Analysis of a dynamic library enabled by 13C-labeling strategies

15 02 2012

Marta Dal Molin,  Giulio Gasparini,  Paolo Scrimin,  Federico Rastrelli and Leonard J. Prins
Chem. Commun., 2011, 47, 12476-12478; DOI: 10.1039/C1CC15295E

By Andrew James Surman

As we saw a few months ago, discussing the Würthner group’s review on self-sorting in non-covalent assemblies [Chem. Rev., 2011, 111, 5784-5814], development and use of increasingly complex supramolecular systems is significantly limited by a lack of tools to characterise and quantify them. HPLC is wonderful for well-behaved covalently bound systems, but where real-time measurements are required or interactions are too weak to withstand chromatography, this option isn’t available to us. While 1H NMR spectroscopy is vital during synthesis, and common for studying interactions in simple supramolecular systems, increasing complexity quickly leads to the overlap of resonances, hampering quantification of species and structural analysis. As a result, its use in complex synthetic systems relies on serendipitous resolution, or very careful (and rather limiting) design of systems to facilitate resolution. In our group we know that even mixing two components (a deoxyguanosine derivative and a salt) can lead to horribly complex forest of 1H resonances, where reasonable assignment requires a considerable amount of extra NMR experiments (time-consuming, inconvenient) and where reliable quantification of species can be impractical or impossible.

Despite these limitations, the authors of this week’s article believe that NMR is the answer: previously that have observed that NMR spectroscopy “is one of the few techniques that potentially allows direct identification and quantification of all species present in a solution as a function of time” [Chem. Comm., 2008, 3034-3036] and here add that “the limited use of NMR spectroscopy as an analytical tool within the context of systems chemistry is quite remarkable, considering that NMR is widely accepted as the tool for studying complex biomolecular structures.” They propose we learn from that field, and use isotopic enrichment of what would otherwise be low-abundance nuclei (13C, 15N) as a means to simplify spectra, and increase sensitivity for nuclei whose resonances are usually better-resolved than 1H. As a demonstration of the approach they apply it to an imine-type dynamic covalent library, closely related to several previously studied in the Scrimin & Prins groups (working towards studying mechanistic mimics of serine protease), setting themselves the task of characterising the contribution of spectator groups to the stability of a compound in the minimum number of experiments.

The complete library was formed from four separate aldehyde ‘scaffolds’ (P1-4), which are able to react with amines (A and A+) or hydrazides (H and H+) to form a range imines (PxA or PxA+) or hydrazides (PxH or PxH+). The aldehydes all bore negatively charged phosphonate groups, a transition state analogue for ester hydrolysis, except the control P1. The amines and hydrazines either bore a pendant quaternary amine (A+, H+), the postitive charge of which was previously shown to interact with phosphonates and bias product distribution, or phenyl groups (A, H) as a control. 13C labels were introduced during aldehyde formation by quenching of a lithiated (o-directing) phenol with carbonyl-13C-N,N-dimethylformamide.

Mixing all the components (P1-4, A and A+, H and H+) in MeOH resulted in a 1H spectrum in which many of the iminic resonances were overlapped in the 8 – 9.5 ppm range. In contrast, as a result of the 13C labelling, each of the 26 components of library (including hydrazone E/Z isomers) was manifested by a single (iminic carbon) resonance in a 1H-decoupled DEPT-90 spectrum (improved sensitivity over standard 13C), and all were resolved, allowing quantification down to 0.1 mM. These resonances were assigned through stepwise addition of components to build up the library, most could be quantified in a reasonably short experiment (the 18 best-resolved could be quantified in a 20 min, all 26 required 12h), and concentration down to 0.1 mM could be detected.

Despite the success of the labelling approach in allowing quantification of all 26 components, it was not possible to obtained thermodynamic stabilities of all the species from analysis of the mixture. Since the imines are far less stable, only stoichiometric amounts of hydrazides were used to allow coexistence of all 26 species (and show off the labelling approach), so ‘cross-talk’ between equilibria was possible (the authors have published on the limitations of dealing with these equilibria before) [Chem. Comm., 2007, 1, 1340-1342]. Instead, the hydrazone and imine libraries were analysed separately, and the energy scales correlates by competition experiments with P1-4A and P1-4H. Findings followed the main trends expected (hydrazones more stable than imines; electrostatic interactions of charged amines with phosphonates lending stability), however significant contributions from ‘spectator groups’ on the aldehydes were less predictable, and the authors are still working on a fuller treatment (treating hydrazone isomers separately).

This communication neatly proves a point which people in many other areas of chemistry might consider obvious (that isotopic labelling helps resolve spectra of complex systems), but which synthetic supramolecular chemists seem rarely to use. The spectra (Fig 2) speak for themselves, and it is seems a marked improvement over the 1H-13C HSQC method they proposed in previous work on these systems; I imagine that it would become more impressive as unlabelled spectra get more crowded. Nonetheless, I will be interested to see if they include an estimate of errors in the follow-up: while they note that DEPT-90 is fairly insensitive to variations in 1JCH values, I would be interested to see some data.




15 responses

15 02 2012
Ana Victoria

In this study it was shown that 13C-labelling permits the identification and quantification of a chemical system with up to 26 components. It was also determined how spectator groups affect the thermodynamic stability of hydrazones and imines in which an intramolecular interaction exists with a phosphonate and an ammonium group. For the study, all compounds P1-P4 contained a 13C labelled aldehyde group. This labelling provides a single characteristic resonance for each compound in the spectrum. This provides a 140-170 ppm range, a larger interval permits the study of mixtures of greater complexity. This was evidenced by all 26 components, which had overlapping signals in the H-NMR. Unfortunately, the thermodynamic stabilities weren’t obtained for all the species of the mixture. The hydrazones and imines were analyzed separately. The hydrazones were found to be more stable than the imines and he spectator groups contributed significantly, but more studies are going to be done.
This is a nice communication that is really useful. It is important to have a better way of analyzing complex systems. Andrew’s synopsis is really good, he points out the purpose, his own opinion, and he integrates past subjects that were discussed on previous group meetings. The picture basically represents the essence of the article.

15 02 2012
Nicole Zoé

Prins and colleagues present in this short but precise communication the advantages of using 13C-isotope for labelling a library of imines and hydrazones for more resolved and less complex NMR spectroscopy characterization. I have to say that the title itself caught my attention and high expectations towards the contents and results of the article emerged. They acknowledge the advantages and disadvantages of H1-NMR spectroscopy and consider the vast power and potential of NMR studies as an indispensable and needy analytical tool for complex structures and systems. Taking these details in account they are able to take a disadvantage of NMR studies and convert it into something useful and with high potential, such as 13C-isotope labelling. They mention the fact that the widespread chemically-shifted Larmor frequencies of 13C combined with broadband decoupling minimize the occurrence of signal overlap, and the fact that 13C is a endogenous label does not affect the thermodynamic stability or the structure of study. These reasons just mentioned promote the use of this type of labelling for the characterization of the systems previously mentioned and therefore present an alternative to common H1-NMR studies, which in many cases make difficult the identification of significant and important signals of a wide variety of systems studied nowadays. The overall results presented suggest the success of using 13C-isotope as a label for the identification of the imines and hydrazones in these studies. Although the use of this specific labelling allowed the quantification of all the species present and of interest, it failed in the interpretation of the thermostability of the systems. They adjudicate this problem to the fact the stoichiometric amounts of the hydrazides were required to allow the simultaneous coexistence of the imines, which were less stable thermodynamically.

I really enjoyed this communication and how the whole information and results were presented and organized. Something I really liked was the fact that they showed the importance of NMR studies and how they are essential for characterization of many synthetic systems. Andrew’s synopsis was really good, as well as the picture. I enjoyed the way he incorporated his points of view along the whole synopsis and a very complete reflection of the paper.

15 02 2012
Rocio Roque

In this article, Molini and other coworkers try to elucidate the importance of NMR when studying complex biomolecular structures. Specifically the use of low-abundance nuclei (more specific 13C-isotope ) which can simplify spectra and avoid resonance overlap. The overlap which tends to happen with the use of H NMR in very complex synthetic systems. They demonstrated their approach and showed that 13C-labelling allows the identification and the quantification of up to 26 components (imines and hydrazones).

Although I am not familiar with many concepts presented in the communication, I understood its central idea and what it was trying to convey. Overall I think the article was well written and well presented. In my case Andrew’s synopsis was very helpful, he was good at covering all the important aspects of the paper. As I have never studied or seen NMR, I look forward to understanding how they work. Very interesting!

15 02 2012
Luis Negrón

A potential tool for supramolecular chemistry and dynamics information for self-assemble materials is presented in this article of Prins, Rastrelli and co-workers. Basically, every organic chemist recognizes NMR spectroscopy of one of the main tools for the development of research in organic and inorganic systems. But every day new challenges emerge and other experimental techniques have to be used to surpass the limits of NMR. But what new features are incorporated in NMR experiments? Not only in expensive new technology but also more over in the development of novel techniques or strategies in NMR. In this article, 13C-isotope labeling of aldehyde groups are performed for the elucidation of dynamic libraries of hydrazones and imines mixtures. This technique is attractive because the iminic carbon is sensitive and can be measured effectively, signal overlap is not present and according with their calculations the thermodynamic stability of the labeled derivatives is not affected.

I was very impressed in how they can identify all the members of that library in those complexes mixture. I was looking if there was some 2D experiment to help in the assignment of those signals. Also, I don’t know is I don’t understand of there is some mistake in Figure 1 when they measure the free energy difference of 2.8 kJ/mol between P2A and P3A+ , I don’t know if is P2A+ instead P3A+. But more than that little detail, for me is very interesting the free energy difference of 4.3 kJ/mol between P3A/P3A+ and P4A/P4A+. It will be interesting a derivative with only one orto to the aldehyde to see the effect of that CH3. Because, I know that the –OCH3 affect the ring, but the solvent is CD3OD and I think that the present of that –CH3 will be more affected than the –OCH3 that can have a favorable affinity for the solvent.

It will be interesting the application of this technique in more complex systems, for example a macromolecule with more complex functional group that have to deal with stereochemical properties. Also, it will be interesting if some kinetic information can be obtained from these experiments. I think that this will be the key for the study of important interactions or the effect of those interactions in other supramolecules. About the procedure for this labeling, how suitable can be? How accessible is this tool for other different systems, in terms of sample preparation and elucidation of complex signals that tend to overlap or are concentration dependent.

About Andrew’s synopsis, I really enjoy to read it, because he explains the origin of the need of an application of C13 labeling to solve this kind of complex mixtures. I agree with him, that there are obvious methods in other fields that can be useful in supramolecular chemistry.

16 02 2012
Diana S.

In this communication the authors point to the use of NMR spectra of an isotopic enriched sample (specifically 13C atoms) to extract simple pieces of information from a sample to complex to be studied through regular proton NMR. The system that they choose is ideal for their purpose: the iminic carbon shows good sensitivity towards structural changes, and the entire system is composed of 26 different molecules, clearly indiscernible through 1H NMR.
Its true that supramolecular chemists do not ignore that isotopic labeling can greatly simplify NMR spectra of complex mixtures. This technique has been mostly used for the elucidation of macromolecule structures, -typically larger than supramolecular complexes- and require a combination of several nuclei and 1D/2D NMR spectra to acquire enough information for structural elucidation. The effectives of such a system has been shown time and time again in the multiple bio-macromolecule NMR structures published to date.
I agree that supramolecular chemists can learn from the experience of complex structural elucidation techniques, however the reason that isotopic labeling is used in the first place is because regular 1D/2D NMR alone is incapable of resolving such a large amount of signals. The simplicity of isotopically enriched 13C or 15N spectra is countered by the fact that you need to synthetically incorporate the isotope, which can be rather expensive. Also, the simplicity of the NMR spectrum comes at the price of less information; because proton-proton coupling, 2D spectra, and the large amount of protons in a supramolecule can also provide structural information of the assembly, whereas the single signal of an isotope may give information on the chemical environment, and be sensitive to changes in the supramolecular state, but will require the proton NMR for evidence of the structure. It’s a truly powerful technique to be used when required, and for what it is required. I am however discussing this considering a supramolecule, as AJS; if its more focused towards a dynamic library, in which case this technique is very suitable.
The blogger makes a very good discussion of the paper, incorporating comments and opinions that help appreciate an otherwise rather simple communication. The figure from the paper used as blog image is a very pictorial summary of what’s discussed in the paper.

16 02 2012

In this communication by Prins, Rastrelli and co-workers, present the study of 13C-isotope labeled aldehyde for the elucidation of dynamic libraries of hydrazones and imines mixtures. First a mixture of all aldehyde scaffolds, P1–P4, hydrazides, H and H+, and amines, A and A+, gave all 26 structures (including the isomers) whit well-resolved signals in the 13C NMR spectrum. To facilitate the assignment of each specie they increased the complexity of the mixture gradually. Competition experiments were used to quantitatively correlate the structure and thermodynamic stability of the imines and hydrazones. Going from P1 to P2 a stabilizing interaction between phosphonate and ammonium groups in P2A+ and P2H+ correlate to an increment in thermodynamic stability. This stability is further enhanced by the addition of a methyl group in the ortho-position to the phosphonate group, going from P2 to P3. On the contraire going from P3 to P4, a methoxy substituent meta-position to the phosphonate group, result in a destabilizing effect.

This type of studies has it advantages and disadvantages, as the authors very well argued the main advantage is that NMR is a non-invasive technique, that allowed the direct analysis of the mixture under equilibration conditions, that the signals are very sensitive to small structural changes and most of all is very available. The main disadvantages are cost and time consume of labeling the samples, since this could be more challenging depending on the synthetic approached. Also as the complexity of the system increased some signal overlap is to be expected.

I enjoy reading Andrew’s synopsis, he makes a really good job, better than the authors (which is understandable due to space limitations), explaining the need for the development of 13C labeling techniques for the study of complex mixtures.

16 02 2012

In this article Prins, Rastrelli and coworkers introduced a novel way of analyzing (quantitatively) a dynamic combinatorial library (DCL) containing up to 26 components without the need of chromatography. The DCL is constructed by the reaction of aldehydes with hydrazydes and amines. The reacting aldehydes carbons were 13C labeled to simplify the analysis of the NMR experiments, as shown in DEPT-90 of Fig. 2a and allowing to compare the thermodynamic stabilities of the components in a relative way. The electronic properties of this 13C are being changed by the formation of a new covalent bond, either a hydrazone or an imine which in part increases spectrum resolution. The acquisition times were moderate ranging from minutes to hours and the total concentration of the mixture still in the NMR scale (~25 mM). Although, NMR is a nondestructive, samples could be recover and kinetics could be time studied if the time resolution is improve.
I like this article for more than one reason: first I like thermodynamic synthesis and dynamic combinatorial chem has being one of my favorite subjects, since I wrote my first self-assembly proposal; second the article presents a methodology (aldehyde labeling, NMR experiments) that could be easily implemented in our lab (after the NMR compressor is fix); the narrative was good and the figures are clear.
In my opinion Andy’s synopsis was excellent. The articles main problem is explained using an analogy with JMR system, which makes it easier to understand.
In contrast the cartoon didn’t help much in the understanding of the communication.

16 02 2012

In this communication Prins and colleagues present how 13C-isotope labeling can be integrated to the use of NMR as a means to study complex chemical systems. They explain that in very complex mixtures, 1H-NMR can have serious overlaps, which make it impossible to identify each of the peaks. For this reason they are trying to incorporate isotope labeling, as a sophisticated tool for studying complex mixtures. They also mention the chemically-shifted Larmor frequencies of 13C can be combines with broadband decoupling so that the occurrence of signal overlap can be minimized. Also the 13C label doesn’t affect the thermodynamic stability of the system. In these studies they use a library of imines and hydrazones to show how this technique can be used to label up to 26 components. They also describe how spectator groups can influence in the thermodynamic stability of a their mixtures. However in these experiments they were not able to determine thermostability of their systems because, according to their analysis, the stoichiometric amounts of they hydrazides had to allow simultaneous coexistence of the imines, since the later was much less thermodynamically stable.

This was a very interesting communication, and the studies performed here seem to be a promising tool for NMR studies. It really is a challenge to study and characterize complex systems, and if scientist want to be able to mimic nature’s work, this could be helpful tool to try to achieve part of this goal. I really enjoyed Andrew’s synopsis, it kind of guides you through the whole article and he give useful information about other subjects described in previous group meetings.

16 02 2012

This communication presented by Prins and colleagues reported the used of 13C-isotope labeling to study a dynamic combinatorial library of compounds. The library includes hydrazones and imines, which are difficult to identify in a complex mixture by simple 1H NMR. This situation prompted the authors to label each of the former aldehydes with 13C-isotope, which allows them to facilitate the identification of the different resulting hydrazones and imines in the complex solution.
I do understand their point and their contribution, because as they mentioned NMR is a non-invasive technique and very helpful to study supramolecular self-assembled systems, including structural details and thermodynamic and kinetic stability/parameters. I also agreed that isotope labeling helps and facilitate the assignment and the study of many of these systems, but the cost effectiveness of this process still a challenge. The author made a very attractive introduction to backup their article, although for me was a little bit too ambitious by pretending to cover supramolecular systems in general. As many of you already mentioned the use of the technique such as isotope labeling contribute a lot to “simplify” NMR analysis, including 2D NMR experiments. This time the author showed how just 13C NMR is sufficient to identify a complex chemical mixture. I think this will be very helpful depending on the system and for a very complex self-assemble system the use of many other NMR experiment will be require. What they presented for this particular library is surprising, if this can be useful for artificial and even more complex supramolecular systems still a question for me. If supramolecular chemists are not using it too much, it is because the application can’t be so easy to implement and it is limited by the synthetic approach. I like the way Andy presents his synopsis I felt he was very impartial and made a good introduction, he gave the overall results and gave his opinion in an elegant manner. As Diana mentioned a figure from the article is always good to have an idea of the work that is going to be discussed.

16 02 2012

Without the appropriate tools only limited work can be done. In this article the authors used 13C-labelling to have better resolution on the identification of a very complex and dynamic mixture of species. Figure 2 reminds me of those many articles in which for the structural characterization of oligo-GQs, researchers have to do site-specific labeling to identify for example the imino signals within many overlap signals in 1H-NMR spectra. However, the use of labeled- oligo-GQs is expensive and helpful only for certain levels of complexity. In this article the use of enriched DMF is a relatively simple and handy strategy which goes along with the author’s main purpose of diminishing “the need for advanced instrumentation or sophisticated experimental protocols”. However even though in this case 13-labeling might not be ultimately significantly expensive its limitation would be the type of systems that can be enriched with the synthetic approach used by the authors. In general the article is a good example of a particular system in which 13C-labeling not only allowed them to characterize the complex mixture of species in solution but also their strategy didn’t empty their pockets so much.
Andrews synopsis was amazingly good since its introduction was engaging while also very instructive, he incorporate additional reference to help the reader, he include his personal criticism/insight and he even include the relevance that this article have regarding our own research. Regarding his figure, let’s say that a picture says more than a hundred words jajaja it was just very good since it summarize the major achievement of the article that then allowed the characterization of their combinatorial library of species.

16 02 2012

The use of simple NMR for complex molecules is very difficult to identify the peaks because of multiple overlapping. In this communication Prins et al. and Rastrelli et al. showed how useful of isotope labeled aldehyde for the elucidation of dynamic libraries of hydrazones and imines mixtures.
According to this communication, we can resolve the overlapping problem by incorporating isotopic atom of specific atom (that atom should be reason for complexity, like iminic carbon) and also it is helpful in quantitative and thermodynamic analysis.
But main flaw in this communication is insertion of isotope. It will be easy for one or two steps in synthesis. If in case we need to run steps more than three will be more expensive. In our case, we have to incorporate the carbonyl carbon of acetyl group of mAG before SUZUKI reaction, so to get final compound it will be about 6 steps, so we need to start at least 500 mg of SM it will be too expensive.
Really, I like this communication, it is very useful technique for supramolecular chemistry.

17 02 2012

With the advance of supramolecular chemistry the level of complexity of supramolecular systems has increased dramatically. Working with supramolecular systems implies complex solutions full of chemicals with varying functions and properties. NMR is the go-to tool that chemists use to analyze these mixtures. NMR is non-invasive so it does not affect the equilibrium of the systems under study. In this article the Prins Group present the facile characterization of a 26-member library of compounds. They do this by using 13C enrichment techniques. Since all the compounds are similar, their 1H-NMR spectra is highly convoluted. 13C enrichment however, enables them to differentiate and quantize compounds whose concentration can be up to 0.1 mM, which is very impressive.

Overall I think this is a very nice article that invites us, supramolecular chemists that there are tools to solve our characterization problems. Andrews synopsis is very good as it focuses on how this article might help us characterize our supramolecular structures. His image captured the essence of the article (which was that particular figure).

17 02 2012
Maxier A. S.

In this short paper of L. Prins et al, they work on simplifying NMR studies due to the complexity that self-assembled systems tend to show on 1H-NMR and 13C-NMR. They work on a library of bencilyc aldehydes which was used as scaffold for amines and hydeazides. They put together all of the components and this showed an overlapping of all the signals in the 1H-NMR, but in case of labeled 13C it exhibit a distinct resonance.
In general this paper was “easy to read” in terms of the general language that it uses but while presenting its library it can get a little bit confusing. But after their studies you then really get what was happening. The full purpose of their studies was not achieved so it can get kind of discouraging.
About Andrew’s synopsis I think it was really good so kudos and the picture seems appropriate.

17 02 2012
Maxier A. S.

I need to add that the discouragement comes from the fact that for me the experiments were very interesting and powerful, so its kind of sad that they couldn’t figure out the thermodynamic stabilities in the full mixture yet they worked around it.

17 02 2012

Prims et al. use 13C-labelling to identify compounds in a complex system of up to 26 components in the mixture. They said the study was aimed at determining how neighboring spectator groups affect the thermodynamic stability of their selection of hydrazones and imines. They decided to use NMR for this, since it is a non-invasive method, and were able to identify each component fairly quickly without the use of 2D correlation methods. However, not much can be said about the thermodynamic stability studies. They mention that the overall picture was more complicated in practice, and that there were some things they hadn’t taken into consideration.

I was kind of annoyed at the fact that they didn’t really do much with the thermodynamic stability studies, since it was kind if the draw at the beginning. I get it, their methods had limitations and it was still pretty impressive that they were able to differentiate between the compounds without the use of 2D correlation in such a complex mixture. It was also impressive that they were able to do so in such low concentrations. My main issue with how the article was written is that since they state that their aim was to obtain the relative thermodynamic stabilities, it’s kind of daunting that that didn’t exactly go anywhere. Overall, I think it was properly written and it’s a very decent article.
As for Andrew’s synopsis, I thought it was very well written, and the picture was ok.

Leave a Reply

Fill in your details below or click an icon to log in: Logo

You are commenting using your account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s

%d bloggers like this: