For Toni Planas, a sabbatical in rural Connecticut has accelerated a sweet collaboration with Mark Peczuh’s research group.
Carbohydrate chemists are readily familiar with the concept of neighboring group participation (NPG), where the electrons of a nearby functional group accelerate reactions at a given center. A sociological version of NPG operates in the everyday world of scientific collaborations. Antoni Planas (IQS, U. Ramon Llull, Barcelona) has just completed a year-long sabbatical at the University of Connecticut in the laboratory of collaborator Mark Peczuh. His close proximity to Peczuh and his research group has hastened the progress on their project to develop glycosidase enzymes that selectively hydrolyze septanose sugars, making Planas the human equivalent of a participatory neighboring group. Planas, who lived with his family in an old farmhouse on UConn’s main campus, previously hosted Peczuh as a Fulbright Fellow at IQS in 2013 – a visit that initiated the collaboration.
With support from the National Science Foundation and the Spanish Ministry of Economy and Competitiveness, the team is working on a project that leverages Planas’ pre-eminence in bioengineering, especially on carbohydrate active enzymes, with Peczuh’s expertise in the synthesis of ring expanded, seven membered-ring sugars called septanoses. Their collaborative effort to develop a septanose-selective glycosidase requires the synthesis of new chromogenic substrates and some aggressive directed evolution strategies. On the impact of the visit, Peczuh remarked, “Toni’s energy and creativity have pushed the project forward this year. He has an endless supply of ideas and he’s been invaluable as an informal advisor to my grad students.” Things really got going when Toni’s student Sergi Pascual visited in the Spring to work side-by-side with Aditya Pote from the Peczuh group. Sergi got to do some synthesis while he also showed some of the finer points of library screens to Aditya.
Just as with reactivity, neighboring group participation is not a one-off phenomenon for the Planas-Peczuh collaboration. Their experience has convinced them that being close by not only facilitates research productivity, it makes for good fun.
Note: This originally appeared in the Fall 2018 newsletter of the American Chemical Society Division of Carbohydrate Chemistry.
An explication of Fischer’s proof for the configuration of sugars is the capstone lecture in sophomore organic chemistry. However, like the “irrelevance” of the Diels Alder reaction that is frequently cited, some might say the Fischer Proof lacks value for the training of today’s students. To the contrary: there’s every reason to think old Fischer still has some magic yet in that beard of his. Here I lay out the argument for why it should be taught. The details of the proof itself are nicely presented here and here.
The Fischer Proof is complicated; carbohydrate structure and reactivity can be daunting. Sugar molecules have similar atoms and functional groups and primarily differ in their precise geometric arrangement. It simultaneously appears like everything is the same, and that there are too many details to keep track of. It takes time, effort, and a significant build-up of context for it to make sense. In general terms, that sounds like many of the world’s difficult problems that need to be solved. Rather than shy away from the topic, then, we should embrace it. If an effort is made to truly understand the proof, lessons that transcend organic chemistry can be found. The intellectual tools that derive from those lessons can then be applied elsewhere.
It wasn’t easy for Fischer either; consider the context for when he was working on this question at the end of the 19th century. The notion of tetrahedral carbon had been introduced and its relevance to stereochemistry was apparent, but the details were still murky. Observations on the composition and reactivity of carbohydrate compounds were not yet unified in any way. Consequently, Fischer was going off into the thicket of the unknown. Paraphrasing Lichtenthaler’s tribute  on the centennial of the Fischer Proof, “Textbooks give the false impression that chemistry as a discipline developed in an orderly way where discoveries followed one after the other in a logical progression. They imply that Fischer achieved his insights as a matter of historical course. They don’t detail the serendipity, the intellectual struggles of a dedicated researcher, nor the persistence that enabled him to succeed.” The tribute further pays homage to, “the creative processes underlying a fundamental discovery and the constructive logic that eventually led to it.”
The first lesson of the proof is that Fischer was able to vary his field of view back-and-forth from specific, detailed observations to general concepts that provided the framework to organize all of those observations. He cataloged the identity of his sugars based on their associations, generating a complex web of relationships.  The specific sugar starting materials as well as the reactions that transformed them were the variables in his investigation. Relationships between compounds were established through painstaking associations that arose from different combinations of starting materials and reaction sequences. Results accrued in a non-linear fashion; so, Fischer had to assemble disparate pieces of evidence into one unifying model that explained all the observations as a whole. He made sense out a large body of information by sweating the details while keeping the big picture in mind. The Fischer Proof is a master class in logical reasoning, a technique that is both valuable and generally applicable.
It is easy to take Fischer projections for granted. They consolidate information so efficiently that they fall victim to their inherent intuitiveness. Fischer’s second lesson, then, is the development of creative ways to organize information. The organizational system is clearly connected to the “field of view” lesson above. Fisher projections are proto-infographics; they put a physical manifestation on all the associations between the sugars he’d established. Because tetrahedral carbons could be asymmetric based on the groups attached to them (courtesy of van’t Hoff), Fischer knew there should be 16 isomers of aldohexose sugars. His initial paper on the configuration of sugars used van’t Hoff’s symbolism, where each asymmetric carbon was given a plus (+) or minus (-) sign to designate its configuration. This symbolism was helpful for indicating what the configurational possibilities of the aldohexoses were, but the rationale for implementing them broke down on molecules bigger than four or five carbons. After some thought and presumably a lot of fumbling with physical models, he came up with a 2D representation of those 3D tetrahedra; the Fischer projection was born. By working with the physical models, he was able to establish the relationships that he had observed in all those syntheses in the lab. He bridged a profound gap between physical observables and molecular structure. Model building is a creative activity essential new discoveries. That’s general.
Fischer was the first to admit that his hard work, planning, and reasoning were not everything. He conceded that, “a piece of good luck led me to the target.” The wisdom of Pasteur here fits perfectly. “In the field of observation, chance favors the prepared mind.” Fischer’s third and final lesson was that serendipity will reward attention to detail and tireless persistence.
 The format and ideas of this post were largely inspired by Lichtenthaler’s Angewandte review, Angew. Chem. Int. Ed. Engl.1992, 31, 1541-1556.
 That’s just the beginning. Just think technical hurdles that Fischer had to overcome in his investigation, which added another layer of complexity to the endeavor.
* Thanks to William Bailey and Jeffrey Moore for reading a early draft of this post.
Some time ago my research group stumbled upon a family of macrocycles whose structure contained a somewhat uncommon stereogenic element, a plane of chirality. A plane of chirality arises when restricted rotation around a planar unit in a molecule leads to the formation of stereoisomers. The textbook example (seriously, look at Eliel) is E-cyclooctene. The chirality arises because of the twisting of the double bond and the asymmetry of the bridge that links its two sides. Individual enantiomers can be referred to either as P & M or pR & pS. The enantiomers of E-cyclooctene were resolved via transient formation of diastereomeric platinum complexes. In our system, we’ve explored in some detail how stereogenic centers at specific locations on the macrocycle backbone dictate which planar chirality it adopts as it forms in an RCM reaction. Kozlowski’s story turns that sequence upside down, or backwards. She leverages the chirality bestowed by a stereogenic axis to set two stereogenic centers on the seven-membered ring.
Dynamic Stereochemistry Transfer in a Transannular Aldol Reaction: Total Synthesis of Hypocrellin A
Erin M. O’Brien, Barbara J. Morgan, and Marisa C. Kozlowski*
Title: I frequently start my graduate synthesis course with a standard introduction. I ask, “What are reasons for why humans do total synthesis?” It’s a roll call of sorts: • to provide material not available from its natural source; • to provide material for biological testing, including mechanism of action studies; • to prove the structure; or • to test an idea. This last one is interesting because it’s uncommon. But that’s what Kozlowski’s synthesis does. The questions to be dealt with are intertwined and revolve around stereochemistry and biosynthesis. The idea plays out over the course of the communication.
P1: Paragraph one contains pro forma items like the introduction of the title compound, a listing of biological activities, and observations on its structural details. Along with Hypocrellin A (1) (Scheme 1), the structures of other pyrelenequinones are shown. Kozlowski observes that three of the four compounds come from one producing organism (Shiraia bambusicola) and two come from another (Shiraia bambusae). An interesting coincidence is that for these two different organisms, each produces one complementary enantiomer of the same compound, where bambusicola produces Hypocrellin A (1) and bambusae makes Hypocrellin (4) (Mind the R/S and P/M notation.). And… And… The compound common to both organisms, Hypocrellin B (3), is racemic. The stereogenic centers have been removed and are replaced by a double bond and the stereogenic axis is of both configurations. She is using these observations to soften the reader up to the biosynthetic question she’ll be posing in a couple of paragraphs. It’s subtle and requires that you really focus in on the details in the structures.
The paragraph continues by listing the antiviral activity, immunotherapeutic properties, and “light induced activity against tumor cell lines,” of the perylenequinones. In the middle of those statements is an important clause that feels like it was just jammed in there. It’s a little awkward. She writes, “and elegant approaches to the calphostins have been reported.” As will become apparent, the calphostins are structurally similar to the perylenequinones and much of what was learned in pursuit of those targets will be leveraged by the team toward 1. Angewandte’s page limit may have precluded the consideration she was going to give to the calphostins here. Wrapping up the paragraph Kozlowski points out the seven membered ring, the quaternary stereogenic center, and the fact that, until this report, a synthesis of Hypocrelllin A was elusive. It ends by dangling the concept of dynamic stereochemistry transfer in front of the reader to keep the manuscript flowing.
P2: What “taut” and twisted story is told in paragraph two? It’s one that goes into greater detail about two specific structural features of the perylenequinones. The first is the tautomerization of the core polycyclic structure. Kozlowski calls Scheme 1 again and links the tautomerization of 1 and 1•taut (present at ~1:1) to other structural features such as the planarity of the core and intramolecular hydrogen bonding. It’s best to take a step back and really process that tautomerization. The overall result is easy to see – the quinol and quinolone rings within each of the napthyl units of the perylenequinone core essentially interconvert. That IS the tautomerization. It documents the delocalized nature of electrons in the ring. Explicitly charting that flow of electrons, however, requires a little effort. The second feature is the atropisomerism of the core; this means stereogenic axis talk! Calphostins come back into the story here too. While the helical configurations of calphostins are stable, those of the perylenequinones are not. The argument is that tying the pendant arms of the perylenequinone together enables it to wriggle more easily, or forces it to wriggle. At first that may seem counterintuitive because cyclization is usually equated with rigidification. Chris Farley’s, “fat guy in a little coat” scene comes to mind in an effort to come to terms with the idea. The analogy can’t be taken too far, though, because that might suggests bond breakage! At any rate, atropisomerism introduces yet another equilibrium, depicted in Scheme 2, between 1 and 1•atrop (Once again mind the P’s, M’s, R’s and S’s.). Thermodynamics places the equilibrium at 4:1, favoring the P-configured isomer, Hypocrellin A (1). There is more subtlety (and conservation of space, perhaps) at the end of this paragraph because Scheme 2 houses the atropisomer equilibrium as well as the upcoming retrosynthesis.
P3: If this paragraph had a title, it would be “The Question.” The question focuses on the origins of asymmetry in the molecule, which is what makes it chiral, and the sequence by which those stereogenic elements are installed in the biosynthesis. Kozlowski ponders whether the stereogenic “helical” axis or the centers are installed first. She envisions, “…a potentially biomimetic, dynamic stereochemistry transfer (DST) reaction,” as the driving force behind the design of her synthesis. As defined by her, a DST reaction takes one stereochemical element (center or axis) and leverages it for a diastereoselective reaction, but loses stereochemical integrity as a consequence (or subsequent to) the transformation. In a nutshell, “The Question,” in two parts, is: 1) Can the configuration of the helix in the perylenequinone drive the stereoselective formation of the centers distal to it via an aldol reaction; and 2) Is this transfer of asymmetry – axis to center – biomimetic? What better tool to answer the question than synthesis? The pursuit of an answer to The Question really dictates a certain pathway for the retrosynthetic analysis that follows.
P4: With an over-riding principle behind the synthesis in place, the issue of HOW to orchestrate it was now required. Paragraph four comes through with Kozlowski’s retrosynthetic analysis. We’re sent back to Scheme 2, now in its capacity of illustrating the retrosynthesis. The reaction to be implemented for the DST is an intramolecular aldol. This disconnection, that takes Hypocrellin A (1) to Intermediate 5 is shown with a clear indication that it is the DST and an aldol in the scheme. Note that the stereogenic axis of 5 is in the P configuration, like 1. With helical configuration set, the argument is that the aldol reaction will be selective for formation of the configurations of the centers as shown, which includes the stereogenic quaternary center. Compound 5 itself looks a bit like a calphostin, which makes sense; disconnection of the “2-propanoyl” pendants of 5 give di-iodo compound 6. If space had allowed, addition of 10 to the retrosynthetic scheme would have been instructive. It would have made the allylation/Wacker strategy readily apparent. Additional contemplation of 5 reinforces the biosynthesis argument because it is akin to the aforementioned calphostins and also a likely intermediate en route to the elsinochromes where a saturated six membered ring is fused to the perylenequinone core. The biosynthesis sub-theme keeps the storyline active within the communication.
P5: As intimated earlier, the extensive calphostin literature (including, logically, earlier contributions from the Kozlowski group) was to be utilized toward the synthesis of key intermediate 5 because of the structural similarities between the two. Attempts at developing diastereoselective oxidative aryl couplings, where the stereogenic centers on the pendants were supposed to drive the formation of atropisomers of a specific configuration were ineffective, even counterproductive. Count those failures as another argument in favor of the axis-to-center biosynthetic model. Kozlowski even adds more fuel to her argument by noting that enzymatic oxidative couplings of 2-napthols can be enantioselective. It’s here, essentially mid-paragraph, where a transition occurs. So far, the paper has really presented ideas and strategies. All that positioning set the foundation upon which the results, the synthesis, will be built. Enantionselective access to 5 was essentially the last piece of the retrosynthetic puzzle.
At this point an, “In the event…” phrase could be expected. Maybe it’s too cliché. Regardless, with the sentence, “In our approach, we employed an enantioslective oxidative coupling of 9…,” Kozlowski all but shouts to the reader, “Enough talk, let’s synthesis!” Getting to 9 requires doing some homework. Scheme 3 shows how compound 9 comes from 7 via 8, but the text makes no reference to the transformations at all. The reader is left to muddle through alone, so those who hadn’t already reached for the scrap paper to push electrons in the tautomerization may be reaching for it now. She then coolly picks out the perfect enantioselective oxidative coupling she needed – from her personal bag of tricks. If there is any self-aggrandizement, it is understated, with a citation and a “biaryl coupling with our copper diaza-cis-decalin catalyst (11) further highlights the utility of this method….” Reference 15b goes a long way in explaining the mechanism of the reaction and a rationalization fo the selectivity. An acetate ester to methyl ether switch followed by Suzuki coupling with allylB(pin) boronate delivers key intermediate 10. As is often the case, there is a nice symmetry between the end of the paragraph and the last compound of the scheme.
P6: “Our attention turned,” that’s another lovely phrase. The forward synthesis is now hitting its stride in paragraph six. The next stop is key intermediate 5 that will be used to interrogate The Question. Scheme 4 gets you from 10 to 16, which is the bis-ketal version of 5, so close. The marquee bond in the whole scheme is the one that closes the perylenequinone system. To begin, compound 10 from the previous paragraph is hydroxylated and then protected as the bis-benzyl ether smoothly giving 12. There are a few transformations throughout this synthesis whose reactivities/selectivities presumably rely on precedents from the calphostin literature. This hydroxylation is one of them. Wacker oxidation (pronounced “Vocker” if you’re German or you’re feeling confident) provides 13, and is followed by bis-ketal protection of the newly formed ketones. It’s noted that the ester units at C3 and C3’ were unreactive and required nucleophilic conditions for deprotection to 14; this cleavage is a minor bump in the road but one that was easily overcome. Bump number two now faces Kozlowski, and it is a little bigger. Those newly revealed acids have to be decarboxylated and standard conditions use temperatures that would allow the beloved perylenequinone enough energy to racemize. The solution: develop a new reaction; so a Pd-mediated carboxylation was consequently developed. This reaction could occurs a temperature low enough to keep the helical configuration intact. That meant 15 was in hand. After uneventful debenzylation of the ethers at C5 and C5’, more tweaking is required (bump number three) to make the oxidative cyclization to 16 efficient. The addition of base is telling when considering the napthol/quinol pKas. Critically, the twist (absolute configuration of the stereogenic axis) in 10 is maintained throughout the steps to 16, nearly setting the stage for The Question.
P7: A good story needs suspense and paragraph seven delivers. It’s like the indoor lacrosse ads that claim, “We’ll sell you the whole seat, but you’ll only need the edge!” So 16 is acid sensitive, having decomposed upon attempted ketal hydrolysis under standard acid conditions. To avoid this problem, Kozlowski resorts to another clever solution. The perylenequinone was reduced to the perylene bis-quinol 16b, (Scheme 5) and the ketals were then trans-ketalized using a mild Lewis acid in acetone; air oxidation of that species returns it to the appropriate oxidation state, perylenequinone 5. Now the reader is thinking, “Ok, here we go [with the DST]!” Instead, Kozlowski remarks that the “1,8-diketone aldol reaction that we have proposed to use had not been employed previously..” and leaves you hanging. Suspense.
P8: The manuscript takes a break from the excitement of the forward synthesis to reveal some additional details behind their confidence in the axis-to-center biosynthetic model based on molecular modeling studies. First, they found that the Z-enolate geometry would deliver the needed syn-aldol product via a closed transition state structure. Incidentally, the E-enolate gives rise to the other diastereomer, Shiraiachrome A (2). Second, energies of the same Z-enolate transition state structures showed that the correct diastereoface of the ketone electrophile of the aldol would also be in position based on the P configuration of the perylenequinone. Structures for the transition states are shown in Figure 1. The chemdraws at the bottom are slightly misleading because they aren’t exactly accurate representations of the structures that were in the calculation, and whose ball-and-stick model is right above them. It takes a little more work from the reader to make the jump here, but the message is clear nonetheless. The paragraph finishes with caveats about the need for a stable helical configuration over the reaction despite the fact that it atropisomerizes afterward. A search for the best Z-enolate selective base was on.
P9: And that base was found, “[a]fter considerable optimization.” One could wonder, “What does that mean in practical terms?” The phrase reads like a euphemism for the experimental equivalent of blood, sweat, and tears. Nonetheless, treatment of 5 with lithium disilazide base at low temp (Scheme 5) mediates enolate formation and ultimately the aldol cyclization. D.S.T. Selective deprotection of the C4 and C4’ methyl ethers (Thank you one more time calphostin.) follows the aldol and, “Voila!” Hypocrellin A (1). As may have been expected from the equilibria presented in the introductory paragpraphs, 1 is the major product, but of a mixture of products. Remember that Hypocrellin A naturally exists as a ~4:1 mixture of 1:1•atrop where the stereogenic axis makes them diastereomeric. Though there isn’t much explanation about why, the synthetic Hypocrellin A (1) sample is compared to a sample of 1 that was isolate from bambusicola. Rather, it is compared to the enantiomer, Hypocrellin (4) from bambusae. The spectra match and the CD in the supporting shows them to be enantiomers.
Also present in the mixture is Shiraiachrome A (2) and its atropdiastereomer, both of which probably arose from the E-enolate. The key ratio is for the yield of 1+1•atrop to 2+2•atrop, which is 10:1. This primarily reflects the selectivity of the aldol step. The details of Shiraiachrome A (2) are revealing. Remember 2 was the minor product in the aldol reaction, having arisen from the E-enolate attack on the “correct” face of the ketone. The helical configuration of the starting material, 5, was P. In the product structure the helical configuration switches to M to the tune of 10:1 (2:2•atrop).
P10: A routine summary paragraph wraps up this communication. Steps: 19; overall yield: 1.6%; average yield per step: 82%. Kozlowski also highlights the reaction development that went into this target including the napthol coupling, the Pd-mediated decarboxylation, and of course the DST aldol reaction. She aptly summarizes the key finding by writing, “In the aldol reaction, the two newly formed centrochiral stereocenters of the seven membered ring were dictated by the stable perylenequinone helical configuration and the enolate geometry.” For her this seals the biosynthetic model. From one perspective, the big twist was that the conversion of 5 to Shiraiachrome A (2) has even greater fidelity to DST as it was defined all the way back in paragraph three. This paper excels it posed The Question related to biosynthesis of the perylenequinones and how the target drove methods development, subtly presented. Kozlowski gives you total synthesis with a twist.
Note: A big thank you to Marisa Kozlowski for looking at the draft version of this post and for sharing the original figures and links to other coverage of this synthesis.
I was at the Natural Products and Bioactive Compounds Gordon Research Conference (GRC) last summer. During the usual free time one afternoon, there was a roundtable discussion about the challenges faced by women in science and strategies to overcome those challenges. The conversation was promoted by the GRC organization, which made sure there was an attendee to serve as a facilitator. (Shoutout to our facilitator Michelle Garnsey from Pfizer. Michelle was smart, prepared, and fair. Because of her, our conversation remained lively but also productive.) Sessions like this have been happening at most GRCs for the past couple of years. They have been branded “The GRC Power Hour, ” described as,
“[an] optional informal gathering open to all meeting participants. It is designed to help address the challenges women face in science and support the professional growth of women in our communities by providing an open forum for discussion and mentoring.”
Although the GRC supports The Power Hour, by no means is there a broader agenda or any top-down pressure to achieve a certain result. Rather, it seems to be the perfect opportunity for attendees within the varied technical groups at GRC meetings to explicitly grapple with the issue, hopefully share some best practices, and also provide another opportunity for attendees to network with each other.
Remember this was The Power Hour at a Natural Products meeting; older folks will sense the apparent paradox there. Natural Products is amongst the oldest of the GRCs, with over 50 years of meetings under its belt, and historically it had a reputation for being somewhat of a good old boys conference. It’s a shame if that perception persists, though, because the organizers have been very effective at making an inclusive meeting in terms of both the technical program and the tone of the social events. Even more important is the fact that the rank-and-file regulars are aware of and sensitive to the challenges that women face in science – in part because many of these regulars are women in science themselves!
The one key take-away from the session was a renewed awareness of unconscious bias. It is the type of thing that is best to be talked about (and heard) over and over. My logic is that the repetition might eventually foster groups to think, “Let’s check ourselves,” so that real progress can be made toward gender parity. There were plenty of participants – probably two-dozen or so – of all genders and from both industry and academia. The tenor of the conversation was also very good from my perspective; there was the mutual respect that you hope to have at this type of meeting and everyone who spoke made thoughtful observations. I applaud The Power Hour and hope that it remains as a staple of GRC meetings. Wouldn’t it be great if ideas from those GRC conversations made it to faculty meetings in academia and board meetings in industry? Even from a position of significant privilege, like mine, it is obvious that there is a lot of work to do.
One question, hyper-topical to Natural Products, revealed a particular shortcoming in the field related to gender representation. At some point, someone asked, “How many female professors are doing total synthesis?” By this it was implicitly clear that we were talking about target oriented, natural product total synthesis. This was originally the bread-and-butter of the Natural Products GRC, although the title of the meeting now includes “and Bioactive Compounds,” another explicit signal of an attempt to be more inclusive. There was short silence and some murmurs, but names didn’t flow in a way that would have if the question had been about male professors doing total synthesis. One name that did come up was Sarah Reisman, who coincidentally was the author of a communication covered in one of my “deconstructed synthesis communication” posts. The fact that a group of a few dozen people, actively engaged in synthesis in one way or the other and attending a synthetic chemistry conference, could come up with only one name was disheartening.
The incident prompted me to do some searching through the literature to find female corresponding authors on total synthesis papers in the last decade or so. It is fair to say that the field of total synthesis is far from achieving convergence with respect to gender parity. Below is a list of female PIs currently active in the field of total synthesis along with a representative citation from each. I’ve also added another list of groups doing synthesis (analogs, etc.) and/or reaction development and methodology. The second list is longer, which is encouraging. Thanks to Matt McIntosh’s organic links page for leads on several of these names. It goes without saying, though, that I have overlooked names I should not have. I’m eager to add them, so let me know names of groups and citations that should be included. It’s a big tent; let’s fill it up.
M. Cristina White – University of Illinois Urbana Champaign
Sheryl Wiskur – University of South Carolina
Dan Yang – Hong Kong University
Barbara Zajc – CCNY
1. I clearly have no first hand knowledge of the challenges faced by women or people of color in science or society in general. I can claim sympathy for, and support of equality for all groups, however. This post attempts to make a simple observation about the representation by women in a very specific sub-discipline of organic chemistry: total synthesis.
2. Thank yous to Michelle Garnsey at Pfizer and Amber Onorato at Northern Kentucky University for reading an early version of the post and suggesting improvements.
With the hope of sharing best-practices for success in introductory orgo lecture, I asked an undergrad in my department (John Ovian) what he recommended. He gave me a sheet that explained how he organized his notebook and how his notebook became a valuable tool for studying. Those notes, plus some design inspiration from the venerable Pee-Chee folder, motivated me to have this graphic built. A million thanks to Aneesa Bey in the UConn Chem front office for translating my scribbles on a sheet into what you see here.
Have you ever unwrapped a piece of chocolate only to find it has a whitish powder surrounding it? It almost stops you from eating the chocolate, right? Chocolate that gets the whitish powdery appearance is said to have undergone a bloom – called chocolate bloom. Fast temperature changes or exposure to moisture can hasten the process. To understand it, you have to know that chocolate is a mixture of several ingredients that include sugars and fats, among other things. The temperature changes or moisture can make these ingredients bunch together allowing them to move around in the mixture all the way to the surface. Once separated, things like sugars (think of sugar crystals) have different properties than the original chocolate mixture. That whitish chocolate is often more crumbly than regular chocolate.
A process akin to chocolate bloom is happening with the concrete used in the foundations of houses in eastern Connecticut. The ingredients for concrete are cement, sand, and aggregate. Sand and aggregate are filler materials held together by the cement. When pyrrhotite happens to be a component of aggregate, it can chemically react with air and moisture over time. Sulfur in the pyrrhotite gets converted into sulfuric acid and sulfate salts. Sulfuric acid is bad because it eats away at the structure of the concrete. Sulfate salts are bad because they can join with the cement to make new crystalline materials that are more brittle than regular concrete. As the crystals form, they push up against the concrete mixture, causing even more stress. These events change the structure of the concrete (chocolate) because some of the components like pyrrhotite have been transformed (like sugar or fat in chocolate), making it weaker. Whereas in the chocolate bloom ingredients that are already present merely separate, the pyrrhotite changes, over time, to a different and now deleterious chemical that makes concrete crumble. In either case, however, the strength of the original material is compromised in the process.
“Bloomed” chocolate is completely edible and tastes almost as good as regular chocolate. Crumbling concrete, on the other hand, is very hard to swallow.
I was at a conference this summer where Patrick Harran gave a lecture on his group’s approach toward the synthesis of callyspongiolide. It was special. Harran has charisma that, as best as I can gather, comes from a combination of a near photographic memory, tremendous creativity, deference to the contributions others in his field, and a great sense of humor. His was the most enjoyable presentation in a program full of fascinating stories. That lecture reminded me of a comment I’d made when collecting opinions on well written total synthesis papers. I had observed that, “The Harran diazonamide synthesis communications are also quite good. They struck me as being classy and respectful in their reassignment of the structure. Also, the synthesis really simplifies a complicated molecule for me.” When I put those observations together I was compelled to revisit his diazonamide synthesis as an “active reading” example for my organic synthesis grad class.
Diazonamide was a hot target during an interesting time – the turn of the century. We were still afloat on the dotcom bubble of the roaring 90’s. And as you well know, that bubble burst. On a much smaller scale (a molecular scale!), Harran helped burst the bubble on the errant structural assignment of the diazonamides. That’s the real subject of the communication to be dissected. That’s the funny thing, it doesn’t actually report the synthesis of a natural product. The “nominal” and “proposed” in Harran’s title are harbingers, though by the time the papers were published I assume those in the game already knew the story. The chase for a “wrong” natural product in organic synthesis has plenty of precedents. Nicolaou, a major player in the diazonamide story, even wrote a review about those examples and some of the emotions that surrounded them. On the subject general, KCN observes, “We know, however, what it is like as a synthetic chemist to be in the midst of a total synthesis or at its “end”, only to find out that the molecule we were chasing was never there!” And on the diazonamides in particular, “Although we certainly admired the beautiful synthesis of Harran and his team as well as the logic behind the proposed structural revision, our initial reaction could only be described as intense disappointment and frustration.” You don’t see a lot of that type of honesty in print. I think it’s a valuable reminder to students and professionals alike: science is a contact sport and its practice can leave scars. What follows is a written version of the explication, paragraph by paragraph, of Part 1 of Harran’s back-to-back communications that popped the diazonamide dot-com structure bubble and reassigned it as the true diazonamide structure.
Total Synthesis of Nominal Diazonamides – Part 1: Convergent Preparation of the Structure Proposed for Diazonamide A1
Jing Li, Susan Jeong, Lothar Esser, and Patrick G. Harran
Paragraph 1: Harran could have stolen the Alan Greenspan “irrational exuberance” phrase to use in this opening paragraph. Here he proceeds quickly to the curious conditions that qualify the “purported” structures of the diazonamides. What had to be reconciled were an X-ray crystallographic structure of diazonamide B analog 2 (Scheme 1)2 that contained an acetal rather than the hydroxy hemiacetal motif of parent compound 1b. A C2 amine was invoked (Using a sound rationale because it meant that the amino acid valine was at this position of the molecule.) to make the proposed structure “more consistent with the available data”. More analysis of the spectral and MS data, along with true structure are in the second communication in the back-to-back set. He closes the paragraph with his Alan Greenspan moment, essentially saying, “We should have known. But in our irrational exuberance, we became mesmerized by the beauty and potency of the diazonamides and we (the community) inadvertently overlooked some important details.” The end of the paragraph suggests that those oversights will be explained and will set the stage for his proposal for the true structures of diazonamides A and B.
Paragraph 2: Though this was early in his career, the second paragraph evidences that Harran was already running with the big dogs in pursuit of the diazonamides. Citation five, at the end of the first sentence, gives a list of groups working on this target: Magnus, Vedejs, Wipf, Nicolaou, Stoltz/Wood, Moody, Konopelski, and Pattenden. Those are all household names, at least at my house. The “word efficiency” of the next sentence must be high. Harran writes, “Our own tactics for constructing a diazonamide ring system have likewise evolved steadily.” He’s sending the reader back to three papers, each interesting in its own right, that chart advancements in their approach to the target in incremental stages. The responsible reader who hasn’t already read those papers would do well to stop and follow the evolution of their approach. If I tried to summarize them, I’d go with: 1- first synthesis of the “western” macrolactam; 3 2- validation the pinacol ring contraction; and 3 – lessons from attempts at forming the indole biaryl linkage. Then it’s the big reveal: the old structures for diazonamide A and B were like pets.com – highly touted but ultimately unsustainable. The true structure (fully rationalized in the second communication in this back-to-back sequence) of diazonamide A is that of 3.
Paragraph 3a: Paragraph three is a behemoth. I’ve chosen to split it in half to make sense of it. The first half does three things. First it gives you another homework assignment in terms of retrosynthesis. Harran assumes you know the general approach because you’ve read the earlier “evolution” papers; if not, you have go do it. Embedded in this reminder is that the synthetic design elements had to include consideration for some of the stereochemical features (especially the atropisomerism of the poly-aryl bit on the eastern macrocycle). There will be more on that shortly. Second, and relatedly, Harran identifies the starting materials for the synthesis: two commercial products (These are not explicitly stated, again it’s put on you to reason through it; they are tryptamine and valine.), and compounds 4–6 in Scheme 2. References 6a and 7 give you details on their synthesis. 4 (ref 7) is readily prepared by protecting iodo-L-tyrosine. 5 (ref 6a) is from condensation of Boc-protected valine and aminomalonitrile using EDC. 6 (ref 7) comes from benzyl protection of 2-hydroxyacetophenone followed by dehydrative chlorination. The third, and main, thing this part of paragraph three does is report on the synthesis of macrolactam 12, a key intermediate in the synthesis. Here styrenyl zirconocene 7 (derived from 6) is cross coupled with bromo-oxazole 5 and then acylated with tyrosine derivative 4 to give 10 (Scheme 2). Intermediate 10 is the acyclic precursor for a Heck macrocyclization they’d established earlier. An observation noted in this section is that the modified Takahashi variant of a Negishi coupling “neither requires nor benefits from a Zr-to-Zn transmetallation.” My interpretation is that he’s referring to the stoichiometric zirconocene that is required for the reaction is less than ideal. On this scale, though, the efficiency of the coupling makes me think, “So what?”. The Heck conditions were largely figured out in that earlier work, so conversion of 10 to 12 via pallado-intermediate 11 (Scheme 3) proceeds as expected. The import of 12 to Harran is that it has the content of the diazonamide core and only needs to be “oxidatively restructured” en route to the target. To me, Scheme 2 would have been more effective if it included structures 11 and 12. Further, this choice could have dovetailed with a paragraph three that ended here too. Just a preference.
Paragraph 3b: The second half of the behemoth takes macrolactam 12 all the way to bicyclic lactone 18. Central to this part of the synthesis is the dihydroxylation of 124, converting it to syn-β-diol 14 in Scheme 3 which is followed by the pinacol rearrangement to deliver 16. There’s a lot going on in those steps. First the dihydroxylation: in reference 12, Harran observes that the inherent diastereoselectivity for dihydroxylation of 12 is to give the syn-α-diol in a 6:1 dr. This is an interesting example of macrocyclic diastereoselection – something our group has explored in the context of some other macrocycles. The macrocycle presumably populates multiple conformers where one face or the other of the olefin is accessible to reagents; remarkably, osmium complex 13 facilities the mismatched dihydroxylation. That’s pretty cool. Next the pinacol; here Harran signals euphemistically, with the flourish of a total synthesis chemist, that the pinacol, while low yielding, is valuable because the “stereochemical communication is near perfect.” Agreed. It’s a good trade. Stereochemical fidelity, they argue, is mediated by phenonium ion 15. Then he drops this pearl, “The axial asymmetries of the diazonamide polycycle can now be made an artifact of their assembly.” I like that sentence. My translation is that they’re going to leverage the asymmetry of the macrocycle in 16 to selectively set the atropisomers of the biaryl linkages that are about to follow. Conversion of 16 to 18 involves protecting group manipulations, reduction of the aldehyde that came about in the pinacol, bromination of the E-ring phenol, and lactone formation. One could wonder about the specifics of the sequence, but Harran doesn’t belabor them.
Press pause for a second and assess what has to happen for Harran to get home from 18. He’s amongst those lofty heights of an advanced intermediate here. Now they have to form the hemiacetal to establish the dihydrobenzofuran and also grow the “ter”-biaryl, “eastern” macrocycle onto the macrolactam of 16.
Paragraph 4: Harran calls 18, now at the beginning of Scheme 4, the right platform to launch attempts on the polycycle. You can feel the blood sweat and tears of his co-workers in the sentence, “While this goal was elusive for some time, nonproductive forays finally gave way to success.” Flash-forward to maoecrystal V, the compound whose synthesis launched 1000 reactions. Success took the form of amidation via aluminum amide opening of the lactone, re-oxidation of the alcohol to an aldehyde, and photolysis of an o-nitrobenzyl protecting group on the C16 phenol that had been installed en route to 18. This sequence gave a hemiacetal that was trapped to give one diastereomer, acetate 21. Oxidation at the benzylic position of the tryptamine followed by dehydration “parlays” this part of the molecule into the (bis)-oxazole-indole of product 22. In this short paragraph, Harran has nearly built the core skeleton of diazonamide. He just has to link the indole to the dihydrobenzofuran to close-down the eastern macrocycle.
Paragraph 5: /Harran, to himself,/ “Of all the configurations of all the atropdiastereomers in all the precursors of diazonamide, Witkop delivered mine.” A Witkop-type reaction now converts a dilute solution of precursor 22 to 25. As with the pinacol, Harran takes an aside to walk the reader through some details of how the reaction is works, complete with structures to illustrate his comments. Intramolecular electron transfer from the indole to bromo-arene, initiated by the irradiation, creates an intermediate akin to 23. This next sentence puts it all in a nutshell (parentheticals are my explanations). “Mesolytic elimination of bromide from the resultant radical ion pair 23 (bromide leaves now creating a neutral radical of the erstwhile bromo-arene), biradical collapse (the radical just formed from the bromide departure plus the indoyl radical that comes about via deprotonation of that moiety; these couple to make the new, sought after bond), and prototropy in 4H-indole in 24 would give 25 (tautomerization – proton)”. His vocabulary enables the smooth synergy between the text and the scheme; it’s enviable. A recent review gives greater insight into Witkop reactions in synthesis. Harran then lists results from other experiments (Li+ ion effects – there are two eq. of LiOAc in the reaction; inability phenol to inhibit the reaction, etc.) that are consistent with the proposed mechanism. Harran shows some of the physical organic underpinnings that must have been inspired by his time in Yale’s Sterling Chemistry Lab. Ultimately, it’s closure, and we’re beginning to feel – gratification dot com!
Paragraph 6: End game. Or is it mind game? With the skeleton set, it’s time for some clean-up. Uneventful chlorination of 25 gave 26 (Scheme 5) was followed by hydrogenolyis of the Cbz group on the C2 amine (and the benzyl group on the F-ring phenol) followed by amidationwith a Cbz-protected valine residue. The α-amino group of 27 of the valine is a key player in the structural reassignment. Hydrolyis of the acetate that protects the hemiacetal and another hydrogenolysis to remove the Cbz on the valine residue gives “nominal” diazonamide A, 1a. Think about it. Harran’s group was the first to achieve the synthesis of “the structure originally proposed for (-)-diazonamide A.” Imagine being Jing, Susan, or Lothar – whichever of them it was to take that sample to the NMR. Were they triumphant? Were they already fearful? Had the storm clouds already been seen on the horizon?
Paragraph 7: “[Peewee], There’s no basement at the Alamo!” Harran realizes that the spectra of the stuff they’ve synthesized does not match the sample of natural diazonamide A procured from Fenical; moreover, he finds that 1a is not particularly stable. Look at footnote 205: “Synthetic 1 a appears (1H NMR) as an ~4:1 mixture of C11 epimers. However, unpredictable degradation prevents detailed characterization of these materials. Protected derivative 27 is serviceable with respect to handling and analysis.” The main degradation pathways are deformylation at C10 and diketopiperazine formation where the free amine on valine attacks the C1 amide carbonyl of the macrolactam. Those pathways are giving hints about the true structure. Wait, that’s part of the traditional role of synthetic organic chemistry – reactivity as one parameter (in addition to spectra) of the target to reveal its structure and function. To gather more information from his intermediates, he synthesizes the “nominal” diazonamide B derivative that contains a p-bromobenzamide as the pendant to the macrolactam in place of valine (Scheme 5); this material, too, is “subtly different” than a sample derived from diazonamide B isolated from nature. Last, 26 was converted to acetal 28 (Figure 1); an X-ray structure of 28 confirms that the skeletons of 1a and 2, are as presented and synthesized. Harran has made it to the end of the Alamo tour and there is no basement. However, he has set the stage for the reassignment of the structures for natural diazonamides which is the subject of the proceeding “Part 2” communication.
The correction of the market – the bursting of the dotcom bubble – was toward companies that had a solid foundation. To belabor the analogy to the diazonamides, the bubble was the errant structures. It was not the compounds themselves, Harran, or synthetic organic chemistry as a discipline. In fact, each of these showed their true value. The details of the diazonamide structures were clarified. Harran leveraged his strategy toward the revelation of the true diazonamide structures. Organic synthesis proved its inherent value in an advanced age of modern spectroscopic techniques. In particular, Harran’s characterization of the degradation pathways of the late intermediates localized the NH-O transposition that led to the true structures. So it was win, win, win for diazonamide dot-com.
1 It’s safe to say that Jing, Susan, and Lothar did most if not all the experiments here. I don’t have a clue about who did the writing, but certainly Harran did the editing at a minimum. Nonetheless I refer to the team throughout as “Harran” mostly for simplicity. We all know the students and post-docs do the labor and the team, together, formulates and refines the strategies and ideas. Thanks to Patrick for sharing the original chemdraws (wow, that’s organization) of the figures and schemes from the manuscript and for doing a read-through of an advanced draft of this post.
2 The “nominal” structure of diazonamide A can be generally described as follows: an oxidized, highly dessicated and consequently crosslinked, polycyclic peptide characterized by two fused, 12-membered ring macrocycles. The “western” macrocycle is a macrolactam that also contains a valine residue pendant attached to the macrocycle via an amide bond. The “eastern” macrocycle has four contiguous aromatic rings linked to each other via single bonds (biaryl linkages); two of the aryl rings in the array – one oxazole and an indole – are chlorinated. The structure is further rigidified by a hemiacetal unit at the interface between the two macrocycles.
3 This was my personal favorite of the three. An eloquent and old-fashioned synthesis paper.
4 actually the C16 bromoethyl ether of 12
5 References end at ref 16 in the pdf version of this paper but all the refs are with the online version. FYI.
Conversion of the C-terminal D-Ala to D-Lac in the bacterial peptide target results in the loss of one of the important ligand-tethering H-bonds, with significant consequences, as mentioned previously. The change in the magnitude in affinity/efficacy is rather surprising, given that Williams et al. concluded, after extensive investigation, that an amide-amide H-bond in ligated VC is worth 0 to 2 kcal/mol (which agrees with the N-acetylglycine versus acetate comparison earlier). For perspective, a 1000-fold reduction in binding affinity corresponds to a ΔGº that is more positive by 4.1 kcal/mol at 298 K. Some reports in the VC field attribute the reduced binding affinity solely to a lost H-bond, whereas others astutely added a destabilizing lone pair-lone pair (lp-lp) electrostatic repulsion between a carbonyl oxygen of VC and the ester O of remodeled peptidoglycan precursor peptides to explain the drop in affinity.
Consideration of the subtle details implicated in the reduced binding affinity of VC to the remodeled precursor, and thereby the abolished efficacy of the antibiotic against VanA resistant bacteria, has largely been neglected in derivatization efforts to reinstate potent antibacterial activity. Attention has concentrated on strategies for increasing the local concentration of individually weakly binding glycopeptide molecules at the surface of a bacterial membrane (e.g., tethering together two or more antibiotic molecules), as opposed to efforts to directly regain bimolecular binding affinity.[1,3,8] A 2003 paper from Dale Boger’s research group (McComas et al.) is a notable exception. These researchers wondered whether the lost H-bond or the introduced lp-lp repulsion was worse for the binding affinity of VC for D-Ala-D-Lac.
When substituting an ester O for the amide NH of the terminal Ala of cell wall precursor peptides, VanA resistant bacteria skip over an element in the periodic table: carbon. Why do resistant bacteria ‘choose’ not to substitute a methylene group for the amide NH? Presumably, substitution of a methylene CH2 relative to an ester O is disfavored by selection pressures on bacteria to evolve resistance, because this substitution would provide inferior protection against VC. McComas et al. had the innovative idea to test just how inferior methylene substitution would be in reducing the binding affinity of VC for its bacterial peptide target. This research team reasoned that a methylene group could neither donate an H-bond, like an amide NH, nor suffer lp-lp electrostatic repulsion, like an ester O in close proximity to an amide carbonyl O of the antibiotic. Methylene substitution, therefore, offered a chance to partition the effects of a lost H-bond and an introduced electrostatic repulsion on the binding affinity of VC. By synthesizing a peptide used by neither VC-susceptible nor VC-resistant bacteria, knowledge about how to derivatize VC to reduce or remove the most important factor contributing to VanA resistance could be obtained.
After devising a synthetic strategy to prepare ligand 3 (Fig. 4) – containing a CH2 group in place of the NH of the natural ligand 2, McComas et al. determined the binding affinity of VC and VC aglycon to this ligand, as well as for ligands 2 and 4. Binding constants were determined by a titration method employing differential ultraviolet (UV) absorption spectroscopy, according to the procedure used by Perkins in 1968 to establish that VC specifically recognized the consecutive D-Ala sequence of uncross-linked cell wall peptides.
Briefly, VC absorbs at ~280 nm due to the presence of the phenolic groups in its structure (Fig. 1). VC complexed with cell wall peptide analogues also absorbs UV light of ~280 nm, but with a reduced extinction coefficient. The binding constant determination method employed by McComas et al. depends on measuring the difference in the extinction coefficient (Δε) between uncomplexed and complexed VC at a particular wavelength (279 nm) as a function of added ligand concentration.[6,16] By assuming a direct proportionality between VC-ligand complex concentration and Δε, binding isotherms were constructed and used to determine binding constants (Fig. 4).[6,16]
To a first approximation, VC was found to have a binding affinity for the methylene-substituted ligand (3, Fig. 4) 10 times less than for the D-Ala-terminal ligand 2, but 100-fold greater than the D-lactate ligand 4. Ligand 4 had a free energy of binding more positive than ligand 2 by 4.1 kcal/mol. These results suggested that loss of the H-bond upon exchange of the terminal amide NH in the ligand for a CH2 group destabilizes the bimolecular association by 1.5 to 1.8 kcal/mol (Fig. 4). The antibiotic-ligand complex is further destabilized by 2.6 kcal/mol with the replacement of the CH2 group with an ester O (Fig. 4). Thus, the lp-lp repulsive interaction appears to be nearly twice as costly as the removal of an H-bond. This analysis is overly simplified, as will be demonstrated in the next section. Nevertheless, the findings suggest an answer to the question of which destabilizing interaction was more adverse for binding of the antibiotic: an introduced lp-lp repulsion, as opposed to a lost H-bond. By the same logic, a modified VC that does not suffer the electrostatic repulsion with D-lactate-terminating peptides should regain a substantial fraction of the binding affinity that the antibiotic had for D-Ala-terminating ligands. Gratifyingly, this possibility was realized in subsequent research. A synthesized VC aglycon with an amidine (C(=NH)NH) in place of the amide functionality (C(=O)NH) spanning residues 4 and 5 binds D-lactate-terminating bacterial peptide analogues with an affinity 600-fold greater than unmodified VC aglycan.
Down and dirty: physical organic chemistry fundamentals
McComas et al. determined the thermodynamic consequences of systematically varying a single atom from nitrogen to carbon to oxygen (not counting attached hydrogens) in the peptide ligand of VC. This classic structure-activity approach assumes that changes in the thermodynamics of binding are due to the structural modification, and not to any secondary consequences. Though they partitioned the effects from a lost H-bond and introduced lp-lp repulsion to yield a productive synthetic target, it is interesting to consider the binding interactions of each ligand examined by these researchers in more detail. The binding of a ligand by VC is an excellent example of how collectively and cooperatively numerous binding forces give rise to molecular recognition. To motivate the discussion, general comments on cooperativity and enthalpy-entropy compensation are in order.
Formation of a strong (short and stiff) covalent bond is more exothermic than formation of a weaker bond. Formation of a strong (tighter) non-covalent binding interaction is analogously more exothermic than for a weaker interaction. Just as bond formation restricts the motion of the newly bonded group, a binding interaction reduces the translational and rotational freedom of the newly interacting group. The stronger the binding or bonding interaction, the greater the restriction in motion. In principle, there is a limit to motional restriction (complete immobilization), but there is no inherent limit to the exothermicity of an interaction. As an interaction becomes increasingly exothermic, the limiting case of complete immobilization is approached. The entropic cost for an interaction per a given enthalpic investment (increase in exothermicity) decreases with increasing levels of investment (greater exothermicity). In other words, the incremental entropic cost is smaller for every additional increase in exothermicity as the maximum entropic cost for the interaction is approached.
Consider the binding of two parts, the carboxylate terminus (C) and the rest of the peptide ligand (P) to VC. If it is assumed that the enthalpies for the association of C and P are additive, then the more exothermically each binds, the more exothermic will be the binding of the whole ligand. The entropic cost for binding C and P is not the sum of the adverse entropies for binding C and P separately, however. By virtue of C-P binding with greater exothermicity than C and P separately, the entropic price per kcal/mol of enthalpy is less. Alternatively phrased, two parts of the ligand nearly bind for the entropic cost of one part because, by linking C and P, the adverse entropy change for binding P is partially prepaid when C binds (i.e., the second interaction is effectively intramolecular). With a greater exothermicity, but less than a proportionally greater entropic cost, the free energy change for binding C-P is more favorable (more negative) than binding C and P separately.
The entropic advantage of binding C-P relative to C and P individually, a phenomenon classically known as the chelate effect, is arguably accompanied by an advantageous enthalpic effect. In reality, the enthalpy for the association of C-P with VC may not simply be the sum of the enthalpies for the associations of C and P separately. The binding of C of C-P can reduce the freedom of motion of P, and thereby strengthen (cause to be more exothermic) the binding of P. The effect is reciprocal in that the binding of P in C-P also reduces the residual motion of the ligand, and strengthens the binding of C. This coupled enthalpic-entropic chelate phenomenon is a form of cooperativity termed the Gulliver effect, “If you wish to keep a close interaction [reduced residual motion] between Gulliver’s shoulders and the ground, then it is clear that an agitated Gulliver would be better constrained if he were also held at the waist and feet [increased number of attractive binding interactions].” A binding event becomes more negative entropically (unfavorable) and enthalpically (favorable) for one part of a ligand as the binding interaction(s) for a different part of the ligand becomes more negative enthalpically. The Gulliver effect is a cooperatively-orchestrated form of enthalpy-entropy compensation.
The Gulliver effect is operative in the D-Ala-terminating ligand complex with VC (2, Fig. 4). Williams et al. found a strong correlation between the strength of the 1H nuclear magnetic resonance (NMR) chemical shift for the amide proton of residue 2, and the number of additional H-bonds and hydrophobic contacts formed by the ligand with the antibiotic (Fig. 5).[17,18] As systematically added interactions made the binding free energy more favorable for a series of ligands in the order of: acetate < N-acetyl-D-Ala < N-acetyl-D-Ala-D-Ala < di-acetyl-Lys-D-Ala-D-Ala, the chemical shift of the residue 2 amide proton shifted downfield from 8.79 ppm to 11.69 ppm.[17,18] A downfield shift suggests participation in a stronger H-bond, because a stronger interaction would deplete the 1H nucleus of shielding electron density. Thus, the amide proton at residue 2 makes an increasingly stronger H-bond to the carboxylate of the ligand complexed with VC as a function of the other interactions formed at the binding interface.
Admittedly, conformational changes of VC associated with ligand binding, or different ligand binding geometries could also account for the downfield shift of the amide proton resonance. VC, however, is fairly rigid on account of extensive cross-linking of aromatic sidechains (Fig. 1). Further, molecular modeling based on NMR-determined through-space proton distances suggests a conserved geometry for the H-bonding network at the binding interface. Differences in antibiotic conformation and/or ligand binding geometry would be variable and therefore explain scatter in the experimental correlation between additional antibiotic-ligand interactions and an increasingly downfield shift of the particular amide proton resonance, not the correlation itself. Moreover, a correlation that holds over a ~3 ppm range with a linear regression R2 value of 0.99 (Fig. 5) seems not to be the artifact of experimental error, which is an alternative explanation for the observation of enthalpy-entropy compensation offered by Houk. Though there is no fundamental thermodynamic basis for enthalpy-entropy compensation, the findings of Williams et al. suggest that enthalpy-entropy compensation is a real physical phenomenon in the context of ligand recognition by VC.[17,19] The implication of the Gulliver effect is that the strength of the amide-amide H-bond determined by McComas et al. (1.5-1.8 kcal/mol) is necessarily an overestimation, because replacement of the H-bond donating amide with a methylene group not only removes an H-bond, but also weakens all the other interactions formed by the ligand to VC. The difference in the energetics of binding the amide- and methylene-containing ligands is not simply equivalent to the strength of the removed amide-amide H-bond. As will be discussed shortly, methylene substitution introduces advantages and disadvantages to binding that further question the assumption by McComas et al. that methylene substitution is a neutral structural alteration.
The VC:D-Lac-terminal ligand complex (4, Fig. 4) offers a clear case of enthalpy-entropy compensation by way of contrast to the preceding comments for the D-Ala-terminal ligand (2, Fig. 4). Replacement of an amide NH with an ester O changes the ligand C-P (2, Fig. 4) such that P (now P’) has a repulsive, instead of an attractive, interaction with VC (4, Fig. 4). For the following argument, it is assumed that the lp-lp repulsion (4, Fig. 4) overwhelms the remaining favorable interactions formed by P’ with the antibiotic, so as to make the net interaction of P’ with VC endothermic. If the binding of P’ was still exothermic, but to a lesser extent because of the lp-lp repulsion introduced in place of an H-bond, there would still be an advantageous enthalpic and entropic effect caused by tethering P’ to C (i.e., tethering would lower the entropic cost, and enlarge the enthalpic reward for bimolecular association, but the binding would not be as favorable as when the repulsive interaction was not present and P’ = P).
For the ligand C-P’, binding of C would reduce the motional freedom of P’ in the proximity of a repulsive interaction. The exothermic binding of C is now opposed by the endothermic binding of P’, and the opposition should be greater than the difference between the enthalpies for the separate binding of C and P’. Just as the exothermic binding of C enhanced the exothermicity of the binding of P when C and P were tethered together, the exothermic binding of C should enhance the endothermicity of the binding of P’ when C-P’ binds to the same receptor. Since the effect is reciprocal, the repulsive or ‘anti-binding’ interaction of P weakens the binding interaction of C.
Crystal structure data supports the notion that the endothermicity of the repulsive lp-lp interaction decreases the exothermicity of neighboring interactions. The H-bond between the final amide proton of the D-Ala-terminal ligand and the carbonyl oxygen of residue 4 on VC (2, Fig. 4) has an N–H⋯O length of 2.95 Å. The corresponding O⋯O distance between VC and the D-Lac-terminal ligand (4, Fig. 4) is elongated by 0.13 Å to 3.08 Å. The adjacent H-bond from the amide proton of residue 3 on VC to the carboxylate anion of the ligand (4, Fig. 4) is also elongated from 2.79 Å to 2.91 Å. The implication is that the estimate for the magnitude of the lp-lp repulsive interaction obtained by McComas et al. (2.6 kcal/mol) is necessarily an overestimation, as it also reflects the weakening of neighboring H-bonds. The energetic destabilization of the D-Lac-terminal ligand, relative to the ligand with methylene substitution, is not solely the consequence of lp-lp repulsion as assumed by McComas et al.
The ligands terminating in D-Ala and D-Lac illustrate the concepts of positive and negative cooperativity, respectively. For both ligands, the binding free energy is not simply the sum of the free energies for the individual interactions between VC and the ligand, because the presence or absence of one interaction (an H-bond or a lp-lp repulsion) strengthens or weakens other interactions. For the D-Ala-terminal ligand, the binding free energy is greater than the sum of the individual exothermic binding interactions, because an additional H-bond reduces the residual motions of the ligand, and thereby strengthens or tightens all the other interactions formed by the ligand with VC. The D-Lac-terminal ligand has a binding free energy less than the sum of the exo- and endothermic interactions in which this ligand participates, because of the presence of an additional repulsive interaction that pushes the ligand and antibiotic apart, elongates and weakens other interactions, and allows the ligand to be held relatively loosely by VC. Positive cooperativity results in tighter binding, and negative cooperativity, looser binding, than would otherwise be expected. Given this perspective, the 1000-fold reduction in the binding affinity of VC for D-Lac, relative to D-Ala-terminating ligands, is more understandable.
Since a methylene group will neither participate in H-bonding nor lp-lp repulsion, the ligand with a methylene moiety in place of the amide NH or ester O can not partake in the cooperative effects discussed so far, it should have an intermediate binding affinity. This is in fact the case (3, Fig. 4), but there are interesting subtleties. A methylene group, in the place of an amide NH or ester O, increases the hydrophobic nature of the ligand, which potentially changes the solvation of the ligand so as to promote its binding into the hydrophobic pocket of VC. The exclusion of hydrophobic solutes from an aqueous medium – the hydrophobic effect – is a significant contributor to the free energy change for a bimolecular association. To an approximation, the hydrophobic effect scales with the surface area of the nonpolar solute exposed to water that will be buried upon aggregation with other nonpolar solutes. Williams et al. found that the contribution of the hydrophobic effect to the free energy of binding was 50 cal/mol/Å2 of exposed nonpolar surface area in the context of peptide ligands associating with VC.15 Given that a methylene group contributes a hydrophobic surface of 29 Å2, the free energy change for binding of the methylene-substituted ligand to VC should be enhanced by 1.4 kcal/mol relative to the D-Ala- or D-Lac ligands (if differences in solvation between these other ligands are neglected). Once bound, the methylene group increases the hydrophobic character of the VC-ligand interface, and thereby lowers the dielectric constant of the binding region. In a less polar microenvironment, nearby H-bonds, which are primarily electrostatic interactions, would be strengthened.
A contrary conclusion is reached if the conformational flexibility of the ligands is considered. Both the D-Ala- and D-Lac ligands have substantially less conformational flexibility about the C-X bond in Fig. 4 compared to the ligand with methylene substitution. Resonance within the terminal amide group of 2 in Fig. 4 imparts partial double bond character to, and thereby hindered rotation about, the central bond. Resonance within the ester group of 4 in Figure 4 similarly imparts rigidity to the carbonyl-O bond, but the rotational barrier is less relative to the barrier for the peptide bond. The resonance structure that would place a partial positive charge on X in Figure 4 is a lesser contributor when X = O, compared to when X = N, because it is less favorable for a positive charge to be on a more electronegative element. By contrast, no resonance is possible when X = C in Figure 4. Though a CH2 π group orbital will interact with the carbonyl π system, this interaction is worth <5 kcal/mol, whereas resonance within the amide group is worth 15 – 20 kcal/mol. The ester group has an intermediate rotational barrier.
Hindered rotation in the ligand when X = N or O in Figure 4 reduces the adverse entropy of bimolecular association relative to the ligand for which X = C, because there is one less bond that needs to be frozen out upon association with the antibiotic. Furthermore, hindered rotation in ligands 2 and 3 of Figure 4 locks the ligand into a preferred conformation in which the substituent on X is syn to the carbonyl. This orientation is critical for establishing hydrophobic contact of a methyl group with the glycosolated aromatic ring of VC, and for positioning of the terminal carboxylate of the ligand for binding into the receptor pocket (Fig. 3b). Free rotation of the methylene, as opposed to the amide NH or ester O, gives the carboxylate-containing substituent on the methylene greater conformational flexibility, which, in turn, would loosen the binding interactions of the carboxylate into the receptor pocket.
All of the foregoing arguments underscore the genuine complexity of ligand recognition by VC. The ligand with a methylene in place of an amide NH or ester O is not simply a neutral reference point from which to judge the worth in binding free energy of a lost H-bond or an introduced lp-lp repulsive interaction. The methylene group introduces its own effects that advantageously, as well as adversely, influence binding to VC.
Vancomycin (VC) has long served as a reliable vanquisher of pathogenic Gram-positive bacteria that have outmaneuvered other antimicrobial defenses.[1-3] This archetypical glycopeptide antibiotic is indispensable in the fight against MRSA, and Enterococcal infections that prey on the immunocompromised, but the clinical utility of the drug is seriously threatened by the relatively recent emergence of bacterial resistance.[1-3] The simple replacement of an amide NH with an ester O reduces the binding affinity of VC by 1000-fold. The dramatic reduction in binding affinity has frequently been attributed to a lost H-bond and/or the introduction of a repulsive lone pair-lone pair interaction, but not until a 2003 study by McComas et al. was the relative importance of these factors quantified.[6,8]The introduced repulsive interaction was found to account for a 100-fold reduction in binding affinity (equivalently 2.6 kcal/mol), whereas the effect of the lost H-bond was less by an order of magnitude (only 1.5 kcal/mol).
In the structure-activity study of McComas et al., the binding constants of peptide ligands having an amide NH, methylene group, or ester O linking the terminal residue to the rest of the ligand were examined (Fig. 4). The ligand with the amide NH formed an additional H-bond to the antibiotic, relative to the other ligands. The additional attractive interaction increased the exothermicity of binding, and reduced the residual motion of the ligand (enthalpy-entropy compensation), thereby strengthening all the other binding interactions (positive cooperativity). The ligand with an ester O had an additional repulsive interaction, relative to the other ligands. The exothermicity of binding for the ligand was consequently lessened, and greater residual motion was retained, which resulted in the weakening of other binding interactions with the antibiotic (negative cooperativity). Because positive cooperativity enhanced the exergonicity of an additional H-bond, and negative cooperativity accentuated the endergonicity of an introduced lp-lp repulsion, there was a 1000-fold difference in the binding affinity of the amide- and ester-containing ligands to VC (Fig. 4). This analysis is more sophisticated than the partitioning of the dramatically decreased binding affinity into a 10-fold reduction due to the loss of a single H-bond upon exchange of an amide NH for a CH2, and a 100-fold reduction because of the introduction of one lp-lp repulsive interaction upon the replacement of a methylene group with an ester O (Fig. 4).
Subtler effects influenced the binding of the ligand with the methylene group. The CH2 unit promoted binding by enhancing the contribution of the hydrophobic effect to the binding free energy of the ligand, and by strengthening adjacent H-bonds through the lowering of the dielectric constant of the microenvironment. The CH2 substitution was adverse for binding because it increased the conformational flexibility of the ligand, thereby increasing the entropic penalty for binding to the antibiotic and weakening other interactions at the binding interface. With these opposing factors, as well as the absence of the cooperative effects of the additional H-bond or repulsive interaction of the amide- or ester containing ligands, the ligand with methylene substitution had an intermediate binding affinity. However, this analysis shows that methylene substitution does not offer the neutral reference point assumed by McComas et al. for the assessment of the free energy worth of an H-bond and lp-lp repulsive interaction. Rather, the differential binding of ligands varying in a single atom (not counting hydrogens) to VC is an excellent example of how collectively and cooperatively numerous forces govern a molecular recognition event.
(1) Williams, D. H.; Bardsley, B. The Vancomycin Group of Antibiotics and the Fight against Resistant Bacteria. Angew. Chem. Int. Ed.1999,38, 1172-1193.
(4) Xie, J.; Pierce, J. G.; James, R. C.; Okano, A.; Boger, D. L. A Redesigned Vancomycin Engineered for Dual D-Ala-D-Ala and D-Ala-D-Lac Binding Exhibits Potent Antimicrobial Activity Against Vancomycin-Resistant Bacteria. J. Am. Chem. Soc.2011, 133, 13946-13949.
(5) Williams, D. H.; Waltho, J. Molecular basis of the activity of antibiotics of the vancomycin group. Pure & Appl. Chem.1989, 61, 585-588.
(6) McComas, C. C.; Crowley, B. M.; Boger, D. L. Partitioning the Loss in Vancomycin Binding Affinity for D-Ala-D-Lac into Lost H-Bond and Repulsive Lone Pair Contributions. J. Am. Chem. Soc.2003,125, 9314-9315.
(7) Breukink, E.; de Kruijff, B. Lipid II as a Target for Antibiotics. Nat. Rev. Drug Discov.2006,5, 321-323.
(11) Rekharsky, M.; Hesek, D.; Lee, M.; Meroueh, S. O.; Inoue, Y.; Mobashery, S. Thermodynamics of Interactions of Vancomycin and Synthetic Surrogates of Bacterial Cell Wall. J. Am. Chem. Soc. 2006, 128, 7736-7737.
(12) Waltho, J. P.; Williams, D. H.; Stone, D. J. M.; Skelton, N. J. Intramolecular Determinants of Conformation and Mobility within the Antibiotic Vancomycin. J. Am. Chem. Soc.1988, 110, 5638-5643.
(13) Pearce, C. M.; Gerhard, U.; Williams, D. H. Ligands which Bind Weakly to Vancomycin: Studies by 13C NMR Spectroscopy. J. Chem. Soc., Perkin Trans. 2.1995, 1, 159-162.
(16) Perkins, H. R. Specificity of Combination between Mucopeptide Precursors and Vancomycin or Ristocetin. Biochem. J.1969, 111, 195-205.
(17) Searle, M. S.; Sharman, G. J.; Groves, P.; Benhamu, B.; Beauregard, D. A.; Westwell, M. S.; Dancer, R. J.; Maguire, A. J.; Try, A. C.; Williams, D. H. Enthalpic (electrostatic) contribution to the chelate effect: a correlation between ligand binding constant and a specific hydrogen bond strength in complexes of glycopeptide antibiotics with cell wall. J. Chem. Soc., Perkin Trans. 1.1996, 23, 2781-2786.
(18) Cristofaro, M. F.; Beauregard, D. A.; Yan, H.; Osborn, N. J.; Williams, D. H. Cooperativity between Non-polar and Ionic Forces in the Binding of Bacterial Cell Wall Analogues by Vancomycin in Aqueous Solution. J. Antibiot.1995, 48, 805-810.
(20) Nitanai, Y.; Kikuchi, T.; Kakoi, K.; Hanamaki, S.; Fujisawa, I.; Aoki, K. Crystal Structures of the Complexes between Vancomycin and Cell-Wall Precursor Analogs. J. Mol. Biol. 2009, 385, 1422-1432.
Some of the most rewarding moments in my professional career have been in the midst of protracted conversation with a colleague about some concept or how to communicate an idea to an audience. At times I’ve been the teacher and other times the learner, but the best is when somehow, almost through the strength of the process itself, something new became apparent to both the other person and me at the same time. It’s like those special moments when musicians uncover some hidden beauty in a song. I’ve heard U2’s The Edge describe it as “when the magic happens.” We repeat a process to increase the chance that the magic will happen. In plain language, the magic is fun and it makes us (at least me) happy.
So when the opportunity for some friendly conversation presents itself, I often go for it. (The antipode of friendly conversation – idle conversation – causes me physical pain, however.) As I read a research paper written by a student in my Physical Organic Chemistry class last semester (Spring 2015) I realized that turning it into a blog post would enable us to meet and discuss the topic and his treatment of it.
Matt Guberman chose to write about the physical basis behind the difference in affinities of D-Ala-D-Ala and D-Ala-D-Lac terminating peptides to vancomycin. His analysis centered on a paper from Dale Boger’s group entitled, “Partitioning the Loss in Vancomycin Binding Affinity for D-Ala-D-Lac into Lost H-Bond and Repulsive Lone Pair Contributions” Casey C. McComas , Brendan M. Crowley , and Dale L. Boger J. Am. Chem. Soc.2003,125, 9314–9315. I was attracted to the prospect of collaborating on this with Matt because the vanco story is fascinating and I had done some work on it when I was a post-doc. Mostly, though, I had come to realize how fun it is to have a conversation with Matt about science. The probability of the magic happening when you talk with him is decidedly above average.
The post, “Resistance: How VanA strains get away with it,” flowed out of Matt’s research paper. We’ve broken it into two parts; the second part will be posted in a week or so. Early drafts were edited with the help of another grad student, Jordan Greco. The rest was my collaboration with Matt. The posts are neither exhaustive nor perfect but they will give a newcomer an appreciation for the importance of vanco and some of the physical organic chemistry fundamentals behind its mechanism of action. It was fun assembling them with Matt.
The longstanding invincibility of vancomycin (VC) – the last line of antimicrobial therapy for life-threatening Gram-positive infections – has been compromised over the past decade. The most common and best understood mode of resistance, the VanA type, consists of the replacement of an ester O for an amide NH in the peptide target recognized by the glycopeptide antibiotic. VC targets the C-terminal D-Ala-D-Ala sequence of the peptide portion of Lipid II, an essential intermediate in bacterial cell wall synthesis; binding of VC eventually results in a compromised bacterial cell wall and ultimately cell death. A single heavy atom substitution by VanA resistant bacteria reduces the binding affinity and efficacy of VC by 1000-fold. A lost hydrogen bond and an attendant repulsive lone pair-lone pair interaction, both of which are brought on by the O for NH substitution, are implicated in the VanA resistance mechanism.
In an effort to determine the relative importance of these effects, McComas et al. in a 2003 JACS paper measured the binding constants for bacterial peptide analogues to VC having the terminal residue connected to the rest of the peptide by an amide NH, a methylene group, or an ester O. The methylene-containing ligand bound to VC with an affinity 10 times less than the amide-containing ligand, but 100-fold greater than the ester-containing ligand. From these results the authors concluded that the repulsive interaction introduced by the ester O is the most important for VanA-type resistance. Further, a cooperatively-orchestrated enthalpy-entropy compensation effect accentuates the exogenicity imparted by the additional H-bond in the amide-containing ligand, and the endogenicity of the repulsive interaction suffered by the ester-containing ligand. A mixture of advantageous and adverse factors for binding are invoked to rationalize the intermediate binding affinity of the methylene-containing ligand. The differential binding of ligands with such subtle variations illustrates how numerous small forces, working collectively, govern this molecular recognition event.
Vancomycin and VanA Resistance: A Primer
Vancomycin (VC), an antibiotic first isolated from soil bacteria inhabiting the jungles of Borneo, has served for decades as the last (and sometimes only) resort of the clinician against life-threatening, opportunistic Gram-positive bacteria.[1-3] VC was named in the 1950s for its ability to vanquish all strains of Staphylococci, and the performance of the antibiotic against the ‘superbug’ methicillin-resistant Staphylococcus aureus (MRSA) in subsequent years has been almost legendary. MRSA has evaded, via natural selection, virtually all other clinically approved antibiotics, including cephalosporins, tetracyclines, aminoglycosides, erythromycin and the sulfonamides.[1,2] MRSA infections accounted for 60% of all S. aureus infections in U.S. intensive care units as of 2003, and MRSA infections are becoming frequent in non-clinical (i.e., community acquired) settings. In addition to combating the MRSA menace, VC is effective against Enterococcal bacterial infections, which are potentially lethal to an expanding population of immunocompromised cancer, AIDS, elderly, and organ transplant recipient patients.[1-3] VC has been used to treat infections for patients allergic to β-lactam antibiotics such as penicillin, post-operative Clostridium difficile infections, and is a frontline therapy for endocarditis (inflammation of the tissue lining the heart).[5,6]
The indispensable bastion of effective antimicrobial defense provided by VC is now under siege by the spread of bacterial resistance in recent decades.[2,3] Intense research efforts have sought to elucidate the structure and mechanism of action of VC to enable the development of derivatives that are capable of forestalling the assault by multi-drug resistant bacteria that would return us to the pre-antibiotic era of untreatable, life-threatening infections.[2,3]
VC (Fig. 1), the archetypal glycopeptide antibiotic, consists of a heptapeptide core composed of some non-standard amino acids.[1,3,7] From the N-terminus, VC has β-hydroxychlorotyrosine residues at positions 2 and 6, p-hydroxyphenylglycine residues at positions 4 and 5, and a 3,5-dihydroxyphenylglycine residue at the C-terminus. Some glycopeptide antibiotics lack the β-hydroxyl and/or chlorine substituents of residues 2 and 6. Variation among glycopeptide antibiotics also consists in the use of aliphatic or aromatic residues at positions 1 and 3 from the N-terminus. VC employs an N-methylated leucine and an asparagine for residues 1 and 3, respectively. Unlike a β-pleated sheet strand, the stereochemistry of the peptide backbone of all known glycopeptide antibiotics is not uniformly R or S; from the N to C termini, it is R, R, S, R, R, S, S. This stereochemical pattern along the backbone endows glycopeptide antibiotics with a bowl-shape (PDB Jmol; PDB 1FVM) that is critical to antimicrobial action. The heptapeptide core of all glycopeptide antibiotics is adorned with sugars to varying extents.[1,8] In the case of VC, the side chain of the fourth residue is glycosylated with an L-vancosaminyl-α(1→2)-D-glucopyranosyl disaccharide.[1,8]
Structural rigidity is imparted to VC and other glycopeptide antibiotics by cross-linking of the aromatic side chains of residues 2, 4, and 6 to form a two biphenyl ethers with a shared central ring.[3,7] Hindered rotation about the ether bonds on the chlorinated aromatic rings of residues 2 and 6 locks them into one configuration, endowing VC with two sites of the uncommon phenomenon of atropisomerism. A third site of atropisomerism results from the cross-linking of the aromatic rings of residues 5 and 7 to form a biaryl moiety. Rotation about the biaryl bond is restricted by the substituents of the aromatic rings, as well as the constrained nature of the 12-member macrocycle that contains the biaryl moiety. In fact, the peptide bond spanning residues 5 and 6 assumes the less stable s-cis (as opposed to the usual s-trans) configuration to accommodate the atropisomer shown for the biaryl unit (PDB Jmol). Consideration of the fact that VC has 18 chiral centers, in addition to 3 elements of atropisomerism, reveals that the antibiotic is a single stereoisomer out of more than 2 million possible structures. As a consequence of these structural features, VC is one of the smallest natural products to exhibit stereospecific molecular recognition.
VC was the first example of an antimicrobial agent that targets a specific bacterial membrane precursor essential for cell wall synthesis. The target, known as Lipid II, is the monomer used to build up the cell wall; VC therefore binds a substrate rather than an enzyme (as many antibiotics) involved in a cellular process critical to survival and replication. All bacterial cell walls are composed of polymeric strands of alternating amino sugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), that are crosslinked for mechanical strength via a peptide framework (Fig. 2a).[7,10] Before cross-linking occurs, peptide chains appended to the MurNAc units have the sequence L-Ala-D-Glu-L-Lys-D-Ala-D-Ala.[7,10] In essence, a bacterial cell is effectively jacketed by a single polymeric molecule, known as peptidoglycan, capable of withstanding the positive osmotic pressure of the cell in order to prevent lysis and bacterial death. The disaccharide-peptide monomeric subunit of the peptidoglycan, connected to a membrane-embedded anchor, is transported to the exterior of the plasma membrane for peptidoglycan assembly (Figure 2a).[7,10] The membrane-anchored peptidoglycan precursor subunit (Fig. 2b) is itself Lipid II. In the extracellular space, a transglycosidase enzyme joins the disaccharide of the monomer to the growing glycan strand.[7,10] A transpeptidase enzyme (PBP1b is a “large penicillin binding protein” that contains both the transglycosidase and transpeptidase domain.) then recognizes the C-terminal D-Ala-D-Ala sequence of the pendent peptide of the former monomer, cleaves off the terminal D-Ala, and couples the peptide fragment to an adjacent peptide of the peptidoglycan, thereby freeing the membrane anchor of its cargo.[2,3,7] The survival of bacterial cells depends on the recycling of the membrane anchor that transports precursor monomers for continued peptidoglycan growth.
VC easily diffuses through the layers of peptidoglycan that enclose a Gram-positive bacterial cell to reach the site of peptidoglycan polymerization (Fig. 2). Unlike Gram-positive bacteria, Gram-negative bacteria have an outer membrane beyond the peptidoglycan that is impermeable to VC, rendering Gram-negative bacteria intrinsically resistant to it. At the site of cell wall synthesis, the bowl-shaped antibiotic recognizes the C-terminal D-Ala-D-Ala sequence of un-crosslinked peptidoglycan precursor peptides (Fig. 3), and binds with high affinity (Ka = ~106 M-1, determined in aqueous buffer with a pH of 4.7-5.1 at 298 K).[6,11] The antibiotic forms five hydrogen bonds as well as an extensive array of hydrophobic contacts (PDB Jmol).[6,10,11] VC sequesters the Lipid II substrate, thereby sterically occluding the approach and action of the transglycosidase/transpeptidase enzyme, and shuts down the recycling of its membrane anchor component.[7,10] Unable to either polymerize and cross-link VC-bound monomeric precursor, or recycle the membrane anchors bearing the VC-precursor complexes to bring new monomeric units to the cell wall, peptidoglycan biosynthesis is halted. Meanwhile, enzymes that remove old layers of peptidoglycan that would ordinarily be replaced continue unabated. As the disassembly of the peptidoglycan decreases its mechanical strength, the bacterial cell becomes susceptible to osmotically-induced lysis.
Roughly at the same time the molecular details of the antibacterial action of VC were finally elucidated after decades of research (see below), the first cases of VC-resistant Enterococci were reported in 1988, and of Staphylococcus aureus in 1997.[1-3] The most common strains of Enterococci and MRSA resistant to VC in the U.S. are of the VanA type, which is the best understood mode of resistance.[3,14] VanA-type bacteria synthesize their peptidoglycan normally in the absence of VC, with precursors for the peptide framework terminating in consecutive D-Ala residues.[1,3] If a VanA cell detects the glycopeptide antibiotic, however, an alarm signal is transduced that results in the production of a trio of proteins (vanHAX) that work in concert to remodel the peptidoglycan.[3,10] The newly minted enzymes reduce pyruvate to D-lactate, hydrolyze the D-Ala-D-Ala dipeptides of the normal cell wall biosynthetic pathway, and condense D-Ala with D-lactate for use as the C-terminal residues of peptidoglycan precursor peptides.[3,10] Overall, an O is substituted for an NH in the conversion of an amide to ester linkage. This transformation results in a remarkable 1000-fold reduction in the binding affinity of VC. Though not without exceptions, the VC literature has attributed the drastic decline in binding affinity (and antibiotic efficacy) to the deletion of an H-bond. In a review of the VanA mode of resistance in Enterococci, Walsh (who, with Courvalin, elucidated the molecular logic of vanA resistance), concludes, “The loss of one H-bond is the elegant and simple solution that can spell life or death to the bacterium and, perhaps, to an infected patient.”
Vancomycin Molecular Recognition Events
An essential step in the molecular recognition of D-Ala-D-Ala by VC in water is desolvation of the ligand, as well as the binding site the ligand will occupy. A computational study, in agreement with some experimental observations, suggested that the amide NH groups of residues 3 and 4 rotate by 180° underneath the bis-diphenyl ether moiety (Fig. 1a) at ordinary temperatures, populating conformers where the NH units of these residues are alternatively on the concave and convex faces of VC. Rotation of the amide NH groups is predicted to distort the macrocycle, and in turn, cause dramatic fluctuations in the shape and size of the binding pocket that destabilize and expel formerly ordered water molecules. This phenomenon of ‘macromolecular breathing’ is accompanied by the stripping away of H-bonded waters from two of the amide groups needed for molecular recognition, because the NH groups pass through a macrocycle too small for solvating waters to follow along. Experimental evidence suggested that the asparagine sidechain of residue 3 assists in the regulation of binding site solvation by serving as an intramolecular flap. The sidechain acts as a surrogate ligand in the absence of the target peptide to limit solvation of the highly polarized pocket, and then swings out of the way to permit ligand recognition.
In this view, as the amide NH groups of residues 3 and 4 rotate from the convex to concave side of VC, the antibiotic welcomes the D-alanyl carboxylate terminus of a peptidoglycan precursor peptide into a relatively desolvated pocket. The pocket is lined with the partial positive poles of the amide NH groups from residues 2, 3, and 4, which are crowded together and aligned to form fresh H-bonds to the anionic moiety of the ligand (Fig. 3).[1,9] The electrostatic interactions that underlie the three normal H-bonds formed between VC and the carboxylate terminus of the ligand are strengthened by the folding inward of the isobutyl side chain of the N-methylleucine residue (PDB Jmol). The hydrophobic wall formed by the isobutyl side chain of VC lowers the dielectric constant of the binding pocket and shields the antibiotic-ligand carboxylate H-bonds from direct competition with water. The three amide NH groups of the receptor pocket successfully compete with water to solvate the carboxylate of the ligand, because water molecules, unconstrained by a template, cannot position their oxygen atoms close together without paying an enthalpic price. For the amides of the binding pocket, the enthalpic price for the close proximity of parallel dipoles is prepaid by the expenditure of energy during antibiotic biosynthesis. The inversion in stereochemistry of residue 3 (S), relative to the all R stereochemistries of residues 1, 2, and 4, destabilizes conformations of the peptide backbone relative to the binding conformation, and rigidity within this conformation imparted by side chain cross-links removes a considerable fraction of the adverse entropy that would be incurred upon bimolecular association.[1,8,12]
VC has additional interactions with peptidoglycan precursor peptides terminating in consecutive D-Ala residues. The amide carbonyl belonging to residue 4 in VC accepts an H-bond from the amide NH of the terminal Ala in the ligand (Fig. 3). The amide NH of residue 7 in the antibiotic donates an H-bond to the amide carbonyl of the antepenultimate Lys residue in the ligand (Fig. 3). It is believed that the direct contribution of these two amide-amide H-bonds to the binding affinity of peptidoglycan precursor peptides and their analogues is minimal. N-acetylglycine, relative to acetate, can form the first of the amide-amide H-bonds being discussed, and only associates with VC with a Gibbs free energy of binding (ΔGº) more negative by ~0.6 kcal/mol at 303 K in an aqueous buffer of pH 6.[1,13] For comparison, VC binds ligands (e.g., di-acetyl-Lys-D-Ala-D-Ala, Fig. 3b) capable of making most of the interactions of the natural target pentapeptide (Fig. 3a), with a ΔGº of –8 kcal/mol at 298 K in an aqueous buffer of pH 4.7 – 5.1.[6,11] The two amide-amide H-bonds between VC and its natural D-Ala-D-Ala target, however, indirectly play important roles. Both H-bonds orient the bacterial pentapeptide such that the methyls of the Ala residues establish favorable hydrophobic contacts with aromatic rings of the antibiotic (PDB Jmol), and these interactions are believed to promote binding by a factor of 103. Furthermore, the two amide-amide H-bonds are thought to tether the ligand to the antibiotic, thereby reducing residual motion and strengthening the H-bonds to the carboxylate. This tethering effect is an example of binding cooperativity that is critical to the antimicrobial activity of VC, as will be discussed later. The complex interplay of H-bonding, hydrophobic contacts, and dynamic motions within an otherwise rigid conformation that is responsible for high affinity binding to the bacterial peptide target attests to the evolutionary fine-tuning of the VC structure. VC is tailored for recognizing its natural ligand, as evidenced by the high amino acid sequence conservation among all known glycopeptide antibiotics; structural conservation implies functional necessity.
(4) Xie, J.; Pierce, J. G.; James, R. C.; Okano, A.; Boger, D. L. A Redesigned Vancomycin Engineered for Dual D-Ala-D-Ala and D-Ala-D-Lac Binding Exhibits Potent Antimicrobial Activity Against Vancomycin-Resistant Bacteria. J. Am. Chem. Soc.2011, 133, 13946-13949.
(6) McComas, C. C.; Crowley, B. M.; Boger, D. L. Partitioning the Loss in Vancomycin Binding Affinity for D-Ala-D-Lac into Lost H-Bond and Repulsive Lone Pair Contributions. J. Am. Chem. Soc.2003,125, 9314-9315.
(10) Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story. Chem. Biol.1996, 3, 21-28.
(11) Rekharsky, M.; Hesek, D.; Lee, M.; Meroueh, S. O.; Inoue, Y.; Mobashery, S. Thermodynamics of Interactions of Vancomycin and Synthetic Surrogates of Bacterial Cell Wall. J. Am. Chem. Soc. 2006, 128, 7736-7737.
(12) Waltho, J. P.; Williams, D. H.; Stone, D. J. M.; Skelton, N. J. Intramolecular Determinants of Conformation and Mobility within the Antibiotic Vancomycin. J. Am. Chem. Soc.1988, 110, 5638-5643.