Harnessing the power of some of nature's most remarkable molecules to enable breakthrough

advances in scientific understanding, medical treatment, anda disease control in humans has
been a recurring theme in this book.
However , it is not only the health of the human population which has benefited from these
processes, as impressive strides have also been made in veterinary science and medicine
Many of the same life saving or enhancing treatments developed for humans, such as insulin
injections, hip replacements, and even pacemakers, are now available to animals, and
therapeutic drugs are no exception in this regard. Many of the pharmaceuticals used by peple
are employed to treat the corresponding ailments in animals, although, of course, the vast
array of physiologies enchountered in the latter means that there are also many more species-
specific medicines
a problem commonly encountered in veterinary medicine is one of precise diagnosis ( or,
often more accurately, the relative cost of establishing a correct diagnosis). THerefore, a
number of prescribed treatments for animals are rather all-encompassing in that they target
several different possible causes of the ailment at once.
Examples include panolog and animax, used as topicaltreatments to manage dermatological
disorders in cats and dogs. Each of these medications contains no fewer than four active
ingredients

Nystatin A1 is an antifungal agent against candida albicans, whilst triamcinolone acetonide is

a semi-synthetic corticosteroid which reduces inflammation and pruritis. The last two
components combine to provide a comprehensive therapy against bacterial infection; they are
neomycin sulfate and thiostrepton, the latter molecule being the focus of this chapter.
Thiostrepton is the flagship member of a remarkable class of natural products known as the
thiopeptide antibiotics. As this name might suggest, this family of compounds, which
numbers in the dozens, is comprised of a variety of sulfur-rich peptide-based, the majority of
which exhibit activity againts gram-positive bacteria

Hal 2
Besides thiostrepton, a number of other representative members of the thiopeptide anti-biotics
are illustrated in box 2. a common structural feature among them is the presence of poly
substituted nitrogen heterocycle ( e.g. a dehydropiperidine or pyridine ring system), onto
which is appended a variety of modified amino acid residues, themselves contained within a
larger macrocyclic framework. these modified residues may take the form of thiazole or
oxazole rings, dehydroaminno acid units, or one of several other structural motifs resulting
from enzymatic post-translational modification of the parent amino acid residue.

Despite being one of the first members of this class of compounds to be discovered, more
than half a century ago, thiostrepton remains the jewel in the crown of the thiopeptide
antibiotics. it is the most structurally complex and most extensively studied, and the only one
yet to have reached commercial application as an antibacterial agent. THe story of
thiostrepton begins in 1954 in the desert of new mexico, usa. inhabiting this innocuous pile of
dirt was a strain of bacteria ( streptomyces azureus) which the scientist cultivated in the
laboratory. subsequent extraction of these cultures resulted in the isolation of a novel
substance, named thiostrepton, that displayed potent antibiotic activity againts a variety of
gram-positive bacteria, in particular against a strain resistant to penicillin, the leading
antibiotic of the era

However, hamstrung by the absence of the high resolution spectroscopic techniques that
chemists today take for granted, the squibb institute team was unable to speculate as to the
structure of this mysterious and promising new antibiotic, other than to identify its
polypeptidic nature and perform a rudimentary analysis of its physicochemical properties. the
latter included the interesting observations that the antibacterial activity of thiostrepton was "
not appreciably decreased in twenty four hours by incubation at room temperature in the
presence of artificial intestinal juice, artificial gastric juice, ten per cent solution of human
feces, or in water
An arduous, and at times rather confusing, international quest to unravel the mystery
regarding the structure of thiostrepton unfolded over subsequent years. painstaking classical
degradative experiments, carried out largely by the groups of miklos bodanszky ( squib
instititute) anda george w kenner ( university of liverpool . UK), revealed the nature of
several of the amino acid components, but left unanswered the question of their connectivity.
Concurrently several varying estimates of the molecular weight and empirical formula of
thiostrepton were made by other groups. matters were complicated further by the isolation, in
1955, of a purportedly novel thiopeptide antibiotic, initially given the trade name byamycin
but later renamed thiactin, by bernard heinemann an co-workes ( Bristol laboratories, USA)
from cultured extracts of soil bacteria ( streptomyces hawaiiensis) colected in hawaii.

this substance was in fact shown to be identical to thiostrepton by the Bodanszky group in
1963, but the somewhat unfortunate use of three different names ( thiostrepton, bryamycin or
thiactin) to describe the same compound persist to this day. as events transpired, the major
breakthrough in dispelling the clouds of uncertainly surrounding the structure of thiostrepton
would not arrive until 1970, when dorothy crowfoot hodgkin ( of penicillin and vitamin B12
fame, see chapters 13 and 16, respectively) and co-workers at the university of oxford
published their x-ray crystalographic structure of thiostrepton. this study established the
stereochemistry and atom connectivity of most, but significantly, not all of the molecule.
although x-ray crystallography is generally considered to be the final arbiter of structure
determination, exceptions can occur, as in this case where disorder in the region containing
the side chain prevented the unambiguous assignment of that domain of the molecule.

in 1976, C-NMR (Carbon Nuclear Magnetic Resonance) Studies by kazuo tori and co
workers at the shionogi research laboratory ini osaka , japan, identified the side chain as
being comprised of two dehydroalanine units. thus, more than twenty years after its isolation,
the complete structure of thiostrepton was finally revealed ( box 3). Confirmation of this
assignment has since been provided by further NMR studies, x-ray analysis on an alternative
crystal polymorph, and as we shall describe later in this chapter , total synthesis.

As shown in box 3, the extraordinary polycyclic architecture of thiostrepton can be divided

into four domains, namely the twenty six membered thiazoline macrocycle, the twenty seven
membered quinaldic acid macrocycle, the dehydropiperidine core, and the bisdehydroalanine
tail. the first three domains form a roughly globular structure, with one macrocycle folded
over the top of the other. this contacts to a water molecule which , although not chemically
bound to thiostrepton , is held within a hydrophilic cleft in the interior of its structure.

Hal 3
the year 1970 proved to be seminal in the history of thiostrepton, for not only was the first x-
ray crystal structure reported, but also the first indications as to the molecular basis of its
antibiotic activity were revealed.
A study by Demohn at the University of Wisconsin Medical School (USA) demonstrated that
thiostrepton inhibits bacterial protein biosynthesis by blocking the function of the 505
ribosome subunit, triggering a sequence of events which ultimately lead to bacterial cell
death. A later study by Yanyang Xing and David E. Draper (Johns Hopkins University,
Maryland, USA) in 1996 more accurately pinpointed the binding site of thiostrepton on the
bacterial ribosome as being at a region known as the L11 binding domain.

The majority of known antibiotics function by targeting and inhibiting the bacterial cell
machinery required for one of four following processes :(1) cell wall biosynthesis, (2) folic
acid biosynthesis (a precursor to DNA/RNA), (3) inhibition of DNA/RNA replication or
repair, and (4) inhibition of protein biosynthesis on the ribosome. The former two pathways
have no parallels in eukaryotic cells, whereas the latter two clearly occur in both prokaryotic
(i.e. bacterial) and eukaryotic (e.g. animal, human) celss. Fortuitously, however, there are
sufficient structural difference between the enzymes required for protein and DNA
biosynthesis in prokaryotic and eukaryotic cells such that selective inhibition is possible.

The expression of the genetic information encoded within DNA occurs ( in the broadest
possible terms) in two stages : transcription, in which messenger RNA ( mRNA) is
synthesized from a DNA template, and translation, in which the mRNA serves as the template
for protein synthesis. In prokaryotic organismes, these two yevents are closely coupled, while
in eukaryotic cells these events are separated in both space and time; transcription occurs in
the nucleus while translation occurs in the cytosol. In eukaryotic cells, the enzyme RNA
polymerase II transcribes DNA to synthesize precursors of mRNA ( in addition to a number
of non-coding RNA molecules). In 2001, Roger D. Kornberg and his group at Stanford
University (USA) reported an x-ray crystallographic snapshot of an actively transcribing
complex of RNA polymerase II in complex with DNA (box 4). THe significance of this , and
other work from the Kornberg laboratories, in enhancing our understanding of the structural
basis of transcription was recognized with the awarding of the 2006 Nobel Prize in Chemstry
to Kornberg " for his studies of the molecular basis of eukaryotic transcription."
The ribosome is the site of protein biosynthesis in the cell, functioning to translate the genetic
information encoded within mRNA to assemble amino acid into polypeptides (Box 5), and is
the largest and most complex of all the enzymes. all ribosomes display a hight degree of
structural and functional homology. Nevertheless, several key differences between
prokaryotic and eukaryotic ribosomes enable selectivity in antibiotic action. It is these
structuraldifferences that result in thiostrepton inhibiting bacterial protein biosynthesis over
one hundred times more potently than eukaryotic protein biosynthesis.
Bacterial ribosomes, which are about 30% smaller than in eukaryotes, are composed of two
subunits of unequal size, known as the 30S and 50S subunits. These subunits are roughly
two-thirds ribosomal RNA (rRNA) and one-third protein by weight, and associate through
noncovalent interactions, The larger 50S subunit is itself a conglomerate of aproximately
thirty individual proteins and two strands of rRNA, the latter known as the 23S and 5S
strands. Binding of thiostrepton to a specific site of the 50S subunit composed of protein L11
and 23S rRNA( the L11 binding domain) prevents conformational changes of the ribosome
required to drive movment of the ribosome along the mRNA. Protein biosynthesis is thus
stalled, ultimately leading to the death of the bacterial cell.

Although thiostrepton has been a mainstay topical antibiotic in veterinary medicine for many
years, its application in humans has been limited by its low solubility in aqueous systems and
its poor bioavailability ( the proportion of an administrered dose of medication that reaches
the systemic circulation) due to low absorption from the gastrointestinal tract. These factors
result in the antibiotic inducting resistance to itself in the proliferating bacteria before its
concentration becomes therapeutically significan. There are, however, a number of clinically
important classes of antibiotics relevant to human health which act by inhibiting various steps
of protein biosynthesis on the bacterial ribosome(Box 6). A 7:3 mixture of the macrolactone
dalfopristin ( semi-synthetic derivatives, with improved water solubilty, of naturally
occurring streptogramins A and B, respectively), marketed as Synercid, was granted
accelerated approval by the Food and Drug Administration , USA, in 1999 for the treatment
of serious or life threatening infections associated with vancomycin-resistant Enterococcus
faecium (VRE) bacteremia.

The two components act synergistically to inhibit bacterial protein biosynthesis, the
combination proving more effective than either component individually. Macrolides, such as
erythromycin A (CHapter 17) and its analoges, form arguably the major class of ribosome-
targeted antibiotics. Telithromycin is one the most recent semi synthetic erythromycin
derivatives approved for use in humans, and is representative of the latest generaton of
macrolide antibiotics that have been optimized for potency, stability to gastric aacids in the
stomach, and activity against macrolide resistant pathogens. The aminoglycosides, which can
trace their esteemed lineage back to the discovery of streptomycin in 1944, are potent drugs
agains Gram-negative bacteria, tobramycin sulfate being one of the most prominent members
in contemporary clinical user.

The tetracycline class of antibiotics has suffered a diminishing role as a front line therapy in
recent decades due to emerging bacterial resistance, but this trend may start to be reversed
following the fast track approval by the FDA of tigecycline (Tygacil, Wyeth) in 2005.
Substitution of the aromatic ring with a glycine amide functionality has been found to restore
activity against tetracycline-resistant Escherichia coli and Staphylococcus aureus, as well as
against methicillin resistant Staphylococcus aureus ( MRSA). Linezolid (Zyvox, Pfizer) is the
first, and to date only member of the oxadolidinone class of antibiotics to be approved for use
in humans, and is also currently the only totally synthetic ( as opposed to being derived from
or inspired by a natural product) antibiotik in clicinal use

Its use is usually reserved for serious Gram-positive bacterial infections where older
antibiotics have failed due to bacterial resistance. Linezolid is also novel in that it inhibits the
initiation of bacterial protein biosynthesis, a stage of protein synthesis previously unexplored
as a target, by binding to the 50S subunit and preventing its association with the 30S subunit
and mRNA. In addition to the small selection described here, a number of other new
antibiotics, which target a variety of aspects of the bacterial cell machinery , are currently in
late stafe clinical development. Thus, the outlook for combating the spread of bacterial
resistance, and the concurrent perceived lack of new antibiotics, is not as bleak as the
periodic sensationalism from the media and other sources might have one believe.

The structural complexity and biological properties of the thiopeptide antibiotics have made
them attractive targets for many synthetic practitioners keen to probe and expand the
boundaries of their field. Christopher J, Moody and his group ( then at the University of
Exeter, now at the University of Nottingham, UK) reported the landmark first total synthesis
of a member of the thiopeptide family ini 2000, namely that of promothiocin A (box 2). It
was also about this time that Nicolaou and co workers at The Scripps Research Institute ( La
Jolla, California, USA) began their march towards the total synthesis of thiostrepton, an
endeavor which forms the basis for the rest of this discussion.
Hal 7
The retrosynthetic analysis for what would ultimately be the successful strategy towards
thiostrepton is illustrated in Box 7, in which the molecule was dissected into smaller
fragments, corresponding to each of the domains of the target. Also illustrated is the order of
coupling of these fragments, namely generation of the thiazoline macrocycle, followed by
attachment of a masked form of the dehydroalanine tail, and finally the appendage of the
quinaldic acid macrocycle. It should be noted that this order of bond constructuion was not
entirely arbitrary, rather, arose from a series of systematic reevaluations of the synthetic route,
which, in turn, resulted from encountering numerous pitfalls and roadblocks on early paths to
the coveted prize.
Of all the prominent structural features encompassed within thiostrepton, it is undoubtedly
the dehydropiperidine core, which lies at the heart of the molecule, that is the most striking
from a synthetic perspective. One of the most pressing problems facing the Nicolaou group
was, therefore, to find an efficient (and, ideally, elegant) method to forge this sector of the
target. Fortunately, a combination of clues provided by nature and insightful studies by a
number of researchers appeared to offer a solution. In 1978, Barrie W. Bycroft and Maxim S.
Gowland at the University of Nottingham suggested that the dehydropiperidine core of
thiostrepton may be derived biogenetically " from the interaction of two dehydroalaine units
in a single peptide chain," although no mention of the nature of this interaction was put
forward. This proposal was further refined into the scheme depicted in Box 8 by Heinz G.
Floss and co workes ( University of Washington, USA) during the course of isotopic labeling
experiments on several members of the thiopeptide family aimed at probing their
biosynthesis. The cyclization event in this scheme reprensents a formal Diels-Alder
cycloaddition, albeit with slightly esoteric reaction partners. With this hypothesis as both their
inspiration and guide, Nicolaou and co workers investigated whether this type of process
coud be replicated in the laboratory. Indeed, they found that under the appropriate conditions
a thiazolidine precursor could be coaxed into following the reaction cascade illustrasted in
Box 9, via the fleeting intermediacy of the aza-diene intermediate shown, to furnish the
desired dehydropiperidine core structure.

The final stages in the total synthesis of thiostrepton are shown in Box 10. Following several
agonizing near misses, it was found necessary to install the reactive dehydroalanine moieties
once all the ring systems had been constructed. Formation of the dehydroalanines was
achieved through the selective oxidation of three selenium atoms, followed by the sontaneous
elimination of the resulting selenoxide species. The final step involved removal of the silyl
protecting groups under suitable conditions, an event which was accompanied, gratifyingly,
by the selective dehydration of just one of the hydroxyl groups, to install the final piece in the
puzzle, namely the trisubstituted (Z)-olefin. Synthetic thiostrepton was in hand on June 29,
2004, delightfully ending a five year syntheic odyssey.
The total synthesis of thiostrepton is representative of the state of the art of chemical
synthesis as this creative discipline moves into the new millennium. It also serves to illustrate
just how far the field has advanced since its formative years in the ninteenth century. Much of
this advancement has come within the last few decades; thirty years ago a synthetic target
such as thiostrepton would have been considered unattainable, yet today it lies conquered at
the hands of synthetic chemist, a feat only made possible by gains in our understanding of
fundamental concepts such as mechanism, reactivity, spectroscopy, and biosynthetic
pathways, to name but a few. It is with this knowledge that chemistry, and science as a whole,
can be driven forward, to address the challengers and opportunities facing humanity in the
twenty first century and beyond

Banyak senyawa yang berfungsi sebagai antibiotik pada hewan. Namun, yang
menjadi perhatian pada bab kali ini adalah senyawa thiostrepton. Senyawa thiostrepton
digunakann sebagai agen antibakteri dalam aplikasi secara komersial. Awal penelitian
terhadap senyawa thiostrepton bermula tahun 1954 di Meksiko dengan berawal dari
penelitian terhadap kotoran. Namun karena kurangnya teknik spektroskopi pada waktu itu,
spekulasi mengenai strukturnya masih belum dapat dijelaskan. Pada tahun 1963 ditemukan
pula senyawa yang identik dengan thiostrepton yaitu bryamycin atau thiactin. Penentuan
struktur thiostrepton dilakukan oleh beberapa ilmuwan melalui X-rays, NMR. Diteliti pula
tentang cara kerja thiostrepton sebagai antibakteri adalah dengan menghentikan biosintesis
protein pada bakteri sehingga menyebabkan kematian sel pada bakteri. Namun aplikasi
senyawa thiostrepton sering digunakan untuk hewan dan penggunaannya kepada manusia
masih dibatasi karena kelarutnnya yang rendah terhadap air. Hingga pada tahun 2004 ,
senyawa thiostrepton berhasil disintesis dan mengakhiri penelitian beberapa tahun
sebelumnya.