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Archive for the ‘inorganic chemistry’ Category

To live and function the human body needs energy, the majority of which is produced by the electron transport chain.  The end products from these enzyme catalyzed reactions are oxidized by the oxygen that you breathe and converted into water.  At the center of this complicated process is an incredible  protein that selectively transports oxygen to tissues in need and releases it on demand.  Hemoglobin is a protein carried by red blood cells that has selective affinity for iron, a metal critical to proper function of the body.  The importance of this protein is exemplified by what happens when it is broken.  Sickle Cell Anemia, for example, is caused by a mutation in the gene that codes for the protein.   In this case, the change of a single amino acid (glutamic acid to valine) damages its functionality such that the average life expectancies of people with this disease are only 42 years in males and 48 in women (S).  During this time, those afflicted also experience a host of quality of life maligning health problems, like chronic pain, increased risk of infection and heart disease and vaso-occlusive crises that occur when the misshapen blood cells block blood vessels.

Considering the structure, hemoglobin is a large protein made of four polypeptide chains, sewn together into a tetramer.  There are 2 β chains and two α chains, each of which having a net-like porphyrin ring system.  In human red blood cells, an iron atom is placed in the center of the ring structure, creating a coordinated system called a heme group.  The iron atom is the star of the system and makes the binding of oxygen possible.  It is in the +2 state and has octahedral symmetry, allowing 6 ligands to bind.  The 4 nitrogen atoms of the ring bond equatorially holding the Fe+2 in place, while from the bottom, a histadine amino acid from the protein super structure locks the Fe+2 atom in place.  The open top position is reserved for O2 binding, but in its absence a molecule of water usually takes its place in normal circumstances.

3D images showing the positions of the porphyrin ring, iron and histidine in the heme group. Nitrogen atoms are in blue, iron in red and carbon in the usual grey-black. (3D images courtesy of http://www.3dchem.com By all means check out the structures for yourself using their tremendously useful java based web tool!)

This same system is common throughout other enzymes and respiratory systems.  In humans, Cytochrome C  of the electron transport chain has a similar structure, except that the 6th (top) ligand is a methionine group.  The protein in this case is designed to transport electrons rather than oxygen molecules.  Myoglobin, which is found in muscles and also used for O2 transport, is structurally different, but utilizes a single porphyrin ring system, rather than four.  Enzymes, such as catalase and peroxidase,  also contain Fe manipulating systems similar to the above.  In other kinds of organisms too, the porphyrin ring is utilized in a variety of situations and complexed to many different metals depending on the conditions and needs of the creature involved.  Photosynthetic plants, for example, utilize the same ring, but the metal is magnesium.  Further, some bacteria are also known to use copper as the porphyrin metal of choice.

To make full advantage of this heme system, the binding of O2 in red blood cells is facilitated by a buffer system.  The concentration of any of the components of it increase or decrease the binding affinity of O2 at the hemoglobin binding site.  Proton (H+), CO2, Cl-, and 2,3-Bisphosphoglycerate (BPG) concentrations all have a role in the binding and release of O2 from hemoglobin.  For example, in tissues where the pH (H+ concentration) is acidic and the partial pressure of CO2 is high, the binding affinity of O2 at the binding site will be lowered and will induce hemoglobin to release its contents.  In the lungs, however, the O2 concentration is high compared to that of CO2, facilitating O2 binding.  Normally, BPG is found in equal amounts to hemoglobin, but in situations where O2 is in short supply this balance is undone by the increased production of BPG.  With BPG, binding to one of the active sites reduces the O2 binding affinity by about 25 times, thus when BPG concentration is high it pushes for a release of O2 into tissues that need it.

The larger Fe staggers the planar porphyrin ring, where as the smaller version fits better, contributing to stability.

Aside from outside effects promoting binding, there are effects coming essentially from the design of the system itself that have to be considered as well.  The question of why iron and not some other transition metal is of special relevance here.  Using iron as the central metal, in this case, yields benefits in terms of performance and binding specificity to oxygen.  Cobalt, for instance, is used in similar systems like those of vitamin B12’s corrin ring, but while both cobalt and iron contain d orbitals that could bind, only iron allows for the perfect balance between size and binding specificity in this system.  While unbound, the Fe+2 atom is just a little too large to fit into the normally planar porphyrin ring.  In this case, it is in a high spin state where the molecular orbitals are further away from the atom, giving it a larger size.  The high spin state is larger, because the Pauli exclusion principle prohibits the atom’s 3d electrons from getting too close to one another due to repulsions.  Thus, additional space is needed to house the electrons in orbitals further away.  However, when the Fe+2 binds to an oxygen molecule the electrons can be shuffled into orbitals that are closer to the atom.  Its orbital symmetry changes to a low spin symmetry and CLICK ! the Fe+2 shrinks just enough to fit snugly into the porphyrin ring system.

A silly, but useful comparison.

This part also contributes to a critically important finding: that the binding of O2 is cooperative, in that the binding of one O2 molecule will facilitate the binding of another until all four spaces are filled.  It has been experimentally determined that the binding energies (the Ka) are increasingly smaller for each molecule of O2 bound to hemoglobin.  This happens, in part, because of the protein’s structure.  When the O2 binds and the Fe+2 clicks into place, it pulls on the histadine residue below it, stretching the other protein superstructure bonds.  This pulls slightly on the other 3 Fe+2-histadine bonds.  Much like those dancing string toys, pulling on the string at the bottom causes the toy’s arms and legs to move in a concerted action, which is similar in a way to the physical reaction of the other binding sites.  The additional pulling on the other histadine residues facilitates binding of the other three, such that the binding energy is progressively reduced with each binding until all four slots are filled.

Other structural contributions also play a large role in binding specificity.  Above the plane of the porphyrin molecule lies another histadine residue that physically blocks the strongest and most effective bonding interactions from occurring.  Since the Fe+2 atom is locked into place, the best bonding interactions would come from ones that provide the most overlap of their molecular orbitals, which are those that are end-to-end.  However, with the histadine in the way, these are prevented from occurring.  This is a good thing, because strong covalent interactions in enzymatic reactions, like those seen in the binding of carbon monoxide (CO) for instance, are usually toxic and are difficult to break under normal conditions.  For head-to-head binding to CO, the interaction is estimated to approximately 1000 times as strong as those between O2, illustrating its toxic potential.  In this case, the CO-Fe binding is still strong, but not so much as to completely block removal.  People who have suffered CO inhalation are often given pure oxygen in an attempt to out pace CO binding and ensure that the person continues to have a supply of oxygen.  This hindered binding is also helpful in normal activity as well, since the binding symmetry to O2 is bent as well it further facilitates it’s release into tissues in need.

Yes, it’s about iron again, but it’s quite interesting stuff.

{sources available upon request}

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Enterobactin

A little over 40 years ago it was discovered that bacteria utilize a class of metal chelators called siderophores to capture iron from their environments.  Iron is an important element for the growth and development of most kinds of bacteria.  It is used as a critical element in electron transport chains for energy production, the removal of dangerous reactive oxygen species and the functional element in the reactive sites of many important enzymes.  The finding was critically important to the field of medicine, as the iron scavenging activities of pathogenic bacteria are most often detrimental to the host.  Even though humans and other animals have developed sophisticated systems to sequester iron and make the body inhospitable to these bacteria, likewise they have developed a variety of means of out-competing their hosts for the valuable element.  Neisseria gonorrheae and N. meningitidis, for example, have evolved the ability to directly capture iron containing lactoferrin proteins from their human hosts, a testament to our continued co-evolution (1).  Still others have relied upon producing their own iron scavenging siderophores to fish out this valuable metal from fluid and protein sources.  Escherichia coli , for example, are known for producing enterobactin and have one of the most effective iron scavenging systems known, which at biological pH easily out competes endogenous defenses for iron.

Recently, it has been noted that these compounds are of critical importance to bacterial growth, especially in the ultra-low iron concentrations of bodily tissues.  Rather than tighten their belts in the toughest of times, some kinds of pathogenic bacteria turn on

'Like dissolves like': different kinds of Mycobactin are used in different mediums. The long aliphatic chain endows fat solubility to the molecule, allowing it to shuttle through the waxy mycobacterial membrane easily.

these scavenging systems to forcibly acquire the iron needed for growth.  Mycobacterium tuberculosis produces several siderophores, called mycobactins, that are medium specific in their activities.  The two differ by a water soluble or fat soluble group attached to the siderophore skeleton.  Water-soluble mycobactin T is released into aqueous mediums, whereas fat soluble mycobactin T is designed to take pirated iron across the waxy mycobacterial cell membrane (2).  When iron is in good supply, however, production of these is unnecessary and is down-regulated to conserve valuable energy and resources.  Below the level of production of these siderophores, however, lies a regulated system of control.

Generally siderophore production is controlled by gram negative bacteria’s “Fur” and gram positive’s “DtxR” systems, which have  transcription elements that are bound by iron.  Thus, when intracellular iron concentrations fall below a threshold (approximately 10^-6 M for many pathogenic bacteria) the iron lynchpin holding back the transcription element is gone, activating the siderophore production system.  In 1999, researchers at the University of Queensland showed that Mycobacteria require the production of siderophores for growth in human tissues and macrophages (2).  They used genetic engineering techniques to remove the instructions for the enzyme MbtB, which is a critical component of mycobactin synthesis.  MtbB effects the final step in mycobactin synthesis, the coupling of a salicylic acid group to the siderophore’s finished structure.  After creating an MtbB knockout strain, its ability to produce siderophores was tested by an assay that measured the absorbance of the growth medium.  They used a colored dye compound that has a known affinity for iron in solution.  If a bacteria were to produce something that has a higher affinity for iron than the dye, this would be measured as a drop in absorbance, as the siderophore would compete with the dye for the iron.  They found that after comparing these bacteria against wild type strains that they were unable to produce siderophores and the deficient strains experienced retarded growth both in plate medium and in live macrophages (in vivo vs. in vitro tests).

The understanding of how important these chemicals are for bacterial growth has led to renewed attempts to develop antibiotics against them.  In the late 80 and early 90’s the ‘trojan horse’ tactic of using siderophore-antibiotic conjugates and piggybacking them into a cell began to bear fruit with successes being discovered in a wide variety human diseases (3)(4)(5).  One group of scientists developed conjugates using the carbacephem antibiotic, loracarbef.  They found that the mixed catecholate-hydroximate siderophore they used gave the conjugate compound 2,000 times more potency than the parent drug alone (6).  Though resistances to this tactic still developed, these are thought to leave the bacteria at a disadvantage, because the mutations hinder the siderophore uptake system rather than attack the antibiotics themselves.  This limits access to a vital nutrient and leaves the mutants more susceptible to iron starvation and hindered growth.

Though the trojan horse tactic has yielded positive results, there are opportunities for even greater exploitation.  Techniques involving small molecules that attack that actual production of siderophores could provide another avenue to beneficial therapy for these diseases.  These drugs, much like statins that block the enzyme HMG-coA, would block a critical piece of the siderophore production pathway.  In the particular case of mycobactins, the final step in biosynthesis is the attachment of a salicylate group to the mycobactin skeleton.  Researchers at Cornell published results in 2005 showing that inhibitors of the enzymes that accomplish this task are potent inhibitors of M. tuberculosis and Yersinia pestis (7).  They synthesized a compound, SAL-AMS, that closely resembled a reaction intermediate and measured its effect upon the growth of bacterial cultures in mediums of low iron concentration.  They found that it successfully inhibited the enzyme and drastically reduced bacterial growth in the cultures.  It was shown to have an IC50(*) of 2.2 ± 0.3 μM for M. tuberculosis and about 51.2 ± 4.7 μM for Y. pestis in an iron limited medium.  Though in mediums with high concentration of iron the chemical was ineffective against Y. pestis, but it was found that the chemical might have unknown inhibitory properties against M. tuberculosis, as:

“Salicyl-AMS (tested at up to 8 X IC 50) was not active against Y. pestis in iron-supplemented medium, in which siderophore production is not required for growth.  Under these conditions, salicyl-AMS (tested at up to 180 x IC50) did inhibit M. tuberculosis growth, albeit with an 18-fold increase in IC50 (39.9 ± 7.6 μM).  This suggests that, in addition to blocking siderophore biosynthesis, salicyl-AMS may also inhibit M. tuberculosis growth by other mechanisms.”

Furthermore, researchers at the university of Minnesota developed similar nucleoside inhibitors of MbtA, one of which ” rivals the first-line antitubercular isoniazid” in activity against the bacteria (8).  This tactic follows the same reasoning as SAL-AMP above, as the nucleoside inhibitors attack the same pathway to inhibit siderophore end production.  The research was centered around making logical functional modifications to known structures of inhibitors and choosing the most effective ones for further testing.

Comparison of the pathway intermediate, the Cornell inhibitor and one of the Minnesota nucleoside inhibitors.

These findings are coming just in the nick of time it seems, as certain drug resistant strains of M. tuberculosis have become big news recently.  Extensively drug resistant tuberculosis is a kind of tuberculosis that is resistant to at least two of the top line drugs used to normally treat it (typically isoniazid and/or rifampicin) and a member of the quinolone antibiotics (ciprofloxacin).  Tuberculosis is generally a challenge to treat in the first place, with treatments typically taking up to a year or more to complete.  The loss of the first line drugs and reliance upon second line increases the risk of side effects and patient noncompliance to the already long course of therapy.    This can further complicate the issue, as it could lead to the obsolescence of the few active  drugs used to treat the disease, because resistances to one drug are usually useful against the whole family of drugs.

β-lactamases typically attack the carbonyl in the β-lactam structure, destroying the ring. Nafcillin has a large group that hinders these enzymes from getting too close.

An example of this can be seen in bacteria that produce β-lactamases, as these strains are often cross-resistant to all unprotected β-lactam antibiotics.  Some  penicillins have been designed with bulky groups attached to the skeleton in an attempt to hinder these enzymes.  Nafcillin, with its large 2-ethoxy-1-naphthoyl group, is very effective at blocking these enzymes for the most part.  However, even this tactic has its limits as methicillin resistant Staphylococcus aureus (MRSA) and oxacillin resistant Staphylococcus aureus (ORSA), both have developed resistances against these drugs such that, “From 1999 through 2005, the estimated number of S. aureus–related hospitalizations increased 62%, from 294,570 to 477,927 (9),” and “MRSA accounts for an estimated 12% of all nosocomial bacteremias, 28% of surgical wound infections, and 21% of nosocomial skin infections.  Infections secondary to MRSA result in excess costs of approximately $4,000 per patient per hospitalization compared with patients infected with methicillin-susceptible S aureus (MSSA)”(10)(11).

Since the discovery of penicillin, Alexander Fleming hypothesized that bacteria would develop resistances to the antibiotics used to treat them.  Now, more than ever, the development of new antibiotics must be pursued.  Human defenses have always provoked a counter-response from our pathogens, the most successful tactic selected out.  Much like the pathogens that infect us, we must ‘evolve’ a newer understanding of bacterial biology, which will offer us a foothold to better, more effective treatments.

——

(1) Genetics and Molecular Biology of Siderophore-Mediated Iron Transport in Bacteria

JORGE H. CROSA

(2) The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages

James J. De Voss, Kerry Rutter, Benjamin G. Schroeder, Hua Su, YaQi Zhu, and Clifton E. Barry III

(3) Design, Synthesis, and Study of a Mycobactin−Artemisinin Conjugate That Has Selective and Potent Activity against Tuberculosis and Malaria

Marvin J. Miller, Andrew J. Walz, Helen Zhu, Chunrui Wu,Garrett Moraski, Ute MÖllmann, Esther M. Tristani, Alvin L. Crumbliss, Michael T. Ferdig, Lisa Checkley, Rachel L. Edwards, and Helena I. Boshoff

(4) Species Selectivity of New Siderophore-Drug Conjugates That Use Specific Iron Uptake for Entry into Bacteria

M. S. DIARRA, M. C. LAVOIE, M. JACQUES, I. DARWISH, E. K. DOLENCE, J. A. DOLENCE, A. GHOSH, M. GHOSH, M. J. MILLER,    AND F. MALOUIN

(5) Siderophore-Based Iron Acquisition and Pathogen Control

Miethke M, Marahiel MA.

(6) Iron Transport-Mediated Antibacterial Activity of and Development of Resistance to Hydroxamate and Catechol Siderophore-   Carbacephalosporin Conjugates

ALBERT A. MINNICK, JULIA A. McKEE, E. KURT DOLENCE, AND MARVIN J. MILLER

(7) Small-molecule inhibition of siderophore biosynthesis in Mycobacterium tuberculosis and Yersinia pestis

Julian A Ferreras, Jae-Sang Ryu, Federico Di Lello, Derek S Tan & Luis E N Quadri

(8) Antitubercular Nucleosides That Inhibit Siderophore Biosynthesis: SAR of the Glycosyl Domain

Ravindranadh V. Somu, Daniel J. Wilson, Eric M. Bennett, Helena I. Boshoff, Laura Celia, Brian J. Beck, Clifton E. Barry, III, and Courtney C. Aldrich

(9) Hospitalizations and Deaths Caused by Methicillin-Resistant Staphylococcus aureus, United States, 1999–2005

Eili Klein, David L. Smith, and Ramanan Laxminarayan

(10) Baquero F. Gram-positive resistance: Challenge for the development of new antibiotics. J Antimicrob Chemother. 1997;39(suppl A):1–6.

(11) Kopp BJ, Nix DE, Armstrong EP. Clinical and economic analysis of methicillin-susceptible and -resistant Staphylococcus aureus infections. Ann Pharmacother. 2004;38:1377–1382.

(*) The IC 50 is the half maximum inhibitory concentration of antibiotics, a typical measure of effectiveness of the drug.

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In 1913, a Swiss chemist named Alfred Werner was awarded the Noble Prize in Chemistry for his work on what would be called

Vanadium rainbow.

coordination chemistry, which would lead to a new understanding of how chemicals bond together. Coordination theory describes the nature of bonding in transitional metals and the formation of complexes, which at the time seemed to follow bizarre and unpredictable patterns. Atoms in groups 1-7A followed somewhat predictable patterns in their bonding as shown by classical experiments. For example, atoms in group I took on +1 charges and bonded once to other more negatively charged atoms like the group 7A halogens, but it remained a mystery how the transition metals bonded and why they had so many oxidation states.  Vanadium, for example, produces a wonderful rainbow of oxidation states when potassium permanganate is added to a Vanadium II solution.  Over time it separates into different states: V +2 is violet, V +3 is green, VO +2 is blue, VO2 +1 is pale yellow, MnO2 is brown and MnO4 –1 is pink.  In Werner’s time, the shape of salts like (CoCl3 * 6 NH3) were still undetermined and throughout much of the 1800’s one popular theory emerged: chain theory. It was supported by some of the most powerful chemist-sorcerers at the time, including Werner’s chief rival: S.M. Jorgensen.

Jorgensen believed that the ligands in compounds like (CoCl3 * 4 NH3) were arranged in chains, that is, bonded to each other in some fashion. The main point being that the atoms would follow known valence rules at the time, especially Kekule’s principle, which abstracted the number of times a compound could bond from known chemical reactions. Though useful, it ran into problems when trying to describe why atoms with larger electronic configurations bonded in so many different arrangements. Transitional metals in particular confounded these rule sets.

Werner, however, proposed a different theory that relied on the concept that cobalt (in the above compound) could have more than the three bonds predicted by Kekule’s theory and that the ligands would be centered around cobalt in an octahedrally arrangement, rather than in chains. According to his theory, a compound like the above due to its structure would have two possible conformations: a cis isomer (with chlorine atoms on adjacent vertices) and a more stability favored trans isomer (with the chlorine atoms on opposite side of one another). Interestingly, the two are identifiable by their color with the trans compound being green and cis being a delightful purple color

Naturally, this caused controversy amongst chemists and the debate began. At the time only the structurally favored green trans compound had been synthesized, while the more difficult cis compound was thought to be non-existent.  Cis compounds are generally less stable and in this case it is due to repulsions between the electronegative chlorine atoms positioned close to each other.  Whenever Werner published results that seemingly confirmed his theory, Jorgensen was there to propose a counter theory in favor of the more popular chain theory.   Chain theory had strength in the fact that there are many possibilities in the way ligands can be arranged in that manner.  Eventually, Werner was able to prove his case conclusively through a variety of methods like optical resolution of the compounds and electrical conductivity measurements. The capstone, as the story goes, was his synthesis of the elusive purple cis isomer of [Co (NH3)4 Cl3] and sending a sample through the mail to Jorgensen.  The flurry of high fives and  chest bumps went unabated for three months afterward and was actually seismically measured in Sweden.


Werner used this clever method to synthesize his purple cis isomer. By adding HCL at 0C, carbon dioxide is released and chlorine atoms in solution replace the oxygen atoms lost. (Note: picture does not show positive charge on Cobalt atom)

Transition metal like many of the blue colored above are used in a variety of reactions ranging from biological (Zn, Co, Cu, etc) to industrial (Os, V, Pb, Pt, etc).

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