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Annual report : National Institute of General Medical Sciences (Volume 1988-89) online

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the superoxide dismutase inhibitable reduction of ferri cytochrome c. Since
the active enzyme is metal -free, the indisputable formation of O2' as the
proximate reducing intermediate suggests the participation of an enzyme-bound
organic cofactor mediating the obligatory 2eVle' conversion as electrons pass
on from NADH to O2 in the catalysis. Substantiating this proposition was the
finding that the purified enzyme alone could accept two electrons from NADH
stoichiometrically in the absence of any electron mediators. The observation
of a characteristic free radical signal (g=2.002) in the^ EPR spectrum obtained
anaerobically with a sample of the enzyme and NADH at 8 K may also support
the existence of an organic cofactor. Since this enzyme is expected to
operate via a single mechanism despite its dual functions as a NADH oxidase
and a 3,4-glucoseen reductase, the unique 2eVle' switching capability found
for this enzyme provides, for the first time, compelling evidence that it may
operate through a radical mechanism. Thus, the C-3 deoxygenation in the
biosynthesis of ascarylose, and possibly the 3,6-dideoxyhexoses in general,
may proceed with CO bond disruption followed by stepwise le'/le" reduction.



"Cycloaddition Reactions of Allyl and Related Cations"
ROl GM 35962 (Gassman, P.), University of Minnesota

Nature is extremely effective in generating a wide variety of monocyclic and
polycyclic ring systems containing only carbon. The exact biosynthetic paths
to many of these systems are unknown. Even in many cases where the
biosynthetic paths are believed to be understood, scientists have been unable
to duplicate the specificity of nature. During the past 2 years. Dr. Gassman
and his colleagues have demonstrated that acyclic hydrocarbons can be
converted to certain monocyclic and bicyclic hydrocarbons in high yield with
high specificity at room temperature or below. These reactions accomplish two
goals. First, they provide examples of cyclization reactions under mild
conditions which will add to the synthetic repertoire of chemists and simplify
the synthetic approaches to a variety of terpene type natural products.
Second, they provide examples of intramolecular cycl izations, which had been
ruled out as possible biosynthetic pathways, because of the high temperature
and high pressures previously thought to be necessary in order to accomplish



434



such transformations. These results should prompt a rethinking of certain
proposed biosynthetic routes.

Dr. Gassman and coworkers have found that protonation of certain acyclic
tetraenes leads to the formation of reactive intermediates having a conjugated
diene in one part of the molecule and an allyl cation in another portion of
the molecule. The allyl cation can be viewed as a vinyl group bearing a
powerful electron-withdrawing group (the carbocation) . As a result, this
vinyl group is transformed into an extremely powerful Diels-Alder dienophiles.
This dienophile adds intramolecularly to the conjugated diene to form a
bicyclic ring system. Many of these reactions are complete in seconds below
room temperature. Thus, they serve as excellent models for nature. In this
way, bicyclo[4.3.0]nonanes, bicyclo[4.4.0]decanes, and bicyclo[5.4.0]undecanes
have been prepared in excellent yield and high specificity.

In an attempt to model successfully nature's formation of I0-, I1-, I2-, and
14-membered carbocyclic skeletons, Dr. Gassman and coworkers have shown that
an alkoxyl stabilized allyl cation can be trapped intramolecularly by a vinyl
cyclopropane group to form 11-membered rings. In one case, a pair of
diastereomers were formed in 92 percent yield at -23°C to ambient temperature
over 17 hours, which illustrated the facile nature of this intramolecular
cycl ization.

''Synthetic lonophores for Cation Regulation"
ROl GM 36262 (Gokel , G.), University of Miami

Cation complexation is fundamental to many biological processes. In
particular, the recognition and transport of alkali metal cations such as Na*,
K^, and alkaline earth metals such as Ca"^, are especially important since
these cations are ubiquitous. The problem of recognizing a cation is often
conceptualized in size terms: if the guest (cation) is a specific size, it
will fit (and therefore presumably be recognized) by the same size but
complementary host. The fit is no doubt an important part of cation
complexation and regulation, but the combination of how rapidly the cation is
bound coupled with the strength of binding also plays a crucial role.

Dr. Gokel 's cation regulation program is based on the notion that both
kinetics and thermodynamics are crucial to the problem. The rates of cation
binding and release define the equilibrium position of the reaction between a
ligand and a cation. His group has designed a large variety of macrocyclic
polyether compounds having one or more side arms. These compounds are
generally flexible and therefore have fast cation complexation rates. They
also enjoy reasonably fast release rates so that transport through a membrane
is possible. This is important for cation transport since compounds having
very slow release rates will be poor cation carriers even though they may have
good selectivity and binding strength.

The lariat ether compounds were devised to be both selective and dynamic.
They are based generally on the crown ether framework but have one or more
flexible arms that can assist in solvating a ring-bound cation. As part of
the present program, this team has prepared more than 200 novel structures
containing one, two, or three side arms. Selectivity and binding strength
have been studied in homogeneous solution and some of the factors affecting



A35



selectivity are now better understood. They have been able, for example, to
rank donors in their efficacy for binding Na^, K"*^, and Ca^"^. They have also
been able to dispel the longstanding myth that selectivity is controlled
exclusively or even primarily by the "hole-size fit" notion. Cation binding
strength, dynamics, and both enthalpic and entropic contributions to binding
have been assessed for many structures. Solid state structures have been
obtained for numerous compounds and this suggests the binding relations that
exist in solution. They have developed a number of dynamic binders that have
20-fold selectivity for Ca"^ over identically sized Na^. They have prepared
lariat ether compounds having steroid side arms which exhibit interesting and
novel aggregation behavior and represent a new class of neutral liposomes.
The work accomplished thus far lays the groundwork for a new thrust in the
direction of synthetic cation-conducting channels.



"The Biological Chemistry of Sulfur and Selenium"

ROl GM 37000 (Rabenstein, D.), University of California, Riverside

Although the importance of sulfur, and more recently selenium, as essential
elements is well established, many aspects of their biological chemistry
remains uncharacterized at the molecular level. Dr. Rabenstein and his
coworkers have made substantial progress in characterizing, at the molecular
level, selected reactions of sulfur and selenium compounds in intact
erythrocytes, plasma, and aqueous solution by nuclear magnetic resonance (NMR)
spectroscopy. He and his colleagues have developed methods with which the
water resonance and interfering hemoglobin resonances can be selectively
eliminated from proton NMR spectra of intact erythrocytes. With the high
sensitivity of their 500 MHz NMR spectrometer, they can obtain a spectrum from
compounds present in erythrocytes at concentrations as low as 50 micromolar in
as little as 15 seconds of instrument time. With this high sensitivity, they
have been able to monitor changes in the redox state of intracellular thiol
compounds after the erythrocytes are subjected to oxidative stress.
Endogenous thiol compounds and thiol -containing drug molecules, e.g.
penicillamine, which were introduced into the erythrocytes by incubation have
been studied. With the high chemical shift dispersion and high sensitivity of
their NMR spectrometer, they were able to detect well -resolved resonances for
intracellular glutathione, oxidized glutathione, penicillamine, penicillamine
disulfide, and penicillamine-glutathione mixed disulfide in penicillamine-
containing erythrocytes which had been subjected to oxidative stress. They
found that, after glucose is added to the stressed erythrocytes, the oxidized
glutathione and the penicillamine-glutathione mixed disulfide are reduced by
an enzyme-catalyzed process, whereas penicillamine disulfide is not. The
results of this study demonstrate that proton NMR spectroscopy is a powerful
method for studying the intracellular oxidation/reduction chemistry of small
thiol -containing compounds.

Dr. Rabenstein and his coworkers have also discovered that thiol/disulfide
interchange reactions, which are important pathways for the metabolism of
thiol -containing compounds, can be quantitatively characterized by NMR
spectroscopy. They have characterized reactions involving glutathione,
cysteine, coenzyme A, captopril, and homocysteine. In a parallel study which
has as a major objective the elucidation of the chemistry which occurs at the
active site of the seleno-enzyme glutathione peroxidase, they have



436



quantitatively characterized a selenol/diselenide exchange reaction. They
have found that selenol/diselenide exchange is some 10^ times faster than
thiol/disulfide exchange at physiological pH. This is an amazing difference
in reactivity for these two otherwise very similar elements, which suggests
that nature may have chosen selenium rather than sulfur for the active site of
glutathione peroxidase because of the combined effects of selenolate being
both a better nucleophile and a better leaving group in nucleophilic
displacement reactions.

^^Structural Models for Molvbdoenzymes^^

ROl GM 37773 (Enemark, J.), University of Arizona

Molybdoenzymes are essential in sulfur metabolism and in several other
biological processes. However, the detailed structure of the molybdenum
center of these enzymes is still not known. Dr. Enemark and his colleagues
have been synthesizing model molybdenum compounds which incorporate chemical
and structural features proposed to be present in the molybdenum centers of
enzymes. Previous work by his group and others have shown that at least two
sulfur atoms must be bound to the molybdenum in order mimic the chemical
reactivity and spectroscopic properties of molybdenum in sulfite oxidase and
related enzymes. Most recently they have synthesized a series of
oxomolybdenum{V) compounds with the same donor atoms but varying chelate
ligand backbones. It was found that the size of the chelate ring and of the
alkyl substituents on the chelate ring backbone of LMoO[S-(CH2)n-S] and
LMoO[0-(CH2)n-0] compounds can lead to changes in the reduction potential of
more that 0.2 volts per CH2 unit. The detailed structural changes at the
molybdenum atom that accompany the changes in ring size are not yet known.
The protecting ligand (L) constrains the molybdenum to six-coordinate
fac- stereochemistry, but changes in the torsional angles of the coordinated
heteroatom can result from changes in chelate ring size. Such torsion angle
changes provide a promising mechanism for transmitting seemingly remote
differences in the ligand backbone to the reduction potentials of the metal
center.

Finally, the significant changes in molybdenum reduction potentials that are
observed upon changing the ligand skeleton may be important for understanding
the properties of the molybdenum cofactor of enzymes, which is thought to
possess a five-membered chelate ring with two side chains.



"The Plane Facts on the Mode of Action of Bicvclomvcin'^
ROl GM 37934 (Kohn, H.), University of Houston

Bicyclomycin is a clinically useful antibiotic possessing a diverse spectrum
of biological activity. This structurally unique drug exhibits moderate to
potent activity against several Gram-negative bacteria including Escherichia
col i , Klebsiella , Shigella . Salmonella , Citrobacter , Enterobacter cloacae , and
Neisseria gonorrheae . Its emerging importance for the treatment of
nonspecific diarrhea in humans and bacterial diarrhea in calves and pigs has
led to the commercial introduction of this drug under the trade name:
Bicozamycin. Unfortunately, efforts aimed at elucidating the mechanism of
action of bicylomycin at the molecular level both in vitro and in vivo have



437



not paralleled the introduction and use of the drug. Little is known
concerning the drug activation process, the energetics of key steps, and the
interaction of the antibiotic with the receptor site. Both chemical and
enzyme -mediated pathways have been invoked to account for the mode of action
of the drug.

The major goals of studies instituted in Dr. Kohn's laboratory have been the
elucidation of the chemical pathways for drug activation, and the
determination of the minimal conditions needed for activation under conditions
which approximate the biological process. Significantly, Drs. Kohn and Abuzar
have discovered that drug activation proceeds over a wide pH range. Of
particular interest, recent studies have demonstrated that treatment of
bicyclomycin with alkyl mercaptans, cysteine derivatives, and select secondary
amines in tetrahydrofuran-water mixtures at near neutral 'pH' led to the
stereoselective formation of a single compound. The reactions proceeded at
the exomethylene group in bicyclomycin with the loss of ammonia. These
transformations represented the first documented examples of the activation of
the drug under mild conditions. Furthermore, the observation that
bicyclomycin binds to cysteine derivatives has commanded special attention in
light of previous suggestions that sulfhydryl -containing proteins present
within the peptidoglycan assembly of the growing bacteria are the primary
targets for the antibiotic. Finally, the exomethylene functional ized product
has been shown to undergo further binding with lysine derivatives to generate
bis-alkylated adducts. These discoveries have permitted Dr. Kohn and
coworkers to suggest that the antibiotic functions as a sequential alkylating
agent and to propose a new pathway for its mode of action.

"Antibodies as Catalysts"

R29 GM 38273 (Hilvert, D.), Scripps Clinic and Research Foundation

The mammalian immune system is the most prolific source of specific receptor
molecules known. Recently, scientists have demonstrated that this marvelously
diverse system can be exploited to prepare antibody proteins with tailored
catalytic activities. This approach to enzyme design involves synthesizing
compounds that mimic the transition state of a particular reaction, eliciting
immune responses against such substances, and characterizing the specific
antibodies generated.

Dr. Hilvert is utilizing this new technology to develop antibody catalysts for
concerted electrocyclic reactions, including Claisen rearrangements and Diels-
Alder cyclizations. Since a presumably low probability exists for generating
an effective constellation of multiple catalytic groups (e.g., general acids,
general bases, nucleophiles) in the binding site of an antibody during
immunization, reactions that are shape- rather than chemoselective should be
especially good targets for catalysis. Concerted reactions generally do not
require chemical catalysis but should be particularly sensitive to the
principal catalytic effects antibodies are likely to impart, i.e. induced
strain and proximity. Moreover, these processes are of enormous theoretical
and practical interest, especially for enhancing our understanding of how
enzymes work and for synthesizing biologically active molecules.

The conversion of {-)-chor1smate into prephenate is an example of a
biologically relevant 3,3-sigmatropic rearrangement. In plants and lower



438



organisms this transformation, catalyzed by the enzyme chorismate mutase, is
the committed step in the biosynthesis of the aromatic amino acids
phenylalanine and tyrosine. Dr. Hilvert used a transition state analog
inhibitor for the natural enzyme to prepare antibodies with chorismate mutase
activity. One of the antibodies catalyzed the rearrangement with a rate
acceleration of more than two orders of magnitude compared to the uncatalyzed
process. Saturation kinetics were observed, and at 25° C the values of k^^t
and K^ were 1.2 X 10"^ s"^ and 51 /iM, respectively. The transition state
analog was shown to be a competitive inhibitor of the reaction with K^ equal
to 0.6 //M. The antibody also exhibited high enantioselectivity, accepting
only the (-)-isomer as a substrate.

These results represent the first example of an antibody-promoted carbon-
carbon bond-forming reaction and demonstrate the feasibility of catalyzing
concerted electrocycl ic processes with antibodies. The strategy utilized in
Dr. Hilvert's experiment is now being extended to the development of other
antibodies that are shape-selective rather than chemoselective. Such
molecules will be valuable as tools to study the importance of strain and
proximity in protein-catalyzed reactions or for the synthesis of complex
natural products. Since the efficacy of many drug molecules depends on the
precise configuration of bonds around a single atom, the superb stereo-
specificity of catalytic antibodies, similar to that of natural enzymes,
should be of great practical value.

"Chemical Interactions of Coral Reef Invertebrates^'
R29 GM 38524 (Paul, V.), University of Guam

Research in Dr. Paul's laboratory is directed towards understanding the
chemical defenses of marine organisms on coral reefs as a way of investigating
bioactivities of marine natural products. Dr. Paul and her coworkers have
developed a field-oriented approach to identifying bioactive natural products
from marine invertebrates on Guam. They use a field assay that incorporates
extracts and isolated metabolites into a carrageenan-based diet to test the
effects of these compounds as feeding deterrents toward natural populations of
coral reef fishes. These methods allow for a bioassay-guided approach to
isolating natural products from marine invertebrates that function as chemical
defenses. The metabolites are further tested for their pharmacological
activities in Dr. Bob Jacob's laboratory. Thus, they can compare feeding
deterrent properties of natural products with potential biomedical activities.

Dr. Paul's research tries to understand ecological factors associated with
invertebrates that produce biologically active natural products. For example,
soft corals produce two types of defenses against predators, secondary
metabolites and calcified sclerites. Concentrations of secondary metabolites
are much greater in the top portions of the colonies, while sclerite
concentrations are much higher in the bases. Top portions are more accessible
to predators but at the same time, high concentrations of secondary
metabolites make the tops better chemically defended. Similarly, high
concentrations of secondary metabolites are found in soft corals and gorgonian
corals that have low sclerite concentrations or very small, powdery sclerites.
The soft coral Sinularia maxima on Guam has relatively low concentrations of
very fine sclerites but produces a potent cembranoid diterpenoid that
functions as a chemical defense against predatory fishes. This same



A39



metabolite shows potent anti- inflammatory activity in pharmacological assays
that test for inhibition of phorbol -induced inflammation of the mouse ear.
This metabolite is one of the best natural feeding deterrents and
anti -inflammatory agents that has been isolated to date.

Other potent feeding deterrents that they have isolated from sponges and
ascidians (tunicates) have also been very active in other pharmacological
assays. Patellazole B from the tunicate Lissocl inum Patella and laulimalide
from the sponge Hvattella s£. were both potent cytotoxins against the KB cell
line, and extracts of each organism were effective feeding deterrents in field
assays. Many other extracts and isolated metabolites have been investigated
in these bioassays and the relationships between natural chemical defenses and
cytotoxicity and anti -inflammatory activities are good. These methods may
provide a useful way of relating chemical ecology with biomedical activities
of marine natural products.



"Novel Ring-Enlargement Chain Extension Reaction"
ROl GM 39825 (Dowd, P.), University of Pittsburgh

In the course of their investigations into the mechanism of action of vitamin
Bi2> Dr. Paul Dowd and his colleagues discovered an unusual rearrangement
reaction involving migration of an a-keto ester group to a methylene radical
center. The essentials of a novel ring expansion/chain extension reaction
were contained in these experiments. The first ring expansion experiments
took the form of the alkylation of five-, six-, and seven-membered cyclic
i3-keto esters, readily available from the Dieckmann condensation, with
methylene dibromide or diiodide. Ring expansion to the respective six-,
seven-, and eight-membered ^-keto esters occurred smoothly in high yields upon
treatment with tri-7-butyltin hydride in refluxing benzene. This set the
stage for development of a new synthetic strategy for ring expansion.

The next step involved extension of the reaction to include ring expansion by
larger increments. In this fashion, attachment of three- and four-carbon
halogen-bearing sidechains to the keto ester followed by treatment with
tri-n-butyltin hydride resulted in ring expansion by three and four carbon
atoms. Thus, entry into the medium-sized rings of 9, 10, and 11 carbons
becomes straightforward. The method is compatible with ketone and ester
groups, so it encourages inclusion of useful functionality for further
synthetic elaboration.

Current efforts involve extension of the method to heterocyclic compounds,
where there are special problems to overcome. For example, a new precursor
was required because the bromomethyl sidechains undergo undesirable side
reactions. Accordingly, the selenophenylmethyl group was used as the
precursor to the free radical. Using this approach N-benzylpiperidone
carboxylate undergoes smooth ring expansion to the corresponding
seven-membered azepinone. Likewise, ring expansions of tetrahydrofuryl and
tetrahydrothiophyl keto esters were readily affected.

The ring expansion method has also been applied to large rings. Dr. Dowd's
method allows expansion of 12- to 13-, 14- to 15-, and 15- to 16-membered
rings. An attractive target molecule in this series is the 15-membered,



440



naturally occurring perfume constituent muscone. By attaching a
2 -methyl -1-iodopropyl side chain to readily available cyclododecalnone and
treating with tri-n-butyltin hydride, (±)-muscone was produced in 20 percent
yield. The optically active sidechain precursor is commercially available so
enantiomerically pure muscone is also accessible by this route



441

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Online LibraryNational Institute of General Medical Sciences (U.Annual report : National Institute of General Medical Sciences (Volume 1988-89) → online text (page 44 of 44)