81%
H
H
OTBS
OTBS
2) NH4OAc
HOAc 100°C
Me
N
80%
Ph
I
Me
TIPS
AllylOCOCl, THF
I
O
N
3 steps
80%
TIPS
BrMg
1) CAN, MeCN
H2O
61
Me
N
64
OTBS
OTBS
65
66
OMOM
O
1) Tf2O, py
2) DMP, DCM
3) CrCl2, THF
Me
Teoc
N
4 steps
N
O
87%
CO2MOM
67
O
69
Me
CO2H
O
OMe
OMe
H
NOMO
H
OH O HN
O
O
OTBS
O
Me
and 3 steps
H
O
68
OH O
OH
Dynemicin A
Shair, M. D.; Yoon, T. Y.; Mosny, K. K.; Chou, T. C.; Danishefsky, S. J. J. Am. Chem. Soc. 1996, 118, 9509-9525.
Joint initiative of IITs and IISc – Funded by MHRD
Page 29 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Schreiber's total synthetic route to Dynemicin A
SnBu3
1)
BTSO
N
Br
Me
MeO
SiMe2thexyl
OMe
70
CO2H
1) TBAF, THF
2) BrCH=CHCO2Me
Pd(PPh3)4, CuI
3) LiOH H2O THF
SiMe2thexyl
O
PyBrOP, Et3 N
MeO
O
Cl
N
N
2) ClCO2Me,
Me
TBSO
OH
71
O
Me
MeO
N
O
H
OMe
OMe
H
H
N
O
O
O
CO2Me
OMe O
O
H
HN
75
N
Br P N
N
CO2H
OMe
OMe
2) K2CO3, MeI, acetone
O
O
H
OMe Br
Dynemicin A
OMe
OMe O
OMe Br AgOTf, 4A MS
Me
OMe
O
H
OMe
Me
O
O
O
CO2Me
74
OMe
Me
N
O
O
OMe
O
MeO2C
N
O
73
O
Me
Cl
72
Me
1)
O
COCl
OMe
OMe
MgBr
MeO
Cl
thexyl =
PyBrOP
76
Taunton, J.; Wood, J. L.; Schreiber, S. L. J. Am. Chem. Soc. 1993, 115, 10378-10379
2.3.5. Total Synthesis of N1999A2
Retrosynthetic Analysis for the synthesis of N1999A2
Ester Formation
OH
O
OH
HO
O
O
HO
OMe
Cl
N1999A2
Sonogashira
Coupling
Intermolecular
Nucleophilic
Addition
Transannular
Reaction
PO
O
I
PO
O
HO
P
OP
OP
PO
(Hirama)
OH
Sonogashira
Coupling
H
NMe
O
O
(Myers)
O
H
OH
Br
OP
PO
Elimination
O
PO
P
M
I
O
O
Glaser Coupling
Intramolecular
Nucleophilic
Addition
OP
O
BTSO
I
H
M
PH2
PO
Joint initiative of IITs and IISc – Funded by MHRD
P
OP
OH
SnBu3
P
Page 30 of 89
OH
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Hirama's Total Synthetic Route to N1999A2
1) [A], Pd(PPh3)4, CuI
iPr2NEt, DMF, r.t.
2) TBAF, THF
3) TESOTf, 2,6-lutidine,
-78 oC
TBSO
TBSO
2 Steps
I
I
TMS
TMSO
O
O
O
O
1) DIBAL, DCM, -78 oC
2) DMP, Pyr, DCM, r.t.
OTES
TBSO
78%
over all yield
OPiV
O
TESO
PivO
O
O
O
LiHMDS, CeCl3
0
OTES THF, - 30 C
TBSO
OH
A
O
TBSO
O
OTES
OTES
68%
O
O
1) MsCl Et3N DMAP
2) TBAF, THF, -15 oC HO
63%
TMSO
OH
TESO
OH
TMSO
O
O
OTBS
O
Cl
B
OH
CO2H
DCM, -85 C
2) Et3N, DMAP, Ms2O
HO
OH
2) TFA, THF, H2O
MeO
OH
OH 1) TESOTf, 2,6-lutidine
1) [B], DCC, THF
HO
TBSO
Cl
OMe
HO
OH
O
O
O
3) TFA, THF, H2O, 30C
35%
HO
Cl
OMe
N1999A2
54%
Kobayashi, S.; Ashizawa, S.; Takahashi, Y.; Sugiura, Y.; Nagaoka, M.; Lear, M. J.; Hirama, M. J. Am. Chem. Soc. 2001, 123, 11294.
Joint initiative of IITs and IISc – Funded by MHRD
Page 31 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Myers' Total Synthetic Route to N1999A2
O
THPO
TES
LiHMDS,
1) n-BuLi, Bu3SnCl, THF
2) Cp2Zr(H)Cl, THF,
Then I2, THF
H
HO
OTHP 5 Steps TBSO
BF3-OEt2
TES
TES
2-Steps TMS
PPh3
TBS
n-BuLi, LiI
O
O
1) (DHQD)2PYR,
K2OsO4, 2H2O
K3Fe(CN)6, K2CO3,
MsNH2, t-BuOH
2) FeCl3.6H2O, MeCN
3)[B], CSA, THF
Br
1)
O
O
MeS
B
Benzene
64%
Then t-BuLi
Then HOAc
Mes
O
OH
OH
SnBu3
TES
1) TBAF, THF
2) DEIPSCl, TBSO
Imidazole
3) NBS, DCM
72%
SnBu3
H
TBS
OSiEt2(i-Pr)
LiHMDS,
THF-Toluene
H
H
O
O
TBS
OMe
H
Mes
O
O Cu(OAC)
2
THF-Pyr
OH
H
OH
Mes
OSiEt2(i-Pr) O
O
OH
75%
Br
OSiEt2(i-Pr)
H
MeS
H
MeS
O
O
OTES
1) Et3N.3HF, MeCN,
2)[C], DCC, THF.
O
O
OH
O
OH
3)TBAF, O-NO2-Phenol
4) TESCl, Et3N
OSiEt2(i-Pr)
MeS
OH
OH
OMe
TBS
O
O
26%
2) K2CO3, MeOH
O
H
H
O
O
H
TBSO
[A], Pd(PPh3)4, CuI,
Et3N,
SnBu3
A
TES
O
I
TBSO
OH
(i-Pr)Et2SiO
30%
Cl
OMe
OH
HO
OH
OH
O
1) TsCl, DANCO
O
2) TFA, THF/H2O
HO
Cl
OMe
N1999A2
CO2H
O
(i-Pr)Et2SiO
C
Cl
OMe
Ji, N.; O-fDowd, H.; Rosen, B. M.; Myers, A.G. J. Am. Chem. Soc. 2006, 128, 14825-14827
Joint initiative of IITs and IISc – Funded by MHRD
Page 32 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.3.6. Total Synthesis of Kedarcidin Chromophore
Joint initiative of IITs and IISc – Funded by MHRD
Page 33 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Joint initiative of IITs and IISc – Funded by MHRD
Page 34 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.3.6.1.
Approaches for the Synthesis of Kedarcidin Chromophore Aglycon
Two Approaches for the Synthesis of Kedarcidin Chromophore Aglycon
i-PrO
OH
O
CH3O
CH3O
HN
O
N
O
Cl
OR2
O
Kedarcidin
Chromophore
R1O O
Aldehyde Addition
Cyclisation
Transannular
Cyclisation
BocHN
ArHN
O
N
O
Cl
N
OPMBM
Cl
O
O
OTIPS
OTES
O
OH
O
TBSO
Br
CHO
OTBS
TBSO
H
M. Hirama 2007
(partial synthesis)
A. G. Myers 2002/2007
Hirama‘s Synthesis of Kedarcidin Chromophore Aglycon
2.3.6.2.
Hirama’s Retrosynthetic Analysis of Kedarcidin Chromophore Aglycon
i-PrO
i-PrO
i-PrO
OH
O
CH3O
CH3O
N
O
CH3O
CH3O
HN
Cl
OMOM
O
Amide Formation
O
Cl
O
Cl
OPMBM
OPMBM
OTES
O
O
Dehydration
Sonogashira
TBSO O
TMS OH
RO O
HO
O
N
OTMS
O
CH3O
BocHN
O
N
OR'
O
CH3O
HN
O
OMOM
TBSO
Cyclisation
O
TES
O
Epoxide Formation
BocHN
Ester Formation
BocHN
OH
N
O
Cl
N
HO
OMOM
HO
H
OH
O
I
TES
O
O
Joint initiative of IITs and IISc – Funded by MHRD
O
OPMBM
O
Cl
OTES
O
Ether Bond
Formation
TBSO
H
I
TES
O
O
Page 35 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Hirama‘s Synthesis of Kedarcidin: Part-1: Synthesis of Naphthalene Aromatic
Core
2.3.6.2.1.
Part-1: Synthesis of Naphthalene Aromatic Core (Hirama's Synthesis)
HO
CO2Me
HO
Ph2CCl2
175oC
O
HO
Ph
OH
i-PrO
H3CO
CO2Me
1. i-PrI, K2CO3,
acetone, 50oC
2. AcOH, H2O,
reflux, 72% (3 steps)
O
Ph
O
CO2Me 1. NaOH, MeOH,
i-PrO
H2O, 60oC
Br
2. (COCl)2, PhCl3 H3CO
OCH3
Br
CO2Me 1. NBS, THF, r.t.
2. MeI, K2CO3,
i-PrO
Acetone, 50oC
86% (2 steps)
HO
OH
CH2N2, Et3N, Et2O,
Cl then PhCO2Ag, Et3N,
i-PrO
MeOH, 75% (3 steps)
H3CO
OCH3
CH2=CHCO2t-Bu, Pd(OAc)2,
P(p-tol)3, Et3N, 100oC, 90%
i-Pr2NEt,
PhMe, r.t.,
62% (3 steps)
i-PrO
CO2Me
H3CO
CO2t-Bu
1. Ba(OH)2.8H2O, t-BuOH
2. (COCl)2, DCM
CH3O
Br
OCH3
i-PrO
COCl
H3CO
CO2t-Bu
OCH3
OCH3
i-PrO
CO2Me
OH
TFA
i-PrO
CO2t-Bu
99%
CH3O
OCH3
Joint initiative of IITs and IISc – Funded by MHRD
OH
CO2H
OCH3
A
Page 36 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Joint initiative of IITs and IISc – Funded by MHRD
Page 37 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.3.6.2.2.
Hirama‘s Synthesis of Kedarcidin: Part-2: Synthesis of β-Amino Acid Core
2.3.6.2.3.
Hirama‘s Synthesis of Kedarcidin: Part-3: Synthesis of Fused 5-Membered Ring
System of Enediyne Core
Part-3: Synthesis of Fused Ring of Enediyne Core (Hirama's Synthesis)
OMe
MeO
1. MeO
O
HO
OH
PMB
MeO
O
X
Amberlyst-15
OH
2. MOMCl
3. PPTS/MeOH
72% (3 steps)
OH
MOMO
OH
OMOM
X = OH
X=I
O
OMOM
MOMO
i-PrMgCl, CH2I2
I
2. I2, Pyridine
89% (2 steps)
MOMO
O
-78 to 0oC, THF
69-71%
1. (Cl3C)2C=O, Pyridine TBSO
2. PPTS, 2-butanone
3. TBSCl, imidazole
77% (3 steps)
TBSO
I
O
Joint initiative of IITs and IISc – Funded by MHRD
CH2Cl2, r.t.
92%
OH
OMOM
MOMO
HClO4
I
O
MOMO
OMOM
MOMO
OH
THF(aq)
61%
OMOM
MOMO
I
MOMO
HO
OH
HO
3. TBAF, THF, 0oC HO
O
MOMO
1 (0.2 mol%)
1. DIBAL-H, -80oC
2. (MeO)MeC=CH2
O
O
I2/PPh3
99%
MOMO
THF, r.t.,
96% (2 steps)
OH
HO
EtOH (aq), reflux
Ph3P=CH2,
MOMO
1. TPAP, NMO
Zn dust
I
O
O
DIAD, PPh3, THF O
72% (4 steps)
I
O
O
O
(C)
Page 38 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Grignard via Iodine-Magnesium Exchange
THF
CH2I2
i-PrMgCl
i-PrI
ClMg
I
O
MOMO
I
ClMg
I
O
O
MOMO
O
O
I
O
MOMO
MOMO
I
I
MOMO
MOMO
O
-
O
I
Hirama‘s Synthesis of Kedarcidin: Part-4: Synthesis of Enediyne Core
2.3.6.2.4.
Part-4: Synthesis of Enediyne Core (Hirama's Synthesis)
O
O
O
HO
OH
O
O
1) NaIO4, SiO2,
DCM, H2O
2) H
TMS
n-BuLi, THF, -78°C
TMS
74% (2 steps)
PDC, MS 3A,
OH
O
O
MgBr
O
O
H
TMS
O
OH
Et2O, -78°C
O 63% (2 steps)
DCM
TMS
H
O
1) n-BuLi, THF,
-100°C,
then, TESCl,
-80°C, 80%
2) LiOH, THF,
H2O, 97%
H
O
HO
1) TFA, THF, H2O
(2 : 10 : 5), 93%
H
OTES
Joint initiative of IITs and IISc – Funded by MHRD
PMBMO
1) PMBMCl,
i-Pr2NEt,DCM, 76%
OH
H
TES
OH
OH
2) TFA, THF, H2O
(1 : 20 : 10), 90%
2) TBSCl, DMAP,
Et3N,DCM, > 99%
TES
OTBS
TES
Page 39 of 89
(D)
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Hirama‘s Synthesis of Kedarcidin: Part-5: Attaching A-D Units with Enediyne
Core
2.3.6.2.5.
Part-5: Attaching A-D Units with Enediyne Core (Hirama's Synthesis)
O
C
BocHN
1. 2, CsF
DMF, 60 oC
I
BocHN
N
O
O
O
Cl
2. TBSCl
90% (2 steps)
1. 0.3 M KOH(aq)
OMe
O
+
CO2Me
I
PMBMO
OPMBM
O
Cl
OTES
O
3.D, TESCl, Imidazole,
DMF, 95%
TBSO
BocHN
O
N
2. 3, EDC.HCl, DMAP
86% (2 steps)
H
I
TBSO
OH
O
TES
O
O
H
N
O
OH
B
Cl
OH
D
TES
Pd2(dba)3.
CHCl3(0.5 equiv.)
CuI (2 equiv.),
i-Pr2NEt (36 equiv.), Cl
i-PrO
BocHN
O
N
O
OPMBM
OTES
O
DMF (0.001M),
r.t., 1h, (44-47%)
TBSO
O
TES
O
1. TBSOTf, 2-6-lutidine,
DCM
OH
O
CH3O
CH3O
2. SiO2, DCM
3. A, HOAt, EDC.HCl,
DCM, 89% (3 steps)
i-PrO
HN
CO2H
TBSO
O
O
OTES
O
R1
O
N
N
N
N .HCl
OH
O
TES
N
EDC.HCl
O
OPMBM
O
TBSO
N C N
O
N
O
TES
O
Peptide Coupling
HN
Cl
OTES
A
OAllyl
CH3O
AllylBr, Cs2CO3,
CH3O
DMF, 0°C, > 99%
OPMBM
O
CH3O
i-PrO
O
Cl
OH
CH3O
O
N
N
P.T.
R1
R1
O
R2 NH2
O
O
R1
N
HO
O HN
O
N
N N
N
N
N
H
N
P.T.
-HOAt R1
HN
R2
Joint initiative of IITs and IISc – Funded by MHRD
N
Page 40 of 89
O
N
H
R2
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Hirama‘s Synthesis of Kedarcidin: Synthesis of Cyclic Enediyne Core: Partial
Synthesis of Kedercidin
2.3.6.2.6.
Part-6: Synthesis of Cyclic Enediyne Core: Partial Synthesis of Kedercidin
i-PrO
OAllyl
CH3O
O
CH3O
TBAF, THF
HN
OAllyl
CH3O
O
CH3O
0°C, 93%
HN
O
N
OTES
OPMBM
OH
O
HO
O
TES
O
O
H
i-PrO
i-PrO
OAllyl
OAllyl
1. TFA, THF, H2O
CH O
(2 : 10 : 5), 50°C, 71% 3
CH3O
2. TBSCl, Et3N,
DMAP, DCE, 85%
O
Cl
OPMBM
O
O
O
N
O
Cl
TBSO
i-PrO
O
CH3O
1. IBX, MS 4A
DCM-DMSO (10 : 3)
CH3O
O
HN
2. TMSOTf, 2,6-lutidine,
DCM, -70°C
O
N
O
Cl
N
Cl
OPMBM
O
O
OPMBM
OTMS
O
OH
O
HN
CHO
TBSO HO
TBSO
OH
H
Unstable, use immediately
H
i-PrO
OAllyl
CH3O
O
Additive 30-50 equiv.
CH3O
YbCl3-LHMDS 30-50 equiv.
THF [1 mM], -25°C, 25h
22% (3 steps), 2/3 (
i-PrO
N
Cl
OAllyl
O
H3CO
HN
THF [1 mM]
CH3O
HN
O
O
OTMS
OPMBM
OTMS
O
TBSO O
TMS OH
N
+
Cl
Kedarcidin
O
O
OPMBM
OTMS
O
TBSO O
TMS OH
Key steps:
(a) Nucleophilic Addition-Cyclisation; (b)1% overall yield from the longest linear sequence (16 steps);
(c)Still 4 steps to achieve the total synthesis of kedarcidin chromophore aglycon
Joint initiative of IITs and IISc – Funded by MHRD
Page 41 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Myers‘ Synthesis of Kedarcidin Chromophore Aglycon
2.3.6.3.
Myers’ Retrosynthetic Analysis for the Synthesis of Kedercidine
i-PrO
OH
O
CH3O
CH3O
i-PrO
OMOM
i-PrO
CH3O
O
CH3O
CH3O
HN
OR'
HN
O
N
O
O
CH3O
HN
O
N
Cl
OMOM
O
N
O
Cl
OTIPS
O
Cl
OTIPS
OH
O
OH
O
Dehydration
O
Br
RO O
HO
TBSO
Transannular Cyclisation
Epoxidation
Glaser Cyclisation
i-PrO
BocHN
O
Cl
O
S
O
Ether
Formation
CH3O
O
Br
Ester Formation
HN
HO
O
O
Cl
HO
O
O
H
O
TBS
OTIPS
OH
H
Br
Amide Formation
O
CH3O
N
TBSO
Br
O
O
O
Cl
HO
O
CH3O
OMO
CH3O
OCH3
N
Br
OMOM
BocHN
OCH3
N
i-PrO
Br
TBSO
TBS
Pd Reation
H
TES
Myers‘ Synthesis of Kedarcidin: Part-1: Synthesis of Naphthalene Aromatic Core
2.3.6.3.1.
Part-1: Synthesis of Naphthalene Aromatic Core (Myers' Synthesis)
HO
i) PPh3, DEAD, i-PrO
i-PrOH, THF
O
ii) NaOH, H2O,
93%
O
O
1) CH(OEt)3, SnCl4,
DCM
HO
OH
i-PrO
2) CH3I, K2CO3, DMF H3CO
79% (2 steps)
i) (CH3)2NCH2CH2N(CH3)Li,
THF, -20°C
CHO
OCH3
O
i-PrO
H3CO
CH3
i) (CH3)2NCH2CH2N(CH3)Li,
i-PrO
THF, -20°C
CHO ii) n-BuLi, -20°C
OCH3
iii) CO2, 88%
i-Pr2NEt,
PhH, r.t.
i-PrO
89% (2 steps)
CH3O
H3CO
OH
1) EtO
CO2H THF, 96%
CHO
OCH3
NaOH, EtOH/H2O,
CO2Et
OCH3
Joint initiative of IITs and IISc – Funded by MHRD
60°C, 99%
Li
ii) n-BuLi, -20°C
iii) CH3I, 92%
O
P OEt
OEt
2) (COCl)2, PhH
i-PrO
COCl
CO2Et
H3CO
OCH3
i-PrO
OH
CH3O
CO2H
OCH3
A
Page 42 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Myers‘ Synthesis of Kedarcidin: Part-2: Synthesis of β-Amino Acid Core
2.3.6.3.2.
Staudinger Reaction
R-N3
PX3
R-N-N=N-PX3
-N2
phosphazide
NN+
N
Cl
MOMO
N
Cl
O
OCH3
aza-ylide
PPh3
N
N
N
PPh3
Cl
N
H2O
R-N=PX3
- X3P=O
N
N
N
PPh3
R-NH2
PPh3
N
-N2
Cl
MOMO
MOMO
O
OCH3
Joint initiative of IITs and IISc – Funded by MHRD
MOMO
N
N
O
O
OCH3
Page 43 of 89
OCH3
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.3.6.3.3.
Myers‘ Synthesis of Kedarcidin: Part-3:
Joint initiative of IITs and IISc – Funded by MHRD
Page 44 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Joint initiative of IITs and IISc – Funded by MHRD
Page 45 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.3.6.3.4.
Myers‘ Synthesis of Kedarcidin: Part-4: Synthesis of Diyne Core
Joint initiative of IITs and IISc – Funded by MHRD
Page 46 of 89
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
Myers‘ Synthesis of Kedarcidin: Synthesis of Cyclic Enediyne Core
2.3.6.3.5.
i-PrO
i-PrO
OH
O
CH3O
CH3O
CH3O
1) [VO(acac)2] (0.2 equiv.),
CH3CPh2OOH (1.3 equiv.), PhH
O
O
Cl
O
CH3O
HN
N
OH
OTIPS
O
OTIPS
OH
O
TESO O
i-PrO
Martin Sulfurane Dehydration
CH3O
PhH, 83%
O
Cl
HO
Martin Sulfurane
Dehydration
CF3
F3C Ph O
Ph
S
Ph
O Ph CF3
F3C
(10 equiv.),
O
N
2) TESCl (50 equiv.),
Imidazole (100 equiv.),
DCM, 32% (2 steps)
OH
HN
CH3O
OH
HN
Cl
R1
R1 R
2
F3C Ph O
S
Ph
O Ph
R3
F3C
OH
R2
R3
Martin Sulfurane
N
F3C
HO
O
TESO
O
CF3
F3C Ph O
Ph
S
Ph
O Ph CF3
F3C
O
O
Ph
CF3
O
OTIPS
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Ph
O
S
F3C
Ph
HO
Ph
CF3
R1
R2
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2.3.6.3.6.
Overview Myers‘ Synthesis
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2.3.6.4.
Summary of Kedarcidin Synthesis
Summary of Kedarcidin Synthesis
Hirama’s Synthesis
16 steps, 1% overall yield
Still 4 steps to go
Key Step: Aldehyde Addition Cyclisation
i-PrO
OH
O
CH3O
CH3O
HN
O
O
N
O
Cl
H3CN(CH3)2
OH
O
O
H3C
HO
H3C
OH
O
O O
Kedarcidin
Myers’ Synthesis
25 steps, 1% overall yield
Synthesis applied to Kedarcidin glycon in 2007
Key Step: Transannular Cyclisation
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2.3.7. Total Synthesis of Maduropeptin
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2.4. Biosynthesis of a Few Members of Natural Enediynes
2.4.1. Introduction
Microorganisms mostly from soil and marine natural source, (such as bacteria, fungi)
produce a large variety of substances (secondary metabolites) with a vast diversity of fascinating
molecular architecture and potent biological activities that are not available in any other systems.
Enediyne class of natural product is of such fascinating examples produced by microorganisms
and isolated. Biosynthesis of secondary metabolites includes (a) finding the reactions available in
nature, (b) study of the enzymatic mechanisms of these reactions, (c) investigating how these
reactions are linked to produce complex architechture, and (d) the regulatory mechanisms of the
pathways they are formed, (e) to manipulate nature's biosynthetic machinery for the discovery
and development of new drugs of microbes origin.
The biosynthesis of the enediynes is intriguing because of the uniqueness of the chemical
structures of these classes of molecules. The origin of the enediyne core was initially studied
using isotope-labeling experiments (by monitoring the production of neocarzinostatin,
dynemicin, and esperamicin) which established the acetate as a precursor unit. However, the
answer of whether the enediyne core was constructed by the degradation of fatty acids or by de
novo biosynthesis with a dedicated fatty acid or polyketide synthase (PKS) was unclear.
Cloning and characterization of the gene clusters for 5 enediynes, [C-1027, neocarzinostatin
(NCS), calicheamicin, dynemicin, and maduropeptin], led the foundation of investigating
enediyne biosynthesis. Since then molecular and biochemical studies are being undertaken for
deciphering enediyne biosynthesis.
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2.4.2. Enediyne Biosynthesis in the Pre-Genomic Era
In early 1988, Schreiber and Kiessling devised concised synthetic routes for the enediyne
cores of calicheamicins and neocarzinostatin. They envisaged that the branched molecule A
might be the common biosynthetic precursor for both the 9- and 10-membered enediynes.
Considering this, they demonstrated that a series of steps including (a) electrocyclic ring closure,
(b) proton transfer and (c) oxidation could transform A into a 9-membered enediyne (pathway I,
Figure 6). On the other hand an intra-molecular Diels–Alder reaction followed by the addition of
one carbon unit at the acetylene terminus could convert structure A into a 10-membered
enediyne (pathway II, Figure 6).
Biosynthetic Mechanism Postulated for Enediynes in early 1988,
by Schreiber and Kiessling
Path I
9-Membered Ring
Enediyne core
A
Path II
OR
9-Membered Ring
Enediyne core
Figure 6. An early biosynthetic mechanism postulated for enediynes.
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Later on, in 1989, Hensens et al. produced 13C-enriched neocarzinostatin by feeding 13Clabeled acetate to Streptomyces carzinostaticus. This study confirmed the structures of
neocarzinostatin chromophores proposed by Edo and Myers. The first clue about the biosynthetic
origins of the molecular building blocks also came from their studies. The experiment with
singly labeled acetate suggested that the bicyclo[7.3.0]dodecadienediyne moiety could be
derived from a linear precursor that consists of seven acetate units (Figure 7). That the acetates
are assembled in a head-to-tail fashion was evident from the study of culturing with doubly- and
mixed-labeled acetate. Most importantly, they envisaged that the two carbons of the -yne group
originate from the same acetate unit, which brings the difference between the 9- and 10membered enediynes. They also proposed that the linear precursor derived from oleate or
crepenynate is truncated on both ends prior to cyclization. Subsequent oxidation, cyclization and
oxygenation would furnish the fully functionalized 14-carbon enediyne core.
Folding Pattern for the 9-Membered
Enediyne Core of Neocarzinostatin
10
11
9
8
7
O
1
2
3
4
6
OR
12
CH3COOH
5
14
13
9-Membered Ring Enediyne Core
Figure 7. Folding pattern for the 9-membered enediyne of neocarzinostatin.
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Another isotopic labeling study by Tokiwa et al. revealed a very different 13C-incoporation
pattern for the bicyclo[7.3.1]-tridecadiynene core of dynemicin A isolated from Micromonospora
chersina M956-1. They proposed that the bicyclic enediyne core and the anthraquinone moiety
of dynemicin A are derived from two heptaketide chains that consist of two sets of seven acetate
units. There are two possible pathways for the linear heptaketide to fold into the final bicyclic
structure (Figure 8). Identification of the incorporated acetate units from the [1,2-13C2] acetate
feeding experiment suggested that the cyclization followed pathway (a) rather than path (b).
From their results, it was also clear that the two carbons of one -yne group in dynemicin are
derived from different acetate units which is contrary to the early finding that the two carbons of
the -yne group in neocarzinostatin originate from the same acetate unit. From the earlier studies
it was postulated that uncialamycin, the other member of the dynemicin-type enediyne subfamily
containing a different enediyne core, is derived from a dynemicin-like precursor.
Folding Pattern for the 10-Membered
Enediyne Core of Dynamicin A
13
11
10
9
12
(a)
14
1
8
2
(b)
7
3
6
4
5
CH3COOH
10-Membered Ring Enediyne Core
Figure 8. Folding patterns for the 10-membered enediyne of dynemicin A.
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A similar isotopic labeling experiment was performed for investigating the biosynthetic route
to the esperamicin producing Actinomadura verrucosospora. This experiment suggested that the
enediyne of esperamicin A1 is also assembled from seven acetate units in a head-to-tail fashion.
In this case, there four possible pathways were proposed for the linear heptaketide to fold into
the bicyclic structure (Figure 9). Identification of the incorporated acetate units, particularly the
starting acetate unit C11–C12, ruled out the pathways a, b and d. It was observed that the
production of esperamicin A1 by A. verrucosospora was significantly reduced by administrating
cerulenin, an inhibitor that targets the β-ketosynthase (KS) domain of both fatty acid synthase
(FAS) and polyketide synthase (PKS), whereas supplementation of the culture with the fatty acid
oleate did not restore the biosynthesis. These observations suggested that the enediyne core is
probably derived from a polyketide precursor, rather than a fatty acid one. Further, the three
isotopic labeling experiments provided clues that linear polyketide precursors consist of head-totail coupled acetate units. The differences in isotope incorporation pattern among
neocarzinostatin, calicheamicin and dynemicin indicated the different origins of the -yne carbons
and hence suggesting the different biosynthetic pathways for these enediynes.
Folding Pattern for the 10-Membered Enediyne Core of Esperamicin A1
14
11
15
11
10
9
12
13
1
14
2
(a)
(b)
14
6
4
11
5
15
13
11
10
9
12
13
1
4
6
4
5
6
4
5
14
(d)
CH3COOH
11
15
10
9
13
1
8
2
7
3
6
12
8
2
3
7
3
(c)
7
8
2
15
8
2
10
9
12
1
14
13
1
7
3
10
9
12
8
15
10-Membered Ring Enediyne Core
5
7
3
6
4
5
Figure 9. Proposed biosynthetic pathways for the linear heptaketide to fold into the bicyclic structure of 10-membered ring enediynes.
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2.4.3. Enediyne Biosynthesis in the Genomic Era
As was revealed from the isotopic labeling experiments, little new insight was gained on the
biosynthesis of the enediynes. Therefore, in the genomic era effort were undertaken to locate the
putative type I or type II PKS gene on the genomic DNA of the enediyne producers
(microorganisms) by DNA hybridization. However these were unsuccessful. Instead, Shen and
coworkers succeeded in locating the DNA fragment that encompasses the enediyne gene clusters
for C-1027. They were able to find out the gene (cagA) that encodes the chromophore-associated
apoprotein CagA and two putatively conserved deoxysugar synthesizing genes. At the same
time, Thorson et al. were able to identify the gene locus for calicheamicin biosynthesis. In 2002,
the completion of the sequencing and partial annotation of the C-1027 and calicheamicin gene
clusters was announced. Since then the study of enediyne biosynthesis entered the genomic era.
Therefore, genetic manipulations of genes related to secondary metabolism now offer a
promising tool to investigate the biosynthetic pathway of formation of and prepare complex
natural products like enediyne biosynthetically. This approach depends on the cloning and
genetic and biochemical characterization of the biosynthetic pathways of the metabolites. Thus,
several research groups are involved in the cloning, sequencing, and characterization of the
several enediyne biosynthesis gene clusters from the producer microorganisms through which
the convergent biosynthetic strategies for C-1027, NCS and other enediynes were developed.
Manipulation of genes governing enediynes biosynthesis allowed one to engineer enediyne
compounds. This approach offers the opportunity to decode the genetic and biochemical basis for
the biosynthesis of enediynes and many other structurally complex natural products and to
explore ways to make more antitumor agents. Here are few examples of biosynthesis of
enediynes
2.4.3.1.
The Apoprotein and The Gene Cluster for Enediyne Biosynthesis
The Apoprotein: All known 10-membered enediyne natural products were isolated as freestanding chromophores. On the contrary, the nine-membered enediynes were isolated as a
chromoprotein complex- a binding protein known as protective apoprotein covering the
dissociable enediyne chromophore. Exception is N1999A2 where no apoprotein was found. The
9-memred enediyne chromophores are extremely unstable in aqueous solution. For example
neocarzinostatin is extremely unstable in aqueous solution in the absence of the apoprotein but
the apoprotein cover greatly enhances the stability of the labile chromophore.
However the apoproteins are not similar for all the enediynes. As for example, the
maduropeptin apoprotein (MdpA) does not share similarity with the apoproteins of
neocarzinostatin, C-1027 and kedarcidin. The 133 amino acid long MdpA represents a new
protein class that does not share significant sequence homology with any protein deposited in the
National Center for Biotechnology Information (NCBI) database.
The structures of neocarzinostatin and C-1027 associated with their apoproteins (NcsA and
CagA) have been determined which reveals that the apoproteins share an immunoglobulin-like
fold that consists of a seven-stranded antiparallel β-barrel and two additional β-strands. The βstrands and the three loops of the apoprotein forms a hydrophobic pocket in which the
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chromophore (the reactive enediyne core) is accommodated. The structure of the protein–
neocarzinostatin complex revealed that the reactive sites of the cromophore such as the epoxide,
the acetylene groups, and the nucleophilic addition site are greatly shielded from the solvent.
Similarly structure determination of the aromatized C-1027 chromophore revealed that the
enediyne core and the the hydrophobic benzoxazolinate moiety interact and reside on the
hydrophobic residues of the protein. These bindings bring stability of the extremely reactive 9membered enediynes.
Role of Binding Proteins: The binding protein directs transport of the reactive enediyne
chromophore to the extracellular environment. It is also found to be essential for self-resistance
by stabilizing the reactive enediyne chromophore. The 9-membered enediynes C-1027 and
maduropeptin readily undergo cycloaromatization in the absence of the binding protein. Thus the
binding proteins help stabilizing the enediyne in their native form.
Establishment of the amino acid sequence of the homologous binding proteins enables the
cloning and the sequencing of the genes-cagA for C-1027 and ncsA for NCS.
Since there is no binding protein for the ten-membered enediyne calicheamicin, a different
strategy was used to clone the gene cluster of ten-membered enediyne calicheamicin. The
strategy utilized to clone and localized the gene cluster of ten-membered enediyne calicheamicin
are-(a) screening of clones that are capable of conferring calicheamicin resistance using PCRbased screens and (b) followed up by DNA-shotgun sequencing.
2.4.3.2.
General Biosynthesis of Enediyne Cores
Though there exists various types of gene clusters for different enediynes, a unified
biosynthetic scheme for the nine- and ten-membered enediyne cores is now possible owing to the
discovery of a shared iterative type I PKS (polyketide synthase). Bacterial polyketide
biosynthesis carries out either by noniterative, modular type I PKS, or a multienzyme complex of
iteratively acting type II PKS, or homodimeric, iteratively acting condensing type III PKS
without an acyl carrier protein (ACP).
In contrast, the enediyne PKSs from the biosynthetic pathways of calicheamicin 1I and C1027 seem to resemble a family of fungal iterative type I PKSs. To date, five gene clusters
cloned only one type of PKS that are all iterative type I PKS. The gene cluster for C-1027
contains a single PKS. This PKS is shared by sequence homology and domain architecture
among the enediyne family (Fig. 4A). Disruption of this SgcE (the PKS for C-1027 synthesis) in
the producer of C-1027 did not produce C-1027. Identical results were observed with the
homologous neocarzinostatin PKS, NcsE. These results provide strong support that the enediyne
core is produced by a polyketide pathway. The enediyne PKSs use a single set of catalytic
domains for polyketide synthesis. Bioinformatic analysis of this PKS family, PKSE, showed that
the enediyne PKS encompass four domains in the following order (N- to C-terminus): (a) a
ketosynthase (KS), (b) an acyltransferase (AT), (c) a ketoreductase (KS), and (d) dehydrogenase
(DH). Enediyne PKS possess closest sequence homology to polyunsaturated fatty acid (PUFA)
synthases involved in the biosynthesis of docosahexaenoic acid in Moritella marina and
eicosapentaenoic acid in Shewanella (Figure 10). The significant sequence identity and
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similarity shared by PKSs, CalE8 (the PKS for calicheamicin synthesis) and SgcE (the PKS for
C-1027 synthesis) indicated that the 9- and 10-membered enediyne cores may share a common
PKS progenitor. Thus, the iterative type I PKS is unique to all of the enediyne family. It is now
evident that the enediyne biosynthetic gene clusters share conserved architecture featuring the
enediyne PKS. These enediyne PKS formed the basis of several expedient strategies to clone
additional enediyne biosynthetic gene clusters such as of NCS, maduropeptin, and dynemicin
gene clusters via a PCR approach.
All the studies towards polyketide biosynthesis by enediyne PKSs established that with selfphosphopanetheinylation of a unique ACP, PKSE catalyses the enediyne synthesis. Therefore,
enediyne biosynthesis follows an ACP-dependent PKSE-catalyzed pathway (Figure 11).
Architecture and domains of the enediyne PKSE
and its relationship with PUFA synthase
Figure 10. Architecture and domains of the enediyne PKSE and its relationship with PUFA synthase (Lanen, S. G. V.; Shen, B. Curr Top
Med Chem. 2008, 8, 448–459.).
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Proposed pathway for biosynthesis of putative nine- or ten-membered enediyne cores by enediyne PKS
Enediyne PKS
Accessary Enzymes
(9-membered specific)
9-Membered
Enediyne
O
9-Membered Enediyne
Core (Neocarzinostatin)
SCoA
Me
ACP
Acetyl-CoA
PKSE
R
+
O
O
O
OH
SCoA
Malony-CoA
(7 x)
CO2
Polyketide
Intermediate
(7 x)
Enediyne PKS
Accessary Enzymes
(10-membered specific)
(Dynamicin A (Esperamicin
Core)
Core)
10-Membered Enediyne Core
10-Membered
Enediyne
Figure 11. Proposed pathway for biosynthesis of a polyunsaturated intermediate from acyl CoA by PKSE and the subsequent
transformation by enediyne PKS associated enzymes into putative nine- or ten-membered enediyne cores that are finally tailored to
individual enediyne natural product. Atoms that were incorporated intact from acyl CoA precursors to the enediyne cores are shown in
bold.
2.4.3.3.
Peripheral Moieties of the Enediyne Chromophores
As was discussed that the enediyne first bind to the DNA sequence specifically and then
breaks the double strand or single strand DNA via the generation of enediyne biradical through
the well known BC or MSC that abstract –H from the sugar phosphate backbone of DNA. Now
the sequence specific binding of enediyne to DNA and the physical properties of the enediyne
chromophores are largely depend on the peripheral moieties encompassing the enediyne
warheads. The peripheral moieties or building blocks can be divided into two groups- (a)
aromatic and (b) sugar moieties. The aromatic moieties are essential for DNA–chromophore
interaction through intercalation. The aromatic unit benzoxazolinate present in C-1027 and
orsellenic acid in calicheamicin are found to intercalate into DNA. Besides these aromatic units,
many enediyne chromophores contain mono- or polysaccharides that play important roles in
fine-tuning
the
interaction
between
the
chromophore
and
DNA.
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Figure 12. Building blocks of enediyne chromophores. The sites on the enediyne cores for peripheral moiety attachment are indicated by
the big dots.
Nature has created the structurally diverse enediyne chromophores, 13 numbers till the
date by using a small collection of aromatic and sugar moieties (Figure 12). Connecting these
moieties at different positions of the enediyne cores contributes to the structural diversity of
enediyne chromophores. More of such aromatic and sugar moieties are expected to discover that
will help deciphering new enediyne chromophores in the future.
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Success of the sequencing and annotation of the enediyne gene clusters and corroboration
of the biosynthetic pathways has discovered a number of novel enzymes and chemical
transformations in the biosynthesis of the aromatic and sugar moieties and the enediyne cores.
These pathways and enzymatic transformations to the aromatic and sugar moieties as well as
enediyne cores are given below (Figure 13). Also the successful stories of exploiting the
biosynthetic pathways for producing C-1027 and calicheamicin 1I are highlighted.
O
SCoA
Me
Acetyl-CoA
PKSE
AviM (for Avilamycin)
or
CalO5 (for Calicheamicin)
Enz S
+
O
Enz S
O
Me
O
Me
O
Avilamycin
O
OH
OH
O
OH
Orsellinic acid
O
CO2
SCoA
Calicheamicin)
(3 x)
Malony-CoA
(3 x)
O
SCoA
Me
Acetyl-CoA
O
PKSE
NcsB (for Neocarzinostatin)
+
O
O
O
OH
S Enz
O
O
O
Me O
O
HO
NcsB
S Enz
O
O
O
OH
Me OH
Me
Naphthoic acid
(5 x)
Malony-CoA
(5 x)
Enz S
O
MeO
OH
NcsB3
O
NcsB
CO2
SCoA
Enz S
Neocarzinostatin
NcsB1
Me
Naphthoic acid
Figure 13. General biosynthesis of peripheral aromatic moieties of enediynes.
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2.4.4. Biosynthesis of C-1027
The C-1027 chromophore can be dissected into 4 biosynthetic building blocks which are
(a) an enediyne core  synthesized via polyketide pathway by enediyne PKSs.
(b) a β-amino acid  annotation of the gene products of the C-1027 gene cluster suggested
that the β-amino acid is derived from L-tyrosine.
(c) a deoxy aminosugar  initial annotation of the gene products of the C-1027 gene cluster
suggested that the deoxy amino sugar is derived glucose-1-phosphate.
(d) a benzoxazolinate moiety The biosynthesis of the benzoxazolinate moiety was
originally proposed to start from anthranilate, a common intermediate from the shikimate
pathway. Subsequent characterization of the enzymes from the putative pathway revealed
an unexpected pathway with chorismic acid as the starting material (Figure 14).
Model of Convergent Biosynthesis of C-1027
O
SCoA
H3C
OH
H2C
Acyl Transferase
+
O OH
Benzoxazollinate
O
SCoA
Acetyl-& Malony CoA
O
MeO
O
O
Enediyne Core
O
O
O
OH
O
O
Me2N
H3C
HO
HO
OH
O
OH
HO OP
Deoxy
Aminosugar
D-Glucose- 1-Phosphate
Glycosyl
transferase
O
HO
OH
Chorismic Acid
PKSE
N
H
OH
O
O
O
NH2
O
NH2
Cl
Condesation
Enzyme
OH
-Amino Acid
L-Tyrosine
Figure 14. A model of convergent biosynthesis for the enediyne C-1027.
Sequencing of the gene cluster revealed homologs for a glycosyl transferase, an
acyltransferase, and condensation enzymes, suggesting a convergent biosynthetic approach is
used in enediyne assembly for C-1027. The initial enzymatic steps in every biosynthetic pathway
for C-1027 have been analyzed using a combination of in vivo gene inactivation and in vitro
characterization of recombinant enzymes. This experiment established the starting metabolite for
each moiety and provided substantial evidence for a convergent approach in enediyne
biosynthesis.
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Enediyne core (4) biosynthesis: There are 13 genes identified within the C-1027 gene
cluster that are consistent with the structure of C-1027 and are essential for the production of
enediyne core of C-1027. These are sgcE, sgcE1, sgcE2, sgcE3, sgcE4, sgcE5, sgcE6, sgcE7,
sgcE8, sgcE9, sgcE10, sgcE11 and sgcF that encode the enediyne core (4) biosynthesis (Figure
15).
Figure 15. Organization of the C-1027 biosynthesis gene cluster ORFs outside the sgcB1 to sgcR3 region are not essential for C-1027
production. Color coding indicates the biosynthesis genes for the enediyne core (red), deoxyaminosugar (blue), β-amino acid (green),
benzoxazolinate (purple), and all other genes (black). (Liu, W.; Christenson, S. D.; Standage, S.; Shen, B.Science 2002, 297, 1170.)
It is not clear whether the enediyne cores are assembled by de novo polyketide biosynthesis
or degradation from a fatty acid precursor. However, feeding experiments with 13C-labeled
precursors supported that both the nine- and 10-membered enediyne cores are derived from a
minimum of eight acetate units organized in head-to-tail. Importantly, only one gene, sgcE,
among all other genes indentified within the C-1027 cluster encodes a PKS. SgcE contains five
domains—(a) the ketoacyl synthase (KS), (b) acyltransferase (AT), (c) ketoreductase (KR), (d)
dehydratase domains that are characteristic of known PKSs, and (e) a domain at the COOHterminus that is unique to enediyne PKSs. Moreover a putative acyl carrier protein domain is
proposed to present in between AT and KR region (Figure 16). Most importantly, the SgcE
enediyne PKS exhibits head-to-tail sequence homology. It has an identical domain organization
to the CalE8 enediyene PKS that catalyzes the biosynthesis of the 10-membered endiyne core of
calicheamicin in Micromonosporaechinospora. Therefore, the nine- and 10-membered enediyne
cores share a common polyketide biosynthetic pathway.
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Figure 16. Comparison between the SgcE PKS catalyzing the nine-membered enediyne core in C-1027 biosynthesis and the CalD8 PKS
catalyzing the 10-membered enediyne core in calicheamicin biosynthesis. aa = amino acid; KS = ketoacyl synthase; AT = acyltransferase;
ACP = acyl carrier protein; KR = ketoreductase; DH = dehydratase; and TD = COOH-terminal domain. (Liu, W.; Christenson, S. D.;
Standage, S.; Shen, B. Science 2002, 297, 1170.)
It is proposed that the SgcE catalyzes the assembly of a nascent linear polyunsaturated
intermediate from acetyl and malonyl coenzyme A (CoA) in an iterative process. Next, upon
action of other enzymes polyunsaturated intermediate is subsequently desaturated to furnish the
two -yne groups and cyclized to afford the enediyne core. An acetylenase has been reported from
the plant Crepisalpina that was characterized as a nonheme di-iron protein. However, no such
homolog was found within the C-1027 cluster. Close comparison of the C-1027 gene cluster with
that of neocarzinostatin gene cluster revealed the presence of a group of ORFs (sgcE1 to sgcE11)
in addition to sgcE. These ORFs are highly conserved.
The functions of the genes are as follows: (a) Genes SgcE6, SgcE7 and SgcE9 function
similar to that of various oxidoreductases. (b) SgcE1, SgcE2, SgcE3, SgcE4, SgcE5, SgcE8 and
SgcE11 show either no sequence homology or homology only to proteins of unknown function.
(c) SgcE10 is highly homologous to a family of thioesterases. These enzymes, together with the
SgcF epoxide hydrolase are responsible for the synthesis of enediyne intermediate 4 from the
nascent linear polyunsaturated intermediate (Scheme 7). All other experimental evidences
support the polyketide route to the biosynthesis of C-1027 enediyne core.
Biosynthesis of Enediyne core of C-1027
O
HO
O
H3C
S PCP
acetyl CoA
+
O
O
SgcE and
sgcE1-E11
OH
SgcF
O
O
S-CoA
malonyl CoA
O
4
Enediyne core
Scheme 7. Biosynthetic hypothesis for the enediyne core and a convergent assembly strategy.
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Deoxyaminosugar (5) biosynthesis: There are seven genes namely, sgcA, sgcA1, sgcA2,
sgcA3, sgcA4, sgcA5, sgcA6 that are responsible for the biosynthesis deoxyaminosugar (5)
moiety in C-1027. The seven deoxyaminosugar biosynthesis genes encode a thymine
diphosphate (TDP)-glucose synthetase (SgcA1), a TDP-glucose 4,6-dehydratase (SgcA), a TDP4-keto-6-deoxyglucose epimerase (SgcA2), a C-methyl transferase (SgcA3), an amino
transferase (SgcA4), an N-methyl transferase (SgcA5) and a glycosyltransferase (SgcA6).
Together, they do the enzyme functions that is essential for the biosynthesis of 5 from glucose-1phosphate (Scheme 8) and the attachment of 5 to 4.
Biosynthesis of deoxyaminosugar peripheral moiety of C-1027
O
OH
OH
HO
HO
O
SgcA1 HO
HO
O SgcA
O
Me
HO
OH
OH
OH
OP
D-Glucose-1-Phosphate
OTDP
OH
OH Me
OTDP
Me2N
O
Me
OH
OH Me
O
OTDP
SgcA4
O SgcA3
O SgcA2
H2N
Me
OH
OH Me
O
SgcA5
OTDP
Me
O
OH
OH Me
OTDP
5
deoxyaminosugar moiety
Scheme 8. Biosynthetic hypothesis for deoxyaminosugar (5, C).
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OTDP
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β-amino acid (6) biosynthesis: There are six genes, sgcC, sgcC1, sgcC2, sgcC3, sgcC4,
sgcC5 that encode biosynthesis of β-amino acid (6) in C-1027. The six β-amino acid biosynthesis
genes encode (a) a phenol hydroxylase (SgcC), (b) a non ribosomal peptide synthetase (NRPS)–
adenylation enzyme (SgcC1), (c) an NRPS peptidyl–carrier protein (PCP) (SgcC2), (d) a
halogenase (SgcC3), (e) an aminomutase (SgcC4), and (f) an NRPS-condensation enzyme
(SgcC5). These enzyme together functions for the biosynthesis of β-amino acid (6) moiety
starting from tyrosine (Scheme 9). Once formed it is activated by SgcC5 as an aminoacyl-S-PCP.
The activated aminoacyl-S-PCP is then ready for attachment to the enediyne moiety 4 by SgcC5.
Biosynthesis of -amino acid peripheral moiety of C-1027
HO
H3N H
H H O
H H O
H H O
O
sgcC
sgcC1
HO
HO
HO
O
H3N H
H3N H
HO
sgcC1
O Ad
sgcC2
PCP
L-Tyrosine
NH3 O
H H O
H H O
HO
HO
S PCP
H3N H
sgcC3
HO
HO
S PCP
sgcC4
H3N H
HO
Cl
S PCP
H
HO
Cl
6
 -amino acid moiety
Scheme 9. Biosynthetic hypothesis for β-amino acid (6, D).
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H
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Benzoxazolinate (7) biosynthesis: Seven genes were identified that are essential for the
biosynthesis of benzoxazolinate moiety of C-1027. These are sgcD, sgcD1, sgcD2, sgcD3,
sgcD4, sgcD5, sgcD6. These seven benzoxazolinate biosynthesis genes encode the followings:
(a) the anthranilate synthase I and II subunits (SgcD and SgcD1), (b) a monoxygenases (SgcD2),
(c) a P-450 hydroxylase (SgcD3), (d) an O-methyl transferase (SgcD4), (e) a CoA ligase
(SgcD5), and (f) an acyltransferase (SgcD6). Action of these enzymes starts with anthranilate, a
common intermediate from the shikimate pathway, for the biosynthesis of benzoxazolinate
moiety (7) (Scheme 10). The co-localization of SgcD and SgcD1 gene along with the other C1027 production genes assures the availability of anthranilate for secondary metabolite
biosynthesis. However, the origin of the C3 unit is not clear. Moreover, how the C3 unit is fused
to the anthranilate intermediate to form the morpholinone unit of benzoxazolinate moiety (7) is
also unclear. However, it is believed that the anthranilate unit of benzoxazolinate moiety (7) is
activated as an acyl-S-CoA for attachment to the enediyne core 4 by the action of gene SgcD6
(Scheme 11)
Biosynthesis of benzoxazolinate peripheral moiety of C-1027
sgcD2
H2N
HO
HO
sgcD3
O
OMe
"C3 Unit"
H2N
O
OH
HO
OH
O
OMe
HO
H2C
O
O
N
H
OMe
N
H
O
OH
H2O
CoA
O
OMe
sgcD5
O
O
OH
O
H3C
O
sgcD4
H2N
H2N
O
OH
Anthranilic acid
OH
N
H
S CoA
O
OH
7
Benzoxazolinate
Scheme 10. Biosynthetic hypothesis for benzoxazolinate (7, E).
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Biosynthesis of C-1027 by attaching the peripheral moieties to the enediyne core:
Decoration of Enediyne Core of C-1027 with Peripheral Moieties: Biosynthesis of C-1027
O
O
O
N
H
OMe
CoA
O
OMe
S
O
N
H
HO
S CoA
O
SgcD6
Enediyne core
7
Benzoxazolinate
SgcD6
Me
OH
Me
O SgcA6
OH
OH Me
HO
Me2N
Me2N
NH3 O
OTDP
HO
O
O
S PCP
H
OH
OH
SgcA6
OH Me
OTDP
5
deoxyaminosugar moiety
SgcC5
O
H
HO
Cl
4
Enediyne core
MeO
O
SgcC5
NH3 O
HO
O
S PCP
H
H
HO
Cl
6
 -amino acid moiety
O
O
N
OH
OH
1
C-1027 Chromophore
O
OH
O
O
O
Cl
Scheme 11. Final step in biosynthesis of C-1027-decoration of enediyne core of C-1027 with peripheral moieties.
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N
H
O
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.4.5. Biosynthesis of Neocarzinostatin
Neocarzinostatin, the first characterized enediyne chromophore, was isolated from the culture
filtrate of Streptomyces carzinostaticus var. E-41 as a 1 : 1 complex with an 11 kDa apoprotein.
It is the in free-standing state is highly unstable upon exposure to heat, high pH or UV-light
irradiation.
In comparison to C-1027, neocarzinostatin consists of
(a) epoxide-derivatized 9-membered enediyne core — synthesized via polyketide pathway
by enediyne PKSs.
(b) a distinct deoxy aminosugar — it is derived from glucose-1-phosphate.
(c) a naphthoic acid moiety
The peripheral moieties of neocarzinostatin are also anchored to the enediyne core but at
different positions in comparison to C-1027. This indicates that the two enediyne cores are
hydroxylated at different sites during the maturation of the enediyne core (Figure 17).
Model of Convergent Biosynthesis of Neocarzinostatin
O
H3C
Napthoic Acid
SCoA
O
?
OMe
+
O OH
SCoA
Acetyl-& Malony CoA
O
O
PKSE
O
O
O
OH
Sodium Bicarbonate
O
Me
O
O
OH
O
MeHN
HO
Me
HO
HO
Me
O
Enediyne Core
O
OH
O
Deoxy Aminosugar
H3C
OH
OP
D-Glucose- 1-Phosphate
or
D-Mannose- 1-Phosphate
Glycosyl
transferase
O
PKSE
SCoA
+ OH
O
SCoA
Acetyl-& Malony CoA
Figure 17. A model of convergent biosynthesis for the enediyne Neocarzinostatin.
Sequencing of the gene cluster ment revealed six complete ORFs (including ncsA) and one
incomplete ORF. Remarkably, the four ORFs encode a dNDP-D-mannose synthase (NcsC),
dNDP-hexose 4,6-dehydratase (NcsC1, a second distinct NGDH gene in this organism), Nmethyltransferase (NcsC5), and glycosyltransferase (NcsC6). These are the enzymes that would
be predicted to be essential for biosynthesis of the deoxy aminosugar moiety of NCS
chromophore.
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Enediyne core (4) biosynthesis: Fourteen genes, ncsE to ncsE11 and ncsF1 to ncsF2, were
identified within the ncs gene cluster all of which play an important role in the NCS enediyne
core biosynthesis. The enediyne core was previously predicted to be synthesized by an iterative
type I PKS with five domains, of which the KS, AT, KR, and DH are characteristic of known
type I PKSs. NcsE shows head-to-tail sequence homology to the SgcE and CalE8 enediyne
PKSs. Consequently, it was proposed that NcsE, in a mechanistic analogy to other enediyne
PKSs, catalyzes the formation of the nascent linear polyunsaturated intermediate from one acetyl
CoA and seven malonyl CoAs in an iterative manner, which is processed to form the enediyne
core by several gene products, including NcsE1–E11 and epoxide hydrolases F1 and F2 (Scheme
12).
All reported experimental findings unambiguously established that NcsE is essential for NCS
biosynthesis. This further supports an iterative type I PKS paradigm for enediyne core
biosynthesis.
Biosynthesis of Enediyne core of Neocarzinostatin
O
H3C
S PCP
acetyl CoA
+
O
O
O
ncsE and
O
O
OH
ncsF and NcsF2 HO
O OH
ncsE1-E11
O
S-CoA
malonyl CoA
HO
Enediyne core
(7x)
Scheme 12. Biosynthetic hypothesis for the enediyne core and a convergent assembly strategy to enediyne core of Neocarzinostatin.
Naphthoic acid biosynthetic: Naphthoic acid moiety originates from a single polyketide
chain of six head-to-tail acetate units. This was revealed from isotopic labeling experiments.
Characterisation of neocarzinostatin gene cluster showed that the following enzymes are
involved in the biosynthetic pathway for the naphthoic acid moiety: (a) an iterative PKS (NcsB),
(b) a CoA ligase and (c) several ancillary enzymes.
The biosynthesis of the naphthoic acid moiety starts with NcsB, an iterative PKS that
contains the domains like — (a) the ketoacyl synthase (KS), (b) acyltransferase (AT), (c)
ketoreductase (KR), (d) dehydratase and (e) acyl carrier protein domain (ACP) and a core
domain with unknown function. It is believed that NcsB uses acetyl-CoA as stating matetial and
malonyl-CoA as extender to assemble a nascent hexaketide with the selective reduction and
dehydration of the keto groups at C5 and C9 (Scheme 13). The hexaketide intermediate then
undergoes aromatization to furnish the naphthoic acid via intramolecular aldol condensation.
The post-PKS modification of the naphthoic acid moiety starts with the incorporation of a
hydroxyl group at C8 carbon which is catalyzed by the cytochrome P450 hydroxylase NcsB3.
Ultimately the methylation of the hydroxyl group is catalysed by an S-adenosylmethionine
(SAM)-dependent O-methyltransferase (NcsB1). Next, NcsB2 ligase catalyzes the adenylation
of 2-hydroxy-7-methoxy-5-methyl-1-naphthoic acid to form its CoA derivative. The discovery of
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NcsB2 as a promiscuous naphthoic acid/CoA ligase with relaxed substrate specificity provided a
great opportunity for producing structural analogs of neocarzinostatin by metabolic engineering.
Finally, the putative acyl transferase (NcsB4) is responsible for the transfer of the naphthoic
group onto the enediyne core.
Scheme 13. Possible biosynthesis mechanism for the naphthoic acid moiety of neocarzinostatin and azinomycin B.
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Deoxyamino Sugar biosynthesis: The deoxyamino sugar moieties of neocarzinostatin and
C-1027 differ in the positions and numbers of the amino, hydroxyl and methyl groups. The
biosynthetic pathway is likely to start with the activation of the monosaccharide as its NDP
derivative by the nucleotidyltransferase NcsC (Scheme 14). Based on sequence homology and
established biosynthetic routes, several gene products have been proposed for the activation of
the sugar ring via the formation of a 4-keto intermediate, the deoxygenation at C6 and the
installation of amino group at C2. Previous labeling experiments with [methyl-3H] methionine
have suggested that the N-methyl of the deoxyamino sugar originates from the methionine of Sadenosylmethionine (SAM). The methylation is presumably catalyzed by the methyltransferase
NcsC5, whereas the predicted glycosyltransferase NcsC6 may transfer the sugar moiety onto the
enediyne core.
Together, the characterization of the enzymes in the naphthoic acid and deoxyamino sugar
pathways by Shen and coworkers provides an entry point for the exploration of the possibility of
producing neocarzinostatin analogs by metabolic engineering and fermentation. The relaxed
substrate specificity of NcsB1 and NcsB2 can be potentially exploited. Likewise, the final step
catalyzed by the glycosyltransferases NcsC6 could represent another opportunity for generating
analogs by glycodiversification.
Biosynthesis of deoxyaminosugar peripheral moiety of Neocarzinostatin
OH
HO
HO
NcsC
(Nucleotidyl
transferase)
O
OH
HO
HO
OH
OH
OP
D-Glucose-1-Phosphate
O
NcsC1
(Dehydratase)
O
Me OH
Me
O
HO
HO
ONDP
O
NcsC2
O (Dehydratase)
ONDP
ONDP
OH
Me
NcsC3
(Aminomutase)
O
Me
O
OH
Me
NcsC4
(Dehydratase)
HO
O
HO
NH2
O
CH3HN
ONDP
deoxyaminosugar
moiety
(Methyl
transferase)
NH2
ONDP
NcsC5
HO
ONDP
Scheme 14. Biosynthetic hypothesis for deoxyaminosugar moiety of neocarzinostatin.
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Final biosynthesis of neocarzinostatin by attaching the peripheral moieties to the
enediyne core:
A convergent strategy could be envisaged for the assembly of the NCS chromophore from
the three individual building blocks of the deoxy-aminosugar, naphthoic acid, and enediyne core
(Scheme 15). While the coupling between dNDP-sugar and the enediyne core is catalyzed by the
NcsC6 glycosyltransferase, that between naphthoyl-S-NcsB and the enediyne core is most likely
catalyzed by the NcsB2 CoA ligase. Although the cyclic carbonyl carbon of NCS has previously
been shown to originate from carbonate, no obvious candidate catalyzing the attachment of
carbonate could be identified within the gene cluster. The convergent biosynthetic strategy for
NCS once again underscores nature‘s efficiency and versatility in synthesizing complex
molecules.
Scheme 15. Final step in biosynthesis of C-1027-decoration of enediyne core of neocarzinostatin with peripheral moieties.
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2.4.6. Biosynthesis of Maduropeptin
Maduropeptin was isolated as a 1 : 1 apoprotein–chromophore complex from the
Actinomadura madurae strain H710–49 in 1991. The maduropeptin chromophore is tightly
bound by a new class of apoprotein that is distinct from the apoproteins of neocarzinostatin, C1027 and kedarcidin.
Maduropeptin contains the following units – (a) a 3,6-dimethylsalicyclic acid, (b) a deoxy
aminosugar and (c) an aryl hydroxypropionic acid moiety along with the enediyne core. The
sequencing and annotation of the maduropeptin gene clusters revealed the presence of
biosynthetically important 42 ORFs and the biosynthetic pathways for the three peripheral
moieties are related to those of C-1027and neocarzinostatin.
Model of Convergent Biosynthesis of Maduropeptin
HO
HO
Me
O
OH
OP
D-Glucose- 1-Phosphate
H3C
SCoA
PKS
+
O O
mdpB
Deoxy Aminosugar
Me
HO
OH
OH
Me
HN
O
Enediyne Core
O
Dimethylsalicyclic
acid
Glycosyl transferase
O
OMe
Me
O
O
O
SCoA
Acetyl-& Malony CoA
Cl
HO
O
PKSE
H3C
O
SCoA
Acetyl-& Malony CoA
N
O
Aryl hydroxypropionic acid
H H O
H3N H
HO
O
L-Tyrosine
Figure 18. A model of convergent biosynthesis for the enediyne, Maduropeptin.
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SCoA
+
O O
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Biosynthesis of Dimethylsalicyclic acid peripheral moiety of Maduropeptine: It is known
that some bacteria and fungi synthesized 6-methylsalicylic acid (6-MSA) via polyketide
synthetic pathway. The presence of a second PKS gene (mdpB) in the maduropeptin gene cluster
hinted that the 3,6-dimethylsalicyclic acid moiety may also originate from a polyketide
precursor. In addition, MdpB is highly homologous to a group of iterative fungal PKSs that
include the 6-methylsalicylic acid synthases (MSAS) from Penicillium patulum, Aspergillus
terreus and Glarea lozoyensis. MdpB also shares the same domain composition with NcsB, the
PKS for the biosynthesis of the naphthoic acid moiety in neocarzinostatin. The catalytic
mechanism involves a partially reduced polyketide intermediate before aromatization (Scheme
16). The selective reduction of the keto group is not clear for these iterative PKSs. The PKS
product might undergo methylation and adenylation after it is off loaded from the PKS (pathway
a, Scheme 16). In an alternative path, the PKS product may be directly transferred to the sugar
moiety with the assistance of a transferase without off-loading (pathway b, Scheme 16).
Interestingly, the neighboring gene mdpB3 encodes a hydrolase that may actually act as a
transferase.
Scheme 16. Possible biosynthetic mechanisms for the 3,6-dimethylsalicyclic acid of maduropeptin.
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Biosynthesis of Aryl hydroxypropionic acid peripheral moiety of Maduropeptine:
Study of the maduropeptin gene cluster reveals that the pathway for the (S)-3-(2- chloro-3hdyroxy-4-methoxyphenyl)-3-hydroxypropionic acid or simply the aryl hydroxypropionic acid
moiety is resemblance to the biosynthetic pathway for the β-tyrosine moiety of C-1027. The first
two enzymes MdpC4 and MdpC1 are highly homologous to the aminomutase SgcC4 and NRPS
adenylation domain protein SgcC1. The gene MdpC2 is similar to SgcC2-like PCP to which the
substrate is attached (Scheme 17). MdpC7 is a pyridoxal phosphate (PLP)-dependent
transaminase that is absent in the biosynthetic pathway of C-1027. It converts the β -amino group
into a keto group. Further modification of the aromatic moiety is carried out by the hydrolase
MdpC, methyltransferase MdpC6 and halogenase MdpC3. The last enzyme MdpC8 is an alcohol
dehydrogenase that furnishes the β-hydroxyl group. In contrast to the ester linkage in C-1027
between the enediyne core and the β-tyrosine moiety, the aryl hydroxypropionic acid moiety is
connected to the enediyne core through an amide linkage. The enzyme involved in amide bond
formation is not known exactly
Biosynthetic pathway for the aryl hydroxypropionic acid moiety of maduropeptin
OH
OH
OH
H3N
NH3
O
O
L-Tyrosin
O
ATP
MdpC7
(Transaminase)
S
O
O
S
OMe
HO
MdpC6
(Methyl transferase)
MdpC
(Hydroxylase)
NH3
AMP
MdpC2
O
OH
HO
OH
MdpC1
(NRPS A domain
-like protein)
MdpC4
(Aminomutase)
O
HO
FAD
OMe
HO
OMe
MdpC3
(Halogenase)
FADH2
MdpC8
(Dehydrogenase)
Cl
Cl
O FADH2
O
S
O
S
O
OH
FAD
O
S
O
S
Figure 17. Proposed biosynthetic pathway for the aryl hydroxypropionic acid moiety of maduropeptin.
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O
Aryl hydroxypropionic
acid moiety
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Biosynthesis of Aminosugar peripheral moiety of Maduropeptine: Maduropeptin has a
different aminosugar compared to C-1027 and neocarzinostatin, with the major difference being
the absence of two methyl groups on C5. The synthesis of the aminosugar begins with Dglucose-1-phosphate. The gene product (MdpA1) is highly homologous to the
nucleotidyltransferase (SgcA1) and helps installing the NDP group on the D-glucose-1phosphate (Scheme 18). The dehydrogenase (MdpA2) and decarboxylase (MdpA3) are
responsible for the removal of the CH2OH group at the C5 position. The sugar nucleotide moiety
is further modified by four more enzymes that include (a) a dehydrogenase, (b) a decarboxylase,
(c) a SAM-dependent methyltransferase and (d) a transaminase to furnish the final aminosugar.
The glycotransferases (SgcA6) finally transfer the sugar moiety onto the enediyne core.
Biosynthetic pathway for the Aminosugar moiety of maduropeptin
MdpA1
(Nucleotidyltransferase)
OH
HO
HO
O
OH
HO
HO
HOOC
MdpA2
O
OH
ONDP
OP
O
HO
OH
OH
MdpA3
O (Decarboxylase)
O (Dehydrogenase) HO
HO
OH
ONDP
ONDP
MdpA4
(Methyltransferase)
H2N
O
O
MdpA4
(Transaminase)
O
Me
OH
Me
OH
OH
ONDP
?
OH
ONDP
Deoxy Aminosugar
Dimethyl
salicyclic
acid
Dimethylsalicyclic
acid
D-Glucose- 1-Phosphate
Deoxy Aminosugar
Me
Me
OH HO
HN
Me
O
Scheme 18. Proposed biosynthetic pathway for the aminosugar of maduropeptin.
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OH
O
ONDP
NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics
2.5. Biosynthesis of 10-Membered Ring Enediynes
Peripheral moieties of the 10-membered enediynes: Based on the structure and folding
pattern of the enediyne core the 10-membered enediyne chromophores are further divided into
two sub group--(i) calicheamicin-like and (ii) dynemicin-like enediynes. Calicheamicin-like
enediynes include (a) esperamicins, (b) namenamicin and (c) shishijimicins. These are decorated
with a variety of sugar and aromatic peripheral moieties. In comparison, the dynemicin-like
chromophores include (a) dynemicin and (b) uncialamycin. These are characterized by the
presence of an anthraquinone peripheral moiety. The calicheamicin and dynemicin gene clusters
have been sequenced and partially annotated. Studies revealed the presence of enzymes involved
in the biosynthesis of the peripheral moieties of calicheamicin 1I are the sugar-modifying
enzymes and glycotransferases.
2.5.1. Biosynthesis of Calicheamicin 1I
Calicheamicins were isolated from Micromonospora echinospora spp. calichensis from
caliche soils. Calicheamicins represent a subfamily of enediyne chromophores and their
structure, mode of action and pharmacological properties have been examined rigorously.
Calicheamicin 1I, the best characterized member of the calicheamicins, contains a complex
aryltetrasaccharide moiety that confers the chromophore its DNA-binding specificity. The
Building blocks of Calicheamicin 1I are (a) enediyne core, (b) aryltetrasaccharide that is
composed of an iodized orsellenic acid moiety and four monosaccharides. The
aryltetrasaccharide unit is characterized by an unusual thiosugar and a hydroxylamino glycosidic
linkage (Figure 19).
Model of Convergent Biosynthesis of Calicheamicin  1I
SSSMe
H3C
O
SCoA
PKSE
+O
O
SCoA
Acetyl-& Malony CoA
NHCO2Me
H
HO
Glycosyl transferase
HO
HO
O
Sugar
OH
Me O
OH N
H
O
Deoxy Aminosugar
D
O
B
Sugar
O
O
MeO
EtHN
OH
A
Sugar
O
O
Me
S
O
Me
OMe
I
OMe
Me
O
HO
MeO
OH
C Sugar
O
Orsellenic acid
O
Enediyne Core
O
H3C
S PCP
acetyl CoA
NADPH
O + O
CalO1, CalO6,
CalO2, CalO3
Deoxy monosugar
O
S-CoA
malonyl CoA
(3x)
OH
HO
HO
OH
O
OH
OP
OP
D-Glucose- 1-Phosphate
D-Glucose- 1-Phosphate
Figure 19. Building blocks and model of biosynthesis of Calicheamicin 1I.
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Biosynthesis of Orsellenic acid of aryltetrasaccharide peripheral unit of Calicheamicin
1 : In addition to calE8, a second iterative PKS (calO5) gene is present in the Calicheamicin 1I
cluster. The predicted gene product (CalO5) shares significant sequence homology with AviM,
the iterative PKS for the biosynthesis of the oligosaccharide antibiotics avilamycins produced by
Streptomyces viridochromogenes. Notably, AviM is also the first type I polyketide synthase
isolated from bacteria to produce an aromatic compound. The fact that avilamycins and the
related evernimicin also contain a chlorinated orsellenic acid group hints that CalO5 could be
involved in the synthesis of the iodized orsellenic moiety in calicheamicin.
I
Sequence analysis suggested that CalO5 contains at least four domains (KS, AT, DH and
ACP) with a ‗core‘ domain apparently located between the DH and ACP domains. The lack of
KR domain suggests that the keto groups remain unreduced in the nascent polyketide chain. A
possible mechanism for the formation of the aromatic ring has been suggested (Scheme 19).
Four predicted enzymes that include a hydroxylase, a halogenase and two methyltransferases
putatively modify the aromatic ring to furnish the final product. The predicted cytochrome P450
(CalO2) and FADH2-dependent halogenase (CalO3) are likely to catalyze the hydroxylation and
iodination respectively, whereas the O-methyltransferases CalO1 and CalO6 install the two
methyl groups. Although the precise timing of the post-PKS steps and the stringency of the
substrate specificity of the enzymes remain to be fully established, biochemical and structural
characterization of CalO2 indicated that the hydroxylation may occur after the halogenations.
Based on the crystal structure of CalO2, MoCoy and coworkers also suggested that CalO2 may
act on a CoA-derivatized or ACP domain-tethered substrate. Thorson and coworkers have also
confirmed that sugar C is installed on the orsellenic group by glycosyltransferase CalG1.
Meanwhile, the FabH/KS domainlike CalO4 is likely to catalyze the formation of the thioester
linkage between the orsellenic acid and sugar B.
Scheme 19. Putative biosynthetic mechanism for the orsellenic acid moiety of calicheamicins.
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Biosynthesis of the sugar moieties of calicheamicin 1I: Biosynthesis of
monosaccharides-The Aryl Tetrasaccharide of Calicheamicin 1I: Calichiamicin 1I contains
four highly modified monosaccharides. Two of the monosaccharides are linked via an unusual
hydroxylamino glycosidic linkage. All four sugars are deoxysugars that exhibit greater
hydrophobicity compared to common sugars. The deoxysugars along with the linked orsellenic
acid group is giving the DNA binding specificity of calicheamicin 1I. The biosynthesis of the
four monosaccharides starts with the conversion of glucose-1-phosphate to NDP-glucose which
is similar to the biosynthetic pathway of sugar for C-1027, neocarzinostatin, maduropeptin and
other glycosides. Analysis of gene cluster of Calichiamicin 1I reveals that CalS7 catalyzes the
formation of NDP-glucose in the early stage of synthesis of sugars A–D. As discussed below, the
pathways for the four sugars would diverge after the formation of NDP-glucose. The 4hydroxyamino-6-deoxy--D-glucose (sugar A) is directly tethered to the enediyne core. The
deoxygenation at C6 position and installation of the amino group are followed similar routes that
involve the NDP-4-keto-6-deoxy--glucose intermediate in case of C-1027 (Scheme 20).
Biosynthesis of Monosugars peripheral moietes of Calichiamicin
AT2433-B1
D-Glucose- 1Phosphate
CalS7
CalS8
CalS3
NDP-4-keto-6deoxy--D-glucose
CalS9
CalS14
CalE10
CalS12/13
Sugar A
CalS10
Sugar B
Sugar C
Scheme 20. Biosynthetic pathways for the monosaccharides of calicheamicin  1I (Liang, Z-X Nat. Prod. Rep. 2010, 27, 499.).
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The hydroxylamino glycosidic bond between sugar A and sugar B is very crucial for
maintaining the correct conformation and rigidity of the oligosaccharide for effective binding to
the minor groove of DNA. A novel aminosugar oxidase, the N-oxidase CalE10, enzyme
catalyzes the hydroxylation of the amine group. CalE10 is a heme protein that exhibits stringent
regiospecificity with limited overoxidation in vitro.
Calicheamicin 1I contains a 4-thio-2,4,6-trideoxy-D-glucose (sugar B) that is connected to
the orsellenic acid moiety through a thioester linkage. The sulfur atom of sugar B contributes to
the binding mode of calicheamicin 1I by forming hydrogen bonds with an exposed amino proton
of guanine in the minor groove of DNA. However the mechanism of installation of sulfur atom is
not known. The closely related esperamicin A1 also contains a similar thiosugar. From the
isotope feeding experiment, it is known that the sulfur is derived from L-methionine or Lcysteine.
The enzymes involved in the biosynthesis of methylated 3-methoxy-L-rhamnose (sugar C)
synthesis remains unclear. However, it is believed that the biosynthesis follow a common
pathway for rhamnose synthesis with the deoxygenation at C6 facilitated by the formation of the
NDP-4-keto-6-deoxy--D-glucose intermediate (Scheme 20). A putative epimerase and a
methyltransferase most probably catalyze the epimerization at C2 and methylation at C3
respectively.
Aminodideoxypentose (4-amino-3-O-methoxy-2,4,5-trideoxypentose, sugar D) present in
calicheamicin 1I is similar to that present in esperamicin A1 and in indolocarbazole antitumor
antibiotic AT2433. Comparison of the calicheamicin and AT2433 gene clusters indicated the
presence of a set of common genes that are expectedly involved in the biosynthesis of the 4amino-3-O-methoxy-2,4,5-trideoxypentose.
It is established that the synthesis of the modified pentose proceeds via a TDP-sugar
pathway. A decarboxylation step is involved at C6 position with the help of a UDP-glucuronate
decarboxylase-like protein (CalS9). The deoxygenation of C2 and installation of amino group at
C4 follow as per the mechanisms shown in Scheme 20.
Assembly of calicheamicin 1I by glycosyltransferases: The orsellenic acid and sugar
moieties are assembled onto the enediyne aglycone in the final stages of calicheamicin
biosynthesis. A set of O-glycosyltransferases are utilized to catalyze the transfer of the sugars
from the nucleotide diphosphate sugar donors to the aglycone acceptor. Glycosyltransferases
CalG3 and CalG2 catalyze the first two glycosylation steps in a sequential fashion (Figure 21).
The crystal structure of CalG3 revealed a typical UDP-glycosyltransferase/glycogen
phosphorylase fold found in many glycosyltransferases. It is also known that CalG3 catalyzes the
formation of the unusual hydroxylamino glycosidic bond by using TDP-4,6-dideoxy-a-D-glucose
as a substrate surrogate. The other two putative glycosyltransferases (CalG1 and CalG4) encoded
by the gene clusters are the rhamnosyltransferase and aminopentosyltransferase which are
responsible for the installation of sugars C and D respectively.
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Figure 21. Assembly of calicheamicin  1I by glycosyltransferases (Liang, Z-X Nat. Prod. Rep., 2010, 27, 499.).
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2.6. Selected References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
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