University of Connecticut University of UC Title Fallback Connecticut

How to Organize your Orgo Lecture Notebook


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.



Taking thorough, well-organized notes is key to understanding the course material and effectively and efficiently studying for exams. While the organization of your notebook is an individual process and no one way is perfect for everyone, this sheet provides some techniques that could be useful if adopted properly. The second semester of organic chemistry is highly reaction based (although there are plenty of reactions throughout) so keeping them organized negates the need resort to memorization of these transformations. Try breaking your notebook into these key sections: (1) Table of Contents; (2) Lecture Notes; (3) Mechanisms; (4) Reaction Summaries; (5) Practice Problems.

Text: John Ovian

Design: M.W. Peczuh & Aneesa Bey

Layout & Rendering: Aneesa Bey

A Chocolate Bloom Analogy for Crumbling Concrete in Home Foundations*

lrzbe_detailHave 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.

* This post was prompted by the release of the “Concrete Expert’s Final Report” regarding crumbling concrete in foundations of homes in eastern CT.

^ photo: bloomed chocolate by Mark Agbuya on Seasoned Advice

Related News Articles 

AG Issues Final Report on Cause of Crumbling Foundations Hartford Courant – November 4, 2016

Financial Relief Eludes Connecticut Homeowners With Crumbling Foundations New York Times – November 14, 2016

With Connecticut Foundations Crumbling ‘Your Home Is Now Worthless’ New York Times – June 7, 2016

Diazonamide dot-com

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

Angew. Chem. Int. Ed. 2001, 40, 4765-4769

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.


Scheme 1. Diazonamide structure: initial and revised assignments.

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.[6]” 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  – 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 46 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.


Scheme 2. Reaction conditions: a) [Cp2ZrCl2], nBuLi, THF, −78 °C, then 6, −78 °C→RT, 3 h; 3.8 mol % Pd(OAc)2, 3.8 mol % P(o-tolyl)3, Ag3PO4, 5, RT, 8 h, (85 %); b) 2.5 equiv BBr3, CH2Cl2, −78→−20 °C, (quant.); c) 4, iPr2NEt, TBTU, DMF, RT, (89 %). Boc=tert-butoxycarbonyl; TBTU=2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate.

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.


Scheme 3. Reaction conditions: a) 3 mol % [Pd2(dba)3], 6 mol % 2-(di-tert-butylphosphanyl)biphenyl, Ag3PO4, THF, 75 °C (82 % based on recovered 10); b) tBuOK, THF, 1.2 equiv 2-bromoethyltriflate, 0°C; c) 1.2 equiv 13, toluene, −78→−25 °C; H2S(g), THF, −50 °C, (67 % from 12); d) 3×1 equiv p-TsOH, toluene, 95 °C, 40 min; e) ZOSu, DMF, RT, (54 % from 14); f) NaBH4, CeCl3⋅7 H2O, MeOH; g) Rieke zinc (excess), 3:1 THF/EtOH, 0 °C, (75 % from 16); h) tBuNH2/Br2 complex, toluene/THF/CH2Cl2, −78→−20 °C, 10 h (86 %, ortho:ortho/para=5:1); i) o-nitrobenzyl bromide, K2CO3, NaI, DMF, (52 %); j) 4.8 equiv Cl3CCO2H, 1 equiv H2O, toluene, 68 °C, (90 %). dba=trans,trans-dibenzylideneacetone; Z=benzyloxycarbonyl; Su=N-succinimidyl.

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.


Scheme 4. Reaction conditions: a) 1.3 equiv 19, toluene/CH2Cl2, 0°C→RT, (83 %); b) 5 mol % nPr4NRuO4, 1.5 equiv NMO, 4 Å MS, CH2Cl2, (78 %); c) hν (350 nm), 0.003 M in degassed dioxane; excess Ac2O, pyridine, DMAP, CH2Cl2, (85 %); d) 2.2 equiv DDQ, 9:1 THF/H2O, (89 %); e) (Cl3C)2, Ph3P, Et3N, THF, (68 %); f) hν (300 nm), 2 equiv LiOAc, 3 equiv epichlorohydrin, 0.005 M in 3:1 CH3CN/H2O, (32–40 %); g) 2 equiv NCS, THF, 32 °C, 10 h, (60 %). NMO=4-methylmorpholine-N-oxide; DMAP=4-dimethylaminopyridine; DDQ=2,3-Dichloro-5,6-dicyano-1,4-benzoquinone; NCS=N-chlorosuccinimide.

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!


Scheme 5. Reaction conditions: a) 1 atm H2, 10 % Pd/C, MeOH, RT, (quant.); b) Z-L-Val-OH, TBTU, iPr2NEt, DMF, (92 %); c) 40 mol % [{Bu2Sn(O)Cl}2], toluene/MeOH 70 °C, (80 %); d) p-BrC6H4CO2Su, DMF, RT, (79 %); e) NBS, iPr2NH, CH2Cl2/THF, (48 %); f) Amberlyst-15, 4 Å MS, 1:4 CH3CN/CH2Cl2 (54–85 %). NBS=N-bromosuccinimide.

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.


Figure 1. Preparation and X-ray structure (ORTEP; 30 % probability thermal ellipsoids) of diphenyl acetal 28. a) Amberlyst-15, 4 Å MS, 1:4 CH3CN/CH2Cl2.

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.

How VanA Strains Get Away With It, Part Two

An evaluation of the thermodynamics of binding between various ligands of Vancomycin (Part 2/2)

by Matthew Guberman-Pfeffer*

Part 1 in this series can be found here on The Offset and also at the Winnower.

Vancomycin Molecular Recognition Events (cont’d)

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).[15] 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.[1]

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.[6] 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.[6]

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.[6] 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.[6] 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.[6] 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.[6]

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.[6] 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.[16]

Briefly, VC absorbs at ~280 nm due to the presence of the phenolic groups in its structure (Fig. 1).[16] VC complexed with cell wall peptide analogues also absorbs UV light of ~280 nm, but with a reduced extinction coefficient.[16] 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]

Figure 4. Representation of the interactions between VC and model peptide ligands, along with the corresponding thermodynamic binding data. Figure from J. Am. Chem. Soc. 2011, 133, 13946–13949.

Figure 4. Representation of the interactions between VC and model peptide ligands, along with the corresponding thermodynamic binding data. Figure from J. Am. Chem. Soc. 2011, 133, 13946–13949.

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.[6] Ligand 4 had a free energy of binding more positive than ligand 2 by 4.1 kcal/mol.[6] 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.[6] 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.[4]

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.[15]

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].”[2] 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.[17] 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.[17] 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.[19] 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.[6] 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.

Figure 5. Correlation of overall ligand binding energy to VC as a function of the chemical shift of the residue 2 amide proton on the antibiotic. As the ligand was extended by each differently colored moiety from acetate (blue) to di-acetyl-Lys-D-Ala-D-Ala (entire structure including the purple extension), both the binding free energy for the ligand and the chemical shift of the particular amide proton increased. Every additional antibiotic-ligand interaction strengthened the ‘pre-existing’ H-bond of the residue 2 amide proton with the ligand carboxylate. Figure adapted from [17].

Figure 5. Correlation of overall ligand binding energy to VC as a function of the chemical shift of the residue 2 amide proton on the antibiotic. As the ligand was extended by each differently colored moiety from acetate (blue) to di-acetyl-Lys-D-Ala-D-Ala (entire structure including the purple extension), both the binding free energy for the ligand and the chemical shift of the particular amide proton increased. Every additional antibiotic-ligand interaction strengthened the ‘pre-existing’ H-bond of the residue 2 amide proton with the ligand carboxylate. Figure adapted from [17].

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.[20] 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.[6] 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.[6] 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.[21] 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).[21] 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.[21] 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.[6] 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).[6]

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).[6] 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).[6] 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).[6]

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.;2-C/abstract


(2) Williams, D.H. The Glycopeptide Story : How to Kill the Deadly ‘Superbugs.’ Nat. Prod. Rep. 1996, 13, 469-477.!divAbstract


(3) Nicolaou, K. C.; Boddy, C. N. C.; Bräse, S.; Winssinger, N. Chemistry, Biology, and Medicine of the Glycopeptide Antibiotics. Angew. Chem. Int. Ed. 1999, 38, 2096-2152.;2-F/abstract


(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.


(8) Loll, P. J.; Axelsen, P. H. The structural Biology of Molecular Recognition by Vancomycin. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 265-289.


(9) Jusuf, S.; Axelsen, P. H. Synchronized Conformational Fluctuations and Binding Site Desolvation during Molecular Recognition. Biochemistry 2004, 43, 15446-15452.


(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.


(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.!divAbstract


(14) Périchon, B.; Courvalin, P. VanA-Type Vancomycin-Resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 2009, 53, 4580-4587.


(15) Williams, D.; Westwell, M. Aspects of Weak Interactions. Chem. Soc. Rev. 1998, 27, 57-64.!divAbstract


(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.!divAbstract


(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.


(19) Houk, K. N.; Leach, A. G.; Kim, S. P.; Zhang, X. Binding Affinities of Host–Guest, Protein–Ligand, and Protein–Transition-State Complexes. Angew. Chem. Int. Ed. 2003, 42, 4872-4897.


(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.


(21) Anslyn, E. V.; Dougherty, D. A. Modern Physical Organic Chemistry; University Science Books: Sausalito, 2006, pp 23, 114, 189.

Friendly Conversation

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.

Resistance: How VanA Strains Get Away With It

An evaluation of the thermodynamics of binding between various ligands of Vancomycin (Part 1/2)

by Matthew Guberman-Pfeffer*

This post is also published on The Winnower under DOI:10.15200/winn.144060.03577

In Brief

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.


Figure 1. Vancomycin, VC. Figure by permission from Nature 1999, 397, 567-568.

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.[3] 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.[4] 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.[1] 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.[3] 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.[3] 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).[3] 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.[3] As a consequence of these structural features, VC is one of the smallest natural products to exhibit stereospecific molecular recognition.[9]

VC was the first example of an antimicrobial agent that targets a specific bacterial membrane precursor essential for cell wall synthesis.[7] 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.[10] 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.[7] 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.[7]

Figure 2. Schematic representation of the bacterial cell wall synthesis cycle. (a) A disaccharide with a pendent pentapeptide is assembled on a membrane-anchored carrier to produce Lipid II, which then transports the monomer for peptidoglycan polymerization from the cytoplasmic to extracellular side of the membrane. The membrane-anchored portion delivers the cargo to the growing cell wall, and is recycled back to the cytoplasmic side to continue the process. (b) Structure of Lipid II. The membrane-anchored carrier is a polyisoprenoid consisting of eight isoprene units in the cis-conformation, followed by two units in the trans-conformation, and the terminal isoprene unit. The cell wall monomer carried by the polyisoprenoid contains a disaccharide with a pendent pentapeptide. The residue 3 lysine of the pentapeptide is coupled to the glutamate at position two via the side chain carboxylate. Figure adapted from [7] and used with permission.

Figure 2. Schematic representation of the bacterial cell wall synthesis cycle. (a) A disaccharide with a pendent pentapeptide is assembled on a membrane-anchored carrier to produce Lipid II, which then transports the monomer for peptidoglycan polymerization from the cytoplasmic to extracellular side of the membrane. The membrane-anchored portion delivers the cargo to the growing cell wall, and is recycled back to the cytoplasmic side to continue the process. (b) Structure of Lipid II. The membrane-anchored carrier is a polyisoprenoid consisting of eight isoprene units in the cis-conformation, followed by two units in the trans-conformation, and the terminal isoprene unit. The cell wall monomer carried by the polyisoprenoid contains a disaccharide with a pendent pentapeptide. The residue 3 lysine of the pentapeptide is coupled to the glutamate at position two via the side chain carboxylate. Figure from [7] and used with permission.

VC easily diffuses through the layers of peptidoglycan that enclose a Gram-positive bacterial cell to reach the site of peptidoglycan polymerization (Fig. 2).[3] 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.[10] 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.[3] As the disassembly of the peptidoglycan decreases its mechanical strength, the bacterial cell becomes susceptible to osmotically-induced lysis.[10]

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.[6] 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.”[10]

Figure 3. VC recognizes the consecutive D-Ala-terminating sequence of the membrane-anchored disaccharide-pentapeptide intermediate of bacterial cell wall synthesis, and binds by (a) forming five hydrogen-bonds as well as (b) hydrophobic contacts with the methyl groups of the D-Ala residues packed against aromatic rings of the antibiotic. Figure from Nat. Prod. Rep. 2002, 19, 100-107 with permission.

Figure 3. VC recognizes the consecutive D-Ala-terminating sequence of the membrane-anchored disaccharide-pentapeptide intermediate of bacterial cell wall synthesis, and binds by (a) forming five hydrogen-bonds as well as (b) hydrophobic contacts with the methyl groups of the D-Ala residues packed against aromatic rings of the antibiotic. Figure from Nat. Prod. Rep. 2002, 19, 100-107 with permission.

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.[9] 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.[9] 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.[8] 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.[9] 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).[8] 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.[8] 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.[1] 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.[1] 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.[1] 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.[1] 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.[1] 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.[1] 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.



(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.

(2) Williams, D.H. The Glycopeptide Story : How to Kill the Deadly ‘Superbugs.’ Nat. Prod. Rep. 1996, 13, 469-477.

(3) Nicolaou, K. C.; Boddy, C. N. C.; Bräse, S.; Winssinger, N. Chemistry, Biology, and Medicine of the Glycopeptide Antibiotics. Angew. Chem. Int. Ed. 1999, 38, 2096-2152.

(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.

(8) Loll, P. J.; Axelsen, P. H. The structural Biology of Molecular Recognition by Vancomycin. Ann. Rev. Biophys. Biomol. Struct. 2000, 29, 265-289.

(9) Jusuf, S.; Axelsen, P. H. Synchronized Conformational Fluctuations and Binding Site Desolvation during Molecular Recognition. Biochemistry 2004, 43, 15446-15452.

(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.

(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.

(14) Périchon, B.; Courvalin, P. VanA-Type Vancomycin-Resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 2009, 53, 4580-4587.


*Thanks to Jordan Greco for help with editing early versions of this work.

Summer Reads 2015 – Total Synthesis Communications

I assembled a list of journal articles reporting total syntheses of natural products based on suggestions from several people on Twitter. In addition to their subject matter, they are considered to be eloquently written. Read them to see if you agree or even make your own suggestion.

The post is actually on Storify at the following URL:



Sympathy for the amide bond

Enantioselective Total Synthesis of (-)-Lansai B and (+)-Nocardioazines A and B

Haoxuan Wang and Sarah E. Reisman*

Angew. Chem. Int. Ed. 2014, 53, 6206-6210 DOI: 10.1002/anie.201402571


Nothing incites apoplexy in your favorite peptide chemist like mentioning how “real” synthetic chemists are dismissive of the challenges that can arise, from time to time, in the formation of amide bonds. C’mon. How hard can it really be? Activate an acid using your favorite alphabet soup reagent and allow an amine nucleophile to take it from there. Done…. But sometimes things get complicated. Sarah Reisman’s#^ hot-off-the-presses Angewandte Chemie communication reporting the syntheses of (-)-lansai B (1) and the “no cardio required” (+)-nocardioazines A & B (2 & 3) reveal that even a card-carrying synthetic chemist can be stymied by amide bond formation, for a while. As it turns out, the forces of amide bond formation were used for good – in the macrocyclization of (+)-nocardioazine B.

This post is the second installment of an annual exercise for my organic synthesis class where I try to deconstruct a total synthesis communication paragraph by paragraph. The strategy and execution of the writing are put on equal footing with that of the chemistry. Our goal is to understand how the manuscript – both its text and its graphics – communicates the ideas behind the science and the agenda/priorities of the author. It is a case study in synthesis AND manuscript writing. The discussion of the manuscript in class emphasizes active reading and what behaviors active reading entails.

So there’s no better place to start than at the beginning. The title “Enantioselective Total Synthesis of (-)-Lansai B and (+)-Nocardioazines A and B” and abstract are straightforward stylistically and they give clear clues about the story that will unfold in the manuscript. Three words/phrases do the work that is needed here. 1 – The term “enantioselective” indicates how the chirality of the natural products will be introduced in the molecule: via chiral catalysts in enantioselective reactions in the synthetic sequence. 2 – “Formal [3+2] cycloadditions” refers to a new and powerful reaction introduced earlier by the group for formation of pyrroloindolines. 3 – By noting that macrocyclization occurred by “intramolecular diketopiperazine formation” the authors tip their hand on the ultimate route for the synthesis of nocardioazine A. Now to the manuscript proper.

Paragraph 1: The introduction is textbook for a synthesis paper. It gives a quick one-two punch in the form of biological activity (1, 2, and 3 in Figure 1 are potential antibacterial and anticancer agents.) and a description of the structure (They are bis(pyrroloindolines) linked via a diketopiperazine.). It then back-fills with some details for each point. First, the star compound, (+)-nocardioazine A (2), is a P-glycoprotein inhibitor. P-glycoprotein is an efflux pump central to multi-drug resistance resistance in several cancers. The remainder of the paragraph points out the subtleties of the structures, especially the stereochemical relationship between the group at the C3 of the pyrroloindoline unit and the carboxamide, giving rise to the exo- and endo- nomenclature throughout the paper. (-)-Lansai B (1) is constructured of two exo pyrroloindolines in the same enantiomeric series, whereas nocardioazines A (2) and B (3) are made from one endo- and one exo- pyrroloindoline of opposite enantiomeric series. Compounds 47 in Figure 1 nicely illustrate the relationships. Reisman emphasizes that these precursors make “appealing synthetic targets for asymmetric catalysis where selection of the appropriate enantiomer of the catalyst dictates the absolute stereochemistry of the pyrroloindoline building block.” That’s a great sentence. She’s building up the value of their approach before she fully reveals it. The paragraph closes by noting that there is one reported synthesis of 3 and none of 1 and 2.


Figure 1. Angew. Chem. Int. Ed. 2014, 53, 6206-6210.

Paragraph 2: Some readers may have realized already that this synthesis will showcase the formal (3+2) cycloaddition methodology based on the hints (really statements) in the abstract and first paragraph. The pyrroloindoline natural product targets in the this paper, among others, likely motivated the development of the transformation. That’s a standard argument made by organic chemists for the development of new reactions. If you didn’t see it coming, it becomes explicit in the first sentence of this paragraph. The method for enantioselective synthesis of pyrroloindolines (box in Figure 1) is a major component of their retrosynthetic analysis. In fact, that IS their retrosynthetic analysis as shown in the figure. Reisman trusts that you can make the DKP disconnection and, if you don’t see it now, she’ll walk you through it shortly. “Utility” in the last sentence of the paragraph rings in your ears – this report on total syntheses exclaims the utility of the formal (3+2) methodology.

Despite the centrality of the reaction to the syntheses, it gets a mere mention and a call of Figure 1 but that’s nearly it as far as explanation. The reader must work through the figure and, if necessary, revisit reference 4a, which is the JACS communication that describes the reaction. The reaction uses C3 substituted indoles and 2-amidoacryates in the presence of SnCl4 and catalytic (R or S) BINOL to deliver pyrroloindolines with high enantioselectivity. Note the cis relationship between the R1, H, and carboxylate groups in structure 10 of Figure 1; this is the exo- relationship defined earlier. The reaction is formally equivalent to a (3+2) cycloaddition where the two olefinic carbons and the nitrogen of the amidoacrylate are the “3” and the two carbons of the indole are the “2”. Scheme 1 from the JACS communication gives some insight into the transformation and to the origin of the enantioselectivity. Conjugate addition by the indole onto the amidoacrylate is followed by protonation of the resulting enolate followed by ring closure. With regard to selectivity, Reisman speculates in the JACS communication, “[conjugate addition] occurs with modest levels of catalyst control, whereas the enolate protonation step occurs with high levels of catalyst control. In effect, the catalyst-controlled protonation step serves to resolve the mixture of enantiomeric intermediates.”


Scheme 1. J. Am. Chem. Soc. 2010, 132, 14418-14420.

With that, they’re finished with introductions and on to the business of the syntheses.

 Paragraph 3: Like a lion after a young gazelle, Reisman aims for (-)-lansai B (1) as her first target. Here she provides a little more detail on the retrosynthesis by stating that formation of the DKP would be the way to unite the two pyrroloindoline fragments, 4 and 5, via “sequential amide bond formation.” Simple. She finishes the paragraph by calling the starting materials for the synthesis of 4 and 5, indoles 13 and 16, both of which are in Scheme 1. If you wondered where the indoles themselves came from, reference 4a is there to guide you. The SI of that paper shows a tandem amination-Heck reaction with substituted bromo-iodo-benzenes and allyl amine to deliver the indoles.


Scheme 1. Angew. Chem. Int. Ed. 2014, 53, 6206-6210.

From here, the organization of the paragraphs communicating the syntheses of 13 largely follows the synthetic strategy. That is, Reisman walks the reader through pyrroloindoline synthesis using the formal (3+2) method and then make use of those advanced intermediates in the formation of DKPs to complete the syntheses. As always, there are minor twists and turns in the storyline though.

Paragraph 4: This paragraph takes the reader through the synthesis of two pyrroloindolines, 15 and 18, from the corresponding indoles. Again calling Scheme 1, the paragraph starts by describing a Suzuki-Miyaura reaction that installed the prenyl group on intermediate 15. To be clear, this group is the only difference in the two pyrroloindoline units in (-)-lansai B (1). “Subjection” of 13 to the cycloaddition reaction provided 14 in 85% yield, 14:1 dr, and 92%ee. An aside: Subjection is a great word – it’s perfectly descriptive but it bears a tinge of that, “Is it really a word?” feel to it. Anyway, on scale up (> 1.0 mmol), she notes that lower yields were obtained and that a scan of protic additives showed that addition of 2,6-dibromophenol maintained yield and selectivity. They suggest that, “…2,6-dibromophenol facilitates turnover of the chiral catalyst, but is not reactive enough to protonate the transient enolate directly in a nonselective fashion.” It’s like a goldilocks acid in a milieu of acids in the cycloaddition reaction. This observation underscores the complexity of the reaction mechanism. Thankfully, the results are easily understood and useful. Conversion of 16 to 17 follows the same protocol (79%, 12:1 dr, 93% ee). The paragraph continues on with some deprotections to end with intermediates 15 and 18. (-)-Lansai B (1) is about to yield DKP formation.

Paragraph 5: The concession paragraph. The plan was to finish (-)-lansai B (1) through DKP formation by sequential amide bond formation. Preparation of intermediate 19 in Scheme 1 was to be the first of the two amide bond forming reactions. Despite scanning a “wide variety of peptide coupling conditions” – queue visions of alphabet soup – they did not see coupling but only decomposition of 18. Reisman then recalls a successful coupling of exo-pyrroloindolines by Danishefsky and coworkers in their synthesis of amauromine. There both nitrogens were protected as t-butyl carbamates whereas 18 has one N-alkyl and one N-trifluoracetamide. She comments, “these findings reveal that the N substitution of the exo- pyrroloindoline significantly influences the stability of the activated ester under peptide coupling conditions.” So peptide chemistry can pose challenges. They then present an alternative strategy involving the completely deprotected amino acids 20 and 21 (Scheme 2). The plan is to take a hit on yield and mix 20 and 21 to form three DKPs in one reaction; in their estimation this was more efficient that several protecting group manipulations. Coupling using BOPCl gave the two homodimers, each in 20% yield, and (-)-lansai B (1) in 38%; the synthesis was six steps with an overall yield of 20%. In hindsight, the concession was a small one and they learned a little about the amide couplings along the way.


Scheme 2. Angew. Chem. Int. Ed. 2014, 53, 6206-6210.

Paragraph 6: On to the nocardios! Paragraph 6 revisits Figure 1 as it presents the retrosynthesis of 2 and 3 in greater detail. Again the strategy makes use of DKP formation but now targets the more complicated pyrroloindolines 6 and 7 as key intermediates. Compound 6 is the sole endo– configured pyrroloindoline in the paper. Note that its C2 and C3 positions are of the same enantiomeric series as 4 and 5, though. Access to it will require cycloaddition as before followed by epimerization of the carbon a- to the carboxylate. Exo-pyrroloindoline 7, on the other hand, is of the opposite enantiomeric series relative to 4 and 5 and it contains an allyl group (rather than methyl) at C3. Its synthesis will be a real test of the formal (3+2) cycloaddition methodology. The paragraph continues down the forward synthetic path by presenting the cycloaddtion reaction of 22 (Scheme 3) with 2-amidoacrylate 9a to give 23. For the preparation of 23, S rather than R-BINOL is used; this is exactly the point of using asymmetric catalysis and nicely illustrates the power of the cycloaddition. They take a bit of hit on yield for this transformation though. They obtain 23 in 52% yield, 19:1 dr, and 90% ee. The modest yield is attributed to an unwanted side product that results from allyl migration (from C3 to C2 of the indole) under the reaction conditions. However, the presence of the allyl group challenges the scope of the cycloaddition beyond what was done previously and fares well overall. Deprotection of the TFA group then gives one coupling partner, exo-24.

Scheme 3. Angew. Chem. Int. Ed. 2014, 53, 6206-6210.

Scheme 3. Angew. Chem. Int. Ed. 2014, 53, 6206-6210.

Paragraph 7: Paragraph 7 contains more pyrroloindoline synthesis. This time it’s N-allyl-C3-methyl-indole 25 that is the starting material; this indole is also a little more complicated than the models used in the development of the method. Of note is that 9b, the benzyl ester version of the 2-amidoacrylate, is used in the cycloaddition reaction (Scheme 3). This is the only instance among the four cycloadditions that uses this acrylate. It’s not discussed, but in the methodology paper, this acrylate gave the best combination of yield, dr and ee. It is up to the reader to surmise that despite the modest yield (due to lower diastereselectivity in this case), 9b must have given better results than 9a for the transformation. Transesterification, the need for which is not really explained, sets up the epimerization of the carbon alpha to the carboxylate of 26. Cleavage of the methyl ester then reveals acid endo-27 which is ready for amide bond formation.

Paragraph 8: The nocardio A/B synthesis is now at the stage where pyrroloindolines 24 and 27 are “poised” for DKP formation. Coupling in this case using BOPCl was successful. The product, 28 (Scheme 3), was isolated in 84% yield. A footnote directs the reader to the SI for details of the optimization of the yield of the reaction at this point. Inspection of the SI shows that the base, equivalents of amine (24), and rate of addition of acid (endo-27) to the mixture were optimized. She then pays additional homage to the amide bond by comparing the failure to couple 15 and 18 with the success with 24 and 27, writing, “…in addition to the N-substituents, the relative stereochemistry of the pyrroloindoline coupling partners is determinate of the ease of peptide formation.” A saponification-acidification sequence closes the DKP to give 29 (74%). Removal of the N-allyl group then provides 30, a versatile intermediate common to both nocardio A and B. The paragraph ends with the efficient cross methathesis of 30 and 2-methyl-2-butene. The reaction completes the (+)-nocardioazine B (3) synthesis; their route delivered it in 21% overall yield in 9 steps. Modestly, the paper defers the opportunity to compare this synthesis of 3 to the one reported by the group of Ye (10 steps, 12%).

Paragraph 9: Reisman’s attention now turns to (+)-nocardioazine A (2). The plan is to advance compound 30, the penultimate intermediate in the synthesis of (+)-nocardioazine B (3). Cross metathesis of 30 with methacrolein, Luche reduction, and mesylation-chlorination proceed without incident to give 33 (Scheme 4). “Gratifyingly,” (Another great word, although I would have stolen a Wood/Stoltz gem, “To our delight,”) macrocyclization via N-alkylation was also successful, giving 34 in 73% yield. This intermediate is so close to the target itself; it is amongst those “lofty heights of an advanced intermediate”. All that’s left is a seemingly mundane epoxidation that should be quite diastereoselective based on the shape of the macrocycle. The writing seems down right despondent when it lists the epoxidation conditions that yielded little more than the unstable N-oxide 35. Upon inspection of the solid state structure of 34, the recalcitrance of the trisubstituted double bond toward epoxidation was argued not to be steric, but rather resulted from the “electron-withdrawing nature of the allylic nitrogen.” No citation, no further explanation. The lack of additional discussion here leaves the reader grasping for a complete understanding of the lack of reactivity.

Scheme 4. Angew. Chem. Int. Ed. 2014, 53, 6206-6210.

Scheme 4. Angew. Chem. Int. Ed. 2014, 53, 6206-6210.

Paragraph 10: This paragraph details one last gasp to push intermediate 34 over the line to (+)-nocardioazine A (2). Dihydroxylation and chemoselective mesylation followed by intramolecular epoxidation delivered the C2’’-epi-nocardioazine A (37). Ultimately this was a dead end and Reisman moves on without any additional comment. Damn.

Paragraph 11: But then she fires it up one more time. Paragraph 11 walks through a revised synthesis of (+)-nocardioazine A (2) in Scheme 5. They intercept an early intermediate from the previous (attempted) route, pyrroloindoline 23, and proceed with a cross metathesis using methacrolein, Luche reduction and then Sharpless epoxidation on the resultant allylic alcohol giving intermediate 40. This was converted to the corresponding mesylate (41) in anticipation of the upcoming alkylation. Compound 43 is the amine nucleophile for the alkylation. It was prepared from 42 via de-allylation. Although not numbered there, 42 was also an intermediate in Scheme 4. The conversion of 26 to endo-27 entailed transesterification, epimerization, and demethylation to reveal the acid. Drop the last (demethylation) step and you’ve got 42. Alkylation of 41 with 43 then proceeds efficiently and without incident. Saponification of the methyl esters and hydrolysis of the trifluoroacetamides of 44, the product of the alkylation, gave 45. Lofty heights. “We were pleased to find that subjection of 45 to PyBroP in DMF promoted intramolecular DKP formation to afford (+)-nocardioazine A (2).” [YAWP! Mic drop.] Rather than exclaim, Reisman chooses to be understated, cool. She moves on to reassign the stereochemistry of 2 relative to the isolation paper; the reassigned structure of 2 is consistent with Ye and coworker’s reassignment of 3. The paragraph ends with the usual metrics (nine linear steps, 11% overall yield) and the observation that the macrocyclization by DKP formation was effective.

Scheme 5. Angew. Chem. Int. Ed. 2014, 53, 6206-6210.

Scheme 5. Angew. Chem. Int. Ed. 2014, 53, 6206-6210.

Paragraph 12: The wrap up hits the points that have been collected along the manuscript. 1 – We made the three related bis(pyrroloindoline) DKP natural products. 2 – The enantioselective formal (3+2) cycloaddition is useful to total synthesis. And finally, validation their respect for the amide bond. 3- “In addition, subtle changes in the relative stereochemistry and nitrogen substitution patterns of pyrroloindolines were shown to significantly influence the ability to prepare bis(pyrroloindolines) by DKP formation.” This finish signals Reisman’s appreciation of the amide bond as a worthy adversary.


Thanks to Sarah E. Reisman for sharing ChemDraw versions of the figures and schemes from the Angewandte paper.

# Professor Reisman has a tenuous connection to UConn Chemistry. Timo Ovaska, her undergrad research advisor, earned his PhD with Bill Bailey in our department. She then traveled west to New Haven to work in John Wood’s lab when he was still at Yale. Though Sarah isn’t a native nutmegger, we’d gladly claim her as our own; the (+)-nocardioazine B synthesis shows a good bit of Yankee ingenuity.

^ Although this post has been written as though Professor Reisman is the sole researcher and writer, it has been done for clarity only. It’s safe to say that she, like all faculty, values the scientific collaboration and training with post-docs, grad students, and undergrads as the most enjoyable part of the job.

The Ride of a Lifetime: a Fulbright Experience

Posted by Mark W. Peczuh

 Note: This is the long-read version of a post that appeared in the UConn Today.

More photos from Barca on flickr.

It was an international roller coaster ride. There were ups and downs. The time went faster than I expected. I returned to the place where I started. I want to do it all again. My Fulbright experience during the spring and summer of 2013 in Barcelona played a transformative role in my life, both personally and professionally. I have returned to my “normal” life with a broader perspective on my research and with a profound appreciation of place, history and especially interpersonal relationships that I had previously dismissed. In much the same way I packed a bit of eastern Connecticut to share with Barcelona, I’ve returned with my story about Catalunya that must be told.

Initially it was a maelstrom of sensations, impressions, and emotions: new sights, new smells, city life, commuting as a contact sport, late lunch and later dinner, Catalan in my ears that was close to the familiar Castilian Spanish but not quite intelligible, the kindness of the locals, joyous solitude filled with reading, writing and thinking, wretched solitude away from my family. My host and his lab were one constant during this time. I had chosen to work at the Chemistry Institute of Sarria (Instituto Quimico de Sarria, or simply IQS) with Professor Antoni Planas. Science is inherently international. Chemical reactions follow the laws of Nature so the lab was a logical place to connect with something that was familiar to me. I had also rented an apartment before I arrived in Barcelona through a company on the internet. I lived in a furnished third floor apartment in a neighborhood called the Eixample near the Diagonal and Passage of San Joan. The Eixample, literally ‘extension’, covers a huge swath of Barcelona. It was created as part of a huge expansion of the physical layout of the city that occurred in the 19th century. As with similar urban expansions of the time (e.g. NYC) the Eixample was laid out in a very logical grid pattern. With the most important locations of the city – my lab and my apartment – organized, I began to fill in the corridor between the two. Within a couple of weeks I had established a daily routine and had the physical lay of the land in my head. I could traverse the route from apartment to lab (by train) without conscious effort. I had coffee at Mr. Pan on most mornings. I did Saturday morning shopping at several stalls in the Market of the Conception near my house. I knew to get to the Poll Bo before three o’clock on Sunday if I wanted a chicken for lunch. The most basic challenges of day-to-day living had been overcome; I was on top of it all. I began to feel like l was really a local.

Research Collaboration: Let’s get the proteins to do the work.

Toni Planas, my host at IQS, is the Chair of the Department of Bioengineering. Work in his lab ranges from fundamental enzymology to the engineering of algae to overproduce beneficial fish oils. He is world-famous for being amongst the original developers of glycosynthase technology. Glycosynthases are engineered proteins that have their origins in sugar metabolism. One type of carbohydrates, the cellulose that makes up paper, for example, are linear polymers of individual sugar monomers. An easy analogy is to beads on a string – the beads are the sugar monomers and the bits of string between are the bonds that link together the monomers. There are enzymes – an enzyme is a protein that lets a chemical reaction occur more rapidly – in all living organisms that break the bonds between those monomers. Enzymes that break down the bonds in sugar polymers are called glycosidases. As Toni was studying the atomic details of how glycosidases actually speed up the cleavage reaction, he realized that a specific amino acid in the protein was critical for activity. More importantly, he reasoned if that you switched that amino acid so it couldn’t break the bond, maybe the enzyme could form the bond rather than break the bond – the enzyme would run “in reverse”. Given the appropriate conditions, these enzymes are remarkably efficient at making the bonds between sugar monomers. In effect, you could build the string of beads rather than tear it apart. The term glycosynthase was coined to indicate that the enzymes could use sugars (“glyco”) to synthesize (“synthase”) polymers.

Glycosynthases caught my eye because my research group makes artificial sugar compounds that contain an extra atom in the monomer ring. We’ve investigated how to synthesize these sugars and characterized how they are bound by proteins. Before the Fulbright investigation, we used a different type of protein called a lectin whose regular function is simply to bind sugar molecules. Our expanded sugars compete with natural sugars for the binding pockets of lectins – this could have implications for preventing bacterial infections, among other applications. Our question was whether glycosynthases could utilize our expanded sugars in their reactions. This question is more complex because the protein must bind AND then perform the bond forming reaction (They “do chemistry.”).

Once in the lab, I felt like I was a post-doc again. I was quickly reunited with an envelop of sugar samples I had sent from my lab before my departure. With my graduate student mentor, Hugo Aragunde-Pazos, I expressed and purified both a glycosidase and glycosynthase we had identified as the first candidates for our investigation. Some of the techniques I learned were completely new; others were old-hat. Hugo and the rest of my labmates were remarkably helpful and patient. I had many many interactions with the students that introduced me to a different perspective on how to think about science and research. Dealing with language slowed me down relative to how quickly I may have worked in a lab where English was the primary language. My lab technique was a little rusty too, I must admit. Despite the fact that the students were happy to speak Castilian Spanish (and when really pressed, English) in place of Catalan, I spent significant mental energy on simple communication. Language barriers may have initially slowed my work, but it went away after a few weeks. Those weeks I felt stressed and apprehensive because of the challenges language presented. The experience made a strong impression on me that I will never forget. I have more respect for all the international students and post-docs I’ve ever known and I will understand and anticipate this emotion in others when they’re in the same position.

It’s not breaking news to report that the mid-day meal is the main meal of the day in Spain. Lunchtime brought a special opportunity to me in addition to nourishment. It was a relaxed, informal atmosphere to discuss just about anything. My regular lunch group was composed of the people with whom I shared desk space: two of the advanced graduate students Victoria Codera and Sergi Abad, and two of the scientists Teresa Pellicer and Patricia Torruella Barrios. This group instantly became my logistical, social and cultural advisors on Barcelona and all of Catalunya. They taught me some Catalan. They explained how things worked within IQS, within Barcelona/Catalunya and within Spain. They kept me up-to-date on Spanish current events, like a brief hubbub surrounding subsidized cocktails in the Spanish parliament. The most productive lunches were when we planned activities for me to do with my family when they eventually joined me in Barcelona. These conversations were a fertile ground for me to get to learn about Catalan food, history, geography, everything. We determined early on that we could easily plan a year’s worth of weekend excursions, but we would have to concentrate that down to a solid month of touring.

Life Outside the Lab: Diversions

I also did extensive “field work” – aka sight-seeing – in Barcelona and Catalunya to prepare for the arrival of my family. I spent a couple of hours waiting in line one Saturday night to see a landmark Gaudí building, “Casa Milà” better know as “La Pedrera”. Entrance was free because it was the “Night of the Museums,” an event sponsored by the Catalan government and local companies. The line wrapped around three sides of a city block; that alone was sight to see. In between occasional forays on the internet afforded by free wifi hotspots, I actually chatted with some of the people who were in line with me. They provided support for my decision on which museum to visit that night (We didn’t actually get into La Pedrera until about midnight.), but they were happy to show their pride in their city and try out a little English too. Once inside I was struck with the brilliance of Gaudí; the attic of the building collects maquettes of several of his other works to showcase his design philosophy. His ability to harness natural themes – even abstractions such as conic sections – into the design of objects is a hallmark. To Gaudí form had to follow function in a way that uplifts one’s human spirit. Occasionally, a one or two of my labmates would join me on an excursion. The most noteworthy of these was a day trip to the Costa Dorada beach town of Sitges. I joined Victoria and her boyfriend Frances along with Anabella, a group alumni from Guatemala who happened to be visiting. We went to Sitges on Corpus Christi, a Catholic holiday in the spring, to see the “carpets of flowers” that are laid out in the streets. The carpets are designed and executed by neighborhood groups and some are really sophisticated. It’s kind of a stationary Rose Parade. The most interesting part comes during the afternoon when series of 8-10 foot puppets “gigantes” dance through the streets while simultaneously destroying the carpets. Victoria and Frances couldn’t explain why the gigantes had to destroy the flower creations, but it seemed symbolic. Regardless, we, along with the other kids and adults watching, shrieked with delight as the gigantes passed by.

Once they arrived in Barcelona, my family and I roamed around Catalunya as unapologetic tourists. We visited the Gothic quarter, the Magic Fountain on Montjuic and all the Gaudí spots like Parc Guell, Sagrada Familia, La Pedrera and Casa Batllo. All of these sights were in the city itself. We also went further afield in Catalunya to Montserrat, the Codorniu vineyards in Sant Sadurní d’Anoia, the beach at Vilanova i la Geltru, and the town of Figueres which is Salvador Dali’s hometown. We would take “breaks” in between excursions that would allow me to catch up on work and let them catch up on sleep. My one-bedroom apartment seemed palatial for two months became cramped when the four of us were living there together. Throughout our journeys my appreciation of the history, culture and people of Catalunya deepened. I was proud to claim temporary residency there.

Food is an essential component of culture and relationships. The dish most often thought of as the national dish of Spain is paella. In its simplest form it is yellow rice colored by saffron with shellfish cooked in a tomato and seafood broth. There are many, many variations but I staunchly defend my mother-in-law’s version as the world’s best. Paella was both the food and the entertainment at a party in honor of my host Toni who had just received the Fischer Prize at the 2013 European Carbohydrate Symposium. It was also a fortunate coincidence that the party was held as my family arrived in Barcelona. It was a perfect opportunity to introduce them to my new friends and colleagues. Preparing the paella was one of the main activities at the party. Three teams cooked one paella each; this was sufficient to serve everyone at the party. My family and I were happily only spectators for the cooking but participants in the eating. There was drama, trash-talking, some minor sabotage and all-out fun afoot as the paella was being cooked. The noted Catalan “gastronomy as performance art” couldn’t hold a candle to Fischer Prize party. Afterward we consumed the mountains of paella, there was a premier viewing of a video prepared in honor of Toni as Fischer Prize winner. It was a music video parody “Planas Style” a Catalan rendition of PSY’s “Gangnam Style” that included some embarrassing singing and dancing by everyone in the lab. While paella may be the national dish of Spain, the staple of the Catalan diet is bread with tomato, pa amb tomaquet in Catalan. It is one of those foods that is deceptively simple. It is the simplicity that requires using the best possible ingredients: bread (often toasted but not always), ripe tomatoes, olive oil and salt. Raw garlic is also used in the most traditional version. At a restaurant, the ingredients are delivered to your table for you to assemble. Start with the toast and rub it with a clove of raw garlic. Add olive oil and salt. Then take a fresh tomato cut horizontally, not longitudinally, and rub it onto the toast leaving only the tomato skin behind. Part of the attraction to me is the ritualistic nature of the preparation. It brings out my inner alchemist.

The Heart of the Matter

In the spirit of the Fulbright program, I arranged interviews with a few scientists in Barcelona whose research interests are similar to mine. Interviews were extracurricular; they were done outside my lab schedule. I originally had high hopes of doing several interviews, but in the end was able to do only three. In hindsight I wish I had made time for more. I caught up with my friend Xavi Salvatella, who had returned to Barcelona from a post-doc in England since I last spent time with him. I also interviewed Ernest Giralt who was an informal advisor to me as a graduate student. I also met Lluis Ribas, a scientist and entrepreneur working on developing new antibiotics. All three of these researchers work at the Institute for Research in Biomedicine (IRB) in Barcelona. In the he interviews I asked about their latest research results and I also asked how they got to the IRB and what it meant for them to be doing research in Barcelona. The latter part of the interviews revealed more about Catalunya than I had expected they would. A theme centered on the importance of relationships with family and friends emerged as they recounted their decision to live and work in Barcelona. Each one is emotionally connected to Catalunya. Their identity as Catalans was clearly distinct from their identity as Spaniards. It struck me that these things – identification with ‘home’ and close personal relationships – make any group unique but it also common to all groups. What makes us different makes us the same. For me this was both profound and bittersweet. Profound because I realized that these were the same reasons that my brothers would have cited if I had asked them why they chose to live and work in the town where we grew up. And bittersweet because I’ve underemphasized those factors in my own career choices. The realization has kindled a desire in me to remember the importance of people and place.

Looking back on my short residence in Barcelona, the ups and downs seem a little less dramatic now than they were along the ride. All of my experiences there have provided valuable lessons and memories. The scientific work was rewarding and productive. Beyond that, I now have a deeper understanding of how Catalans see the world and themselves. I am attached to both the specifics of this perspective – in terms of their uniqueness in the context of Spain – and the broader concept that emphasizes relationships. I hope that I have revealed some small insights of my Nutmegger’s perspective of the world to my new friends and colleagues in Barcelona. I eagerly anticipate my next ‘ride’ there.


Deconstructing a JACS communication: Winkler’s Ingenol synthesis

The First Total Synthesis of (±)-Ingenol

Jeffrey D. Winkler,* Meagan B. Rouse, Michael F. Greaney,Sean J. Harrison, and Yoon T. Jeon

J. Am. Chem. Soc. 2002, 124, 9726-9728 DOI:10.1021/ja026600a

Note: Many, many thanks go to Jeff Winkler for looking over the post and also sharing an original copy of the manuscript.

Reading a research article is an active process. It’s completely different from leisure reading like a novel or magazine. It’s more like a special – extreme – case of textbook reading. The information has to be unpacked, worked-over and constantly grappled with. During the first meeting of my Organic Synthesis class this year, my students and I worked through the Winkler synthesis of Ingenol. The objectives of the activity were three-fold: 1 – I wanted to compare the strategy used by Winkler to that the recent Baran synthesis. (Which was itself spurred by Carmen Drahl’s reporting on Baran’s synthesis.) 2 – I wanted to analyze how the authors used text and figures to relay the information about natural product synthesis. This helps me develop dos and don’ts for my own writing and a strategy I wanted to share with my students. 3 – I wanted to illustrate how much work an “active” reading of a manuscript should actually be. Below is a play-by-play of the manuscript. It analyzes how Winkler et al. communicated their results in the manuscript and how the reader should interact with the text.

Paragraph 1: Straightforward opening for a total synthesis paper. The authors mention the textbook reasons of “biological activity” and “structural complexity” that inspired their efforts. The inside-outside, C8/C10 trans intrabridgehead stereochemistry linking the BC rings is the most noteworthy feature. Structure 16 is also identified as an important intermediate in this paragraph. It is likely singled out because its preparation completes the carbon skeleton of Ingenol 1, a milestone in the synthesis.

scheme 1

Paragraph 2: This paragraph details the retrosynthetic strategy of the authors and is accompanied by Scheme 1. “Dioxenone photoaddition-fragmentation product 2” is the first intermediate offered in the retrosynthesis. The authors don’t elaborate on what is required to convert 2 to 1, so it is up to the reader to work it out. Inspection of the structures shows that, regardless of order, that: • the A ring of must be methylated at C2 and the C1-C2 alkene must be formed; • both C4 and C5 must be oxygenated; • the C6-C7 alkene must be formed; and • the ester attached to C6 must be reduced down to the corresponding alcohol. Additionally, the authors’ description of 2 leaves a little bit to be desired. It describes how the intermediate was prepared, but little else. To understand, the active reader has to look forward to Scheme 2 to see the intermediates 13/14 to see the photoaddition product and also back at the earlier work from the laboratory where the tandem photoaddition-fragmentation strategy was developed. Once the reader is familiar with the two-step sequence, intermediate 3 becomes a logical precursor for 2. The dioxenone unit in 3 can be simplified to the keto unit in 4 via a retro-carboxylation and enolization process. Reductive enolate formation and alkylation of 5 would lead to 4. The origins of enone 5 are not completely explained in the main text, but reference 6 states, “We thank Professor Phil Eaton (Univeristy of Chicago) for providing an unpublished procedure for the conversion 6 to 5.” Compound 6 is the deoxygenated version of 5; it lacks Ingenol’s C3 hydroxy group. The note in reference 6 points the reader in the right direction, but it still requires work to understand where 6 itself came from. In this case, reference 4 leads the way. Tetrahedron 1968, 24, 553 (ref 4c) shows that 6 can be synthesized via elimination and acylation of lactone X or its corresponding hydroxy acid using polyphosphoric acid. The precursors were probably prepared from beta-keto ester Y and methyl/ethyl methacrylate based on the J. Org. Chem. 1999, 64, 3770 reference (4a).




fig1_JWParagraph 3: The forward synthesis of intermediate 16 from 5/6 depicted in Scheme 2 begins here. The paragraph is dedicated to one reaction in the synthesis: the reductive alkylation of 6 using methyl crotonate as the electrophile. In the reaction, three contiguous stereocenters are set including a quaternary center right in the middle. Two relative stereochemical relationships arise from the three centers. The authors deal with each separately. The cis relationship between the  bridgehead carbons in 7/8 is because the beta face of the enolate is less sterically hindered than the alpha face. This argument should probably conjure an image of a bis-envelope conformation of a cis-[3.3.0]bicyclooctane. The C10-C11 stereochemistry requires transition state structures to rationalize the diastereoselectivity. Figure 1 shows transition states, labeled A and B, and they are “called” in the paragraph; that is, the writing makes specific reference to the items in the figure by name or letter. This connection between text and figures is common and effective technique to facilitate reader understanding. Figure 1 uses arrows to draw attention to the interaction that disfavors TS B; it’s the C4 methine and the alpha hydrogen of crotonate (both TS have a gauche interaction between the electrophile and the enolate). This paragraph is a good example of using a figure to rationalize a key principle that supplements the reaction sequence presented in a scheme. A structure for the product of alkylation discussed in paragraph 3 is not actually shown in Scheme 2. Instead, the product of a two-step sequence, alkylation and silyl enol ether formation, is shown. When 6 was used as the starting material of the sequence, 7 was the product. The sequence yielded a mixture of diastereomers in a 14:1 a:b (C11 methyl) ratio.


Paragraph 4: If the same two-step sequence was conducted on the more highly functionalized enone 5, an unacceptably low yield and diastereoselectivity was obtained. The authors consequently elected to continue forward from compound 7. The paragraph continues on to describe the reactions that converted 7 first into ketone 9 and ultimately to dioxenone. Conversion of 9 to 10 included carboxylation of an in situ generated lithium enolate with Mander’s reagent. This reagent gave the beta keto (methyl) ester as the product. It is converted to the PMB ester in the proceeding step before dioxenone formation without explanation. We reasoned that the transesterification was necessary (Why else would they add a step?) and that it implicated the ester oxygen as a nucleophile in the dioxenone formation. In the case of a PMB ester, the resulting carbocation would be relatively stable especially when compared to a methyl carbocation. The paragraph ends with the formation of compound 10. This makes good sense because they have taken the starting material forward to an intermediate that secures the functionality (the dioxenone) of one half of the photocycloaddition reaction.

Paragraph 5:  The manuscript switches focus to the other end of the molecule that will participate in the upcoming photocycloaddition. They install a hydroxyl group onto C14 of 10 by an allylic oxidation to give compound 11. One unanswered question is why that couldn’t have been done earlier, perhaps before the sequence that formed dioxenone 10. The allylic oxidation is necessary to introduce functionality into this segment of the molecule that can be parlayed toward the cyclopropane unit of Ingenol. Photocycloaddition of 10 was low yielding so they refunctionalized to the corresponding allylic chloride. The chloride was considerably more efficient in the cycloaddition, providing 14 in 60% yield. Compound 14 is the first of the two showcase reactions that will ultimately deliver the carbon skeleton of Ingenol. The enthusiasm of the authors is apparent by the exclamation point used to finish the sentence, “…we were delighted to find that photocycloaddition of the derived allylic chloride 12 proceeded in 60% yield to give the desired photoadduct 14, accompanied by the C13 chloro-isomer (5:2 ratio)!” They are pleased with the reaction. In reference 8 they also promise to explain the origins of the C13 chloro isomer, which here goes without further comment.

Compound 14 is amongst the “perilous heights of an advanced intermediate” as has been stated about so many compounds in natural product synthesis. There is an abundance of functionality waiting to be unleashed en route to key intermediate 16. The cyclobutane, just formed in the cycloaddition, will be unraveled in the formation of the challenging inside-outside bicycle ring junction between rings B and C. The carboxylate will eventually become the C6 hydroxymethyl group. The C14 chloride will enable the cyclopropanation via the intermediacy of the alkene. Based on the group’s previous work and the enthusiasm about the success of the cycloaddition, they are confident that the fragmentation would be successful. They give the fragmentation a clause and then continue tidying up the structural elements until they arrive at 15. In the footnotes there is the comment on the C6 stereochemistry – remember that that gives an indication of the electron motion of the fragmentation.

Stylistically, it may have been nicer to highlight the two-step sequence by making them the sole subjects of one paragraph. Perhaps paragraph 4 could have simply ended with the synthesis of the chloride, 12. I would have relegated the cycloaddtion of 11 to the notes and then let paragraph 5 shine in the brilliance of the photocycloaddition-fragmentation sequence.

Paragraph 6: The key steps necessary for the synthesis of the carbon skeleton of Ingenol are now behind them. A dihalocyclopropanation of alkene in 15 is followed by bis-alkylation with methyl cuprate to give the landmark intermediate 16. This paragraph wraps up the presentation of Scheme 2. To get to this point in the synthesis, there have been 18 linear steps.

Comparing structure 2 and 16 shows that they are very similar. The main differences are the oxygenation at C3 and the redox state of the group attached to the C6 carbon. It could even seem that the rest of the synthesis is endgame.


Paragraph 7: The payment for using 6 in place of 5 has come due. In paragraph 7, the authors quickly move through a series of transformations that convert 16 to 22. The key player that enables these transformations is the hydroxymethyl group attached to C6. Oxidation of that group to the corresponding aldehyde allows sequential eliminations that create the diene in 22. The authors report flatly in this paragraph that the seven steps reported are “to introduce the A ring functionality present in ingenol”. They don’t put emphasis on it, but it’s logical to think that they’d have preferred to carry an oxygenated C3 up to this point and done only one or two steps to be in a much better position than they presently are. So it goes.

Paragraph 8: The alkenes have been introduced into 22 so they can be oxygenated. It must be a “controlled burn”, however. They first reduce the aldehyde attached at C6 and then do a dihydroxylation on the C5-C6 alkene. The regioselectively is ascribed to greater steric accessibility of this alkene relative to the C3-C4 alkene. Similarly, the beta selectivity is also due to the steric accessibility of reagents from the beta face according to the authors. Building a model is an obvious way to get a sense of the 3D shape of the molecule as well as the associated selectivities of reactions associated with intermediates along the way. Unfortunately it’s not always put into practice. It definitely counts as “active” reading and will help understanding.

Paragraph 9: This paragraph finishes up the B ring synthesis. Dihydroxylation of the C3-C4 alkene and protection of the secondary hydroxyl as the benzoate give 28 and set up the linkage of the two tertiary alcohols into cyclic sulfate, 29. Elimination of the cyclic sulfate to provide the C6-C7 alkene was based on previous work in a closely related system. The remainder is some typical plug-and-chug to get to structure 31. As the authors write at the end of the paragraph, “it remained only to introduce the requisite A ring functionality to complete the synthesis of Ingenol.”

Paragraph 10: Structurally the paragraphs in the manuscript are a little clunky. They seem to tease the subject of the proceeding paragraph at the end of a given paragraph and then jump straight into the details at the start of that next paragraph. I don’t know if it’s accidental or by design. The authors walk the reader through C4-C5 diol protection, oxidation, Pd(0) beta-keto ester formation, alkylation, decarboxylation/oxidation to give enone 35. Whew. Steppy? They follow with Luche reduction of the enone and deprotections to ultimately deliver the title compound, Ingenol 1. They remind the reader that this was a racemic synthesis by reporting that the material prepared by them was identical to an authentic sample except for optical rotation.

Paragraph 11: The wrap-up. The synthesis was 43 steps from 6 with an average yield of 80%. That’s an 0.0068% overall yield using their numbers. It would be nice to have the authors report that number themselves. They complete the paragraph with the highlights of the synthesis. The best of these highlights is the penultimate sentence, “The establishment of the C8/C10 trans intrabridgehead stereochemistry serves as a testament to the utility of the intramolecular dioxenone photoaddition-fragmentation approach to the synthesis of structurally and stereochemically complex natural products.” Agreed. This paper showcases that tandem approach to the carbon skeleton AND continues to complete the total synthesis of Ingenol 1.