CatalyCEST

This week we'll be discussing the following recent JACs communication:

A CatalyCEST MRI Contrast Agent That Detects the Enzyme-Catalyzed Creation of a Covalent Bond

by

Dina Hingorani, Edward Randtke and Mark Pagel

http://dx.doi.org/10.1021/ja400254e

http://www.ncbi.nlm.nih.gov/pubmed/23601132

I am a sucker for NMR-based bioanalytical assays.  These experiments appeal to my desire to move NMR beyond its role in structural biology and into other arenas of biology.  Additionally, I have watched these CEST methods with a passing interest, but I have never had a chance to sink my teeth into this kind of work.

In this JACs communication Hingorani et al. describe a MRI contrast agent designed to detect catalysis by the enzyme transglutaminase (TGase).  The authors call this technique CataylCEST.  Here is a rough sketch of how it works.  The authors design a paramagnetic tag with a moiety that looks like the substrate to an enzyme.  (In this case the substrate is lysine and the enzyme is TGase).  Then the enzyme covalently attached the paramagnetic tag to a second substrate (in this case a protein or peptide via glutamine residues).  Once the tag is attached, protons (and 13C, 15N, etc) in the neighborhood to it broaden and resonate at dramatically different frequencies due to hyperfine contact shift.  Let's assume that some of these protons are in slow exchange with the water.  If you saturate these spins using a presat-type experiment, then during the saturation pulse, some of this saturation is transferred to the water by chemical exchange and the integral of the water signal will decrease relative to a control.  Figure 1A of the paper (below) describes this experiment:

 

The specific enzyme the author's query, TGase, catalyzes covalent bond formation between side chains of glutamine (Q) and lysine (K).  TGase forms cross-links in the extracellular matrix in tissues and in cancer.  The authors do not discuss their motivation for monitoring TGase in the introduction and only touch on it in the conclusion.  I can only surmise that their long-term goal is to develop in vivo CEST MRI with exogenous CEST agents that do not suffer from rapid pharmacokinetic washout.  

The results of this study - 

The author's synthesize TM-DO3A-cadaverine paramagnetic CEST agent and couple this molecule to 5 different substrates using inexpensive microbial TGase.  The substrates are: Boc-Gln-OH, CBZ-protected QG peptide, QR peptide, GQR peptide and bovine serum albumin (BSA).  They acquire their NMR data on a 600 MHz NMR at 37 C using (essentially) the presat experiment with a saturation time of 4 s and a field of 20 uT (or (20 uT/14.1 T)*600 MHz = 850 Hz field).  The saturation offset is arrayed in 1 ppm steps from -30 to 30 ppm, relative to water, which is set at 0 ppm.  The data is presented as a saturation profile showing the intensity of the water signal as a function of the saturation offset.  Finally, the authors fit Lorentzian line shapes to assess the chemical shift of the species in slow exchange with water.  The authors do not report error bars, which I find troubling, because the peaks are quite broad.  As we'll see later, the authors will argue that a difference of 1.8 ppm is significant, but a difference of 1 ppm is not.  I am not sure what to make of this difference, myself.

The most convincing result is with BSA.  The authors make an NMR sample with 25 mM CEST agent, 0.75 mM BSA and 10 mM glutathione (to maintain the reducing environment required by the eznyme) in pH 7 Tris buffer.  Then they react with 0.327 uM TGase for 24 h under aerobic conditions.  The authors repeat this experiment in triplicate.  Before catalysis, the saturation profile shows CEST at +4.6 ppm upfield from water.  After cataysis, the saturation profiles shows a CEST at +4.6 ppm and -9.2 ppm.

 

  

The interpretation is that the signal at +4.6 ppm is diamagnetic amines and amides in BSA.  TGase catalyzes the formation of a covalent bond between the CEST agent and Q side chains on BSA.  Hyperfine contact shift of Tm(III) induces an upfield shift in the chemical shift of the amides to -9.2 ppm.  (By the way - do you notice how much easier it is to look at the Lorentzian fits on the right than the saturation profiles on the left!)

If the publication ended right here, I'd be sold.  This paper confuses me, though, when the authors report their controls.  Their results reinforce a long held suspicion about CEST: how do you assign the peaks in your saturation profiles?

Let's discuss their controls.  The most logical control is each of the components individually.  The CEST agent alone shows a CEST at -5.2 ppm, BSA alone shows a CEST at +5.6 ppm and glutathione alone shows a CEST at +5.4 ppm.  By the way, I'll note that the text says that BSA and glutathione have a CEST at +4.6 ppm, but the figure caption says +5.6 and +5.4 ppm, respectively.  You see what I mean about error bars!  I guess 1 ppm is not significant.  So why isn't the CEST profile of the reaction mixture before catalysis equal to a superposition of the reactants?  (I think it probably is, if you correct for concentration, but the authors do not do this for the readers and we are left wondering).  Next the authors try control peptides.  The authors mix 25 mM CEST agent, 25 mM peptide, 10 mM glutathione in pH 7 Tris buffer with and without TGase.  For GQR and QR, there is a CEST at -9.0 ppm before catalysis and CESTs at +5.8 ppm and -10.8 ppm after.  (In the discussion, the authors assign the signals at -9 ppm and -10.8 ppm to a supramolecular adduct and paramagnetic amides, respectively, implying that they can tell a difference between signals the differ by 1.8 ppm.  See what I mean about error bars!)  For CBZ-protected QG, there is no CEST signal before catalysis and CESTs at 4.6 ppm, 9.8 ppm and 22.5 ppm after.  Finally for Boc-protected Q there is a CEST at +7.2 ppm before and a broad CEST between -10 ppm and -20 ppm after catalysis.  Why do none of the controls show the same signal before catalysis as BSA?  (Once again, I presume the issue is concentration, but the authors leave it up to their reader to discern).  Also, after covalent attachment of the CEST agent why does the CEST differ depending so dramatically for different substrates?    

The author's interpretation of their results falls into two categories (they don't make this explicit, I am interpreting).  1) Aggregation/Heterogeneity - There is a noncovalent supramolecular adduct or some type of conformational heterogeneity that dramatically alters the chemical shift of the water exchangeable protons.   This effect is responsible for CESTs at -5.2 for the CEST agent alone, at -9 ppm for GQR and QR peptides prior to TGase catalysis, at +4.6 ppm, +9.8 ppm and +22.5 ppm for the CEST agent-linked ZQG peptide and at -10 to -20 ppm for the CEST agent-linked Boc-Gln-OH.  2) Change in chemical exchange rate contants -  The authors assert that chemically modifying the substrate alters the rate constant and rates of chemical exchange.  This effect is responsible for the CEST at +5.8 ppm for GQR and QR peptides after TGase reaction.

As you can tell, I am skeptical of these explanation.  I am not saying the authors are incorrect.  They know a lot more about this subject than I do.  I only mean to say that I believe more justification is needed.  For example, there are a few additional controls the authors could do to validate their assignments.  If they are concerned about "noncovalent supramolecular adducts", why not reduce concentration or increase ionic strength to break up these interactions?  If they are concerned about hydrophobicity causing conformational heterogeneity or heterogeneous ligand conformations, then perhaps the peptides they are using are not good controls?  If they are concerned about rate constants, why not play with temperature or field?  

In the end, this paper DOES convince me of its main objective: the authors have a catalyCEST MRI contrast agent that can be used to detect the formation of a covalent bond in BSA mediated by TGase.  After studying this paper, though, I am concerned that the CEST varies from substrate to substrate - from -9.2 ppm for BSA to 22.5 for ZQG.  Let me end my critique with a question: If you wanted to check for TGase activity using a new protein and you were not sure if it was a substrate or if you wanted to test TGase activity in vivo, then what results do you expect from the CEST assay?  The fact that you do not know shows how far we have to go. 

Dissecting the stereocontrol ...

My blog post this week will critique the paper:

Dissecting the Stereocontrol Elements of a Catalytic Asymmetric Chloroactonization: Syn Addition Obviates Bridging Chloronium.

By

Yousefi, Ashtekar, Whitehead, Jackson and Borhan

http://pubs.acs.org/doi/abs/10.1021/ja4072145

http://www.ncbi.nlm.nih.gov/pubmed/24025085

Funny story about this JACs communication (JACs 2013 135, 14524): I was reading this paper while riding a stationary bike at my campus gym, when a certain faculty member approached (name withheld to protect the guilty).  He asked "What are you reading?".  I showed him the paper and he said "That looks brutal!"  I laughed.  This science in this paper is excellent, but it was a poor choice by me for a blog that focuses on NMR.  By the time I realized that the NMR methodology was not novel, I was too deep into the paper to pull back.  Nevertheless, I think this publication is worth discussing, because it presents an interesting solution to a difficult diastereospecific assignment problem.

As an exercise for myself, I'll see if I can briefly describe the problem and why this paper is relevant to people interested in NMR.  Then I'll show the author's data and interpretations.

Yousefi et al. are interested in the reaction below: an enantioselective halocyclizations of alkenes.

This type of chemistry is important as "a robust, versatile route a wide range of heterocycles."  The specific question that the authors address is the following:  Why is this reaction enantioselective?  To address this question, the author use NMR spectroscopy of isotopically labeled precursors to assess the mechanism.  Figure 1 describes the two proposed intermediates: path a - three-membered chlorenium ion intermediate; path b - carbocation intermediate.  SPOILER ALERT (this part is in the title): It is path b.

These two mechanism offer some predicable consequence.  If the reaction proceeds by path a, the "enantioselectivity would be controlled in parallel with the initial asymmetric chlorenium delivery, yielding anti addition".  If the reaction proceeds by path b, "the reaction's enantioselectivity would be determined at the (presumably catalyst controlled) ring-closing step."  If the author's can show syn addition, then path a can be ruled out.  The problem demonstrating syn addition is that "the chlorine resides on a nonstereogenic carbon with no record of its attach path on the alkene."  So it is not easy to determine the addition, unless you can somehow label the product!    

The authors synthesize a E-deuterated analog of the alkene.

Then they perform the halocyclization reaction and analyze the products by NMR.

At this point I'll note that the publication itself has ZERO NMR spectra in any figure.  So you'll have to dig through the SI to see any of their data.  Here is the spectrum of their products.

No integrals or peaks picked, but I see 2 methylenes from the lactone at 2.5 and 2.8 ppm and the 5 phenyl protons at 7.4 ppm.  At 3.75 ppm is the CH adjacent to the Cl.  To remind you, below is the expected molecule for the protonated starting material:

  
One relevant factor to consider is that their deuterated analog is not perfect.  They report 94:6  E:Z and 88% D-incorporation.  So they have a bit of protonated product in with their deuterated product.  Looking at this data I see a "roofed" non-first order CH2 from the protonated product (peaks at 3.815, 3.805 and 3.772 ppm with the final leg hidden under the big peak at 3.74 ppm) overlapping with two CHD diastereomers at 3.805 and 3.74 ppm. 

The author's interpretation is explained in the following figure from the SI:

   
You may be asking how they know that c is the R,R or S,S diastereomer and f is the R,S or S,R diastereomer?  This gets to the heart of the difficult problem in diastereospecific resonance assignment.  Let me quote directly from the paper: "The absolute stereochemistry of the CHDCl group in the major isolate was straightforwardly established via NOE analysis of epoxide 3-D obtained from the chemically transformed chloroactone product 2-D"

To translate, the deuteratd epoxide (3-D) has a peak at 2.98 ppm in 1D 1H NMR spectrum.  The protonated epoxide (3) has two peaks at 2.98 and 2.73 ppm.  Using NOE, the peak at 2.98 is established as trans the phenyl group.  (This data is nowhere to be found in the body of the paper or SI, by the way.)  Hence the deuteron in 3-D must be cis.  Retrosynthetic analysis can be used to establish the stereochemistry of the major product 2-D.

I'll leave it to the organic chemists to fight through their mechanistic arguments.  Their NMR argument is interesting to me, though.  Basically, the authors cannot complete diastereospecific assignment of 2-D using conventional approaches, so they modify the molecule in a stereochemically predictable manner to simplify the problem.  I find this approach to be clever.

Like I said at the beginning, this article is not for the faint of heart.  I don't know that I am really all that interested in mechanistic details of this reaction.  My interest is in the NMR.  Frankly, the authors do not wow me with their NMR, but I am intrigued by their solution to the diastereospecific assignment problem.