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Oxandrolone Wikipedia

In a typical photoredox‐catalyzed transformation the "redox partner" is simply the molecule (or molecules) that takes part in the single‑electron transfer (SET) step with the excited photocatalyst – i.e., it is the species that donates an electron to, or accepts one from, the photoexcited catalyst.




What it means in practice





Excitation of the catalyst


The photocatalyst (often a Ru(II) or Ir(III) polypyridyl complex, a Ru(bpy)₃²⁺ derivative, an organic dye such as eosin Y, or a semiconductor like TiO₂) is irradiated. In its excited state it becomes either a stronger oxidant (if it accepts an electron) or a stronger reductant (if it donates an electron).



Electron transfer step


The substrate that will be transformed—the reactive partner in the reaction—is the molecule that receives (or gives) the electron from the excited photocatalyst. This is the "reactive partner" mentioned. It could be a simple alkene, a tertiary amine, an aryl halide, a nitrile, etc.

For example:
- Oxidative quenching: The excited photocatalyst (e.g., Ir(III)) donates an electron to a substrate such as a tertiary amine. The amine becomes oxidized (radical cation), while the catalyst is reduced to Ir(II). This radical cation can then undergo β-scission or deprotonation, leading to a neutral radical that participates in further transformations.
- Reductive quenching: A substrate such as an aryl halide accepts an electron from the excited photocatalyst (after it has been oxidatively quenched by another species). The resulting aryl radical can then add to alkenes or undergo other reactions.





Propagation


In the example of β-scission following amine oxidation, the neutral radical formed after proton abstraction can be trapped by a Michael acceptor such as methyl vinyl ketone (MVK). This leads to an addition product that contains the newly introduced alkyl group and sets up a new radical center at the terminal position. That radical can then undergo further steps:
- It may add again to MVK, extending the chain; or
- It may abstract a hydrogen from a suitable donor (e.g., another amine), thereby propagating the chain reaction.





Termination


Termination occurs when two radicals combine (radical–radical recombination) or when a radical reacts with an electron acceptor, effectively removing reactive intermediates from the system.


2.3 The Role of Electron Transfer and Radical Generation


The key to this strategy lies in leveraging electron transfer between the photoexcited molecule and its environment to generate radicals. In photosystem II, light absorption leads to a charge separation that ultimately results in a highly oxidizing state capable of extracting electrons from water (the "oxidative side"). In our synthetic analogue, the photoexcited state can either donate an electron to an acceptor or withdraw one from a donor, depending on the redox potentials involved. By selecting appropriate donors/acceptors—e.g., organic dyes with suitable HOMO/LUMO levels—we can direct the flow of electrons and thereby generate specific radicals (e.g., phenoxy radicals, radical cations).



Furthermore, by embedding the photoactive center within a supramolecular scaffold that contains reaction sites for oligomerization or polymerization, we can spatially confine the generated radicals. This confinement reduces recombination and enhances selectivity, mirroring the enzyme’s active site architecture.




4.2 Extending to Artificial Photosynthetic Systems


Beyond synthetic chemistry, these principles can inform artificial photosynthesis. For example, in designing a system that captures sunlight and converts it into chemical fuels (e.g., hydrogen from water splitting), one could engineer a photoactive unit coupled with catalytic centers for proton reduction or oxygen evolution. The key is to ensure efficient charge separation, rapid transfer of electrons/holes to the catalytic sites, and minimization of recombination.



In such a design:





Light Absorption: A chromophore (e.g., a porphyrin or quantum dot) absorbs photons, generating excited states.


Charge Transfer: Electrons are transferred from the excited chromophore to an electron acceptor; holes remain on the chromophore or are passed to a hole acceptor.


Catalytic Reaction: Electrons reduce protons at a metal catalytic site (e.g., platinum) forming hydrogen; holes oxidize water at another catalytic site (e.g., iridium oxide).



The spatial separation of electron and hole pathways, analogous to the enzyme’s active site geometry, ensures that each charge carrier participates in its respective reaction before recombining.





3. Comparative Summary



Parameter Enzymatic Catalysis Photocatalytic Systems


Active Site Structure Protein pocket (hydrophobic core, charged residues) Catalyst surface (semiconductor facets, co-catalysts)


Substrate Binding Mode Specific orientation via hydrogen bonds, hydrophobic contacts Adsorption via surface hydroxyls or dangling bonds


Transition State Stabilization Electrostatic field from active site residues Surface-induced polarization; ligand field effects


Reaction Coordinate 1D: bond breaking/formation in substrate (e.g., C–H) Multi-dimensional: electron transfer, proton movement


Driving Force Enzyme catalysis lowers activation energy External potentials or illumination provide driving force


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4. Hypothetical Scenario: Modifying an Active Site Residue


Case Study: Alanine-to-Serine Mutation in a C–H Activation Enzyme




(a) Expected Effects on Reaction Coordinate and Energy Landscape



Reaction Coordinate Shift: The hydrogen abstraction step typically involves a transition state where the H atom is partially transferred to the catalytic metal center. Introducing a serine hydroxyl group could form a new hydrogen bond with the substrate’s C–H bond, potentially stabilizing the transition state.


Energy Barrier Reduction: Stabilization of the TS may lower the activation energy (ΔG‡), shifting the reaction coordinate leftward (towards earlier TS) and steepening the potential energy surface near the TS.


Altered Kinetics: A lowered barrier translates to increased rate constants (k_cat). Conversely, if serine’s side chain sterically hinders substrate binding, the overall turnover may decrease.




Scenario B: Replacing a Cysteine with an Aspartic Acid Residue


Background: In enzymes like glutathione S-transferase (GST), cysteine residues often participate in forming disulfide bonds or serve as nucleophilic centers. Aspartic acid introduces a negatively charged side chain.





Effect on Disulfide Formation:


- Cysteine can form interchain disulfides essential for dimerization. Replacing it with aspartic acid removes the thiol group, abolishing disulfide formation.
- This could lead to monomeric GST lacking proper quaternary structure, potentially reducing catalytic efficiency.





Electrostatic Influence on Substrate Binding:


- Aspartic acid’s negative charge may repel negatively charged substrates (e.g., glutathione) or attract positively charged ones. If the active site requires neutral or positively charged residues for optimal binding, introducing a negative charge could hinder substrate access.



Structural Destabilization:


- Disulfide bonds often contribute to protein stability by crosslinking distant regions. Removing such a bond can increase conformational flexibility, potentially increasing entropy and reducing folding stability (ΔG_fold increases).





3. Comparative Assessment



Property Alanine (Ala) Valine (Val)


Side‑Chain Size Small, methyl Medium, isopropyl


Branching None One tertiary carbon


Hydrophobicity Moderate Strong


Flexibility High Lower due to bulk


Steric Hindrance Low Higher


Potential for Van der Waals Packing Limited Greater (more contacts)






Alanine is often used as a "minimalist" substitution: it preserves backbone geometry, reduces steric clashes, and can sometimes relieve strain. However, it may diminish hydrophobic core packing or disrupt side‑chain–side‑chain interactions critical for stability.



Valine provides more hydrophobic surface area and better van der Waals interactions with neighboring residues, potentially stabilizing the fold if the local environment tolerates increased bulk. Yet, it can also introduce steric clashes or destabilize regions that require flexibility.







3. Hypothetical Scenario: A Key Residue in a Different Protein



3.1 Contextualizing the Mutation


Consider a scenario where a functional residue—for example, a catalytic aspartate involved in enzyme activity—is replaced by valine (Asp → Val) in a different protein domain. This substitution could have distinct consequences depending on:





Protein Context: If Asp is part of an active site or substrate-binding pocket, replacing it with Val removes essential acidic chemistry and introduces hydrophobic bulk.


Structural Role: Asp may form salt bridges or coordinate metal ions; loss of these interactions can destabilize the fold.




3.2 Predicted Structural Consequences




Local Destabilization:


- Loss of electrostatic interaction leads to a void where Asp’s negative charge once contributed to stabilizing the local environment.
- Valine’s side chain may sterically clash with neighboring residues or displace water molecules, perturbing hydrogen bond networks.





Altered Dynamics:


- The region around the mutation becomes more flexible due to the loss of rigid interactions.
- Enhanced mobility can propagate as increased B-factors in X-ray structures.





Global Effects:


- If the mutated residue participates in a network of interactions spanning the protein, local destabilization could lead to subtle shifts in domain orientations or overall folding topology.
- In extreme cases (e.g., highly strained native conformations), such a mutation might tip the balance toward unfolding or misfolding pathways.



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4. Experimental Design: Probing Local vs Global Effects of Small Mutations



4.1 Overview


To validate the theoretical predictions, we propose an integrated experimental approach combining:





High-resolution X-ray crystallography (or cryo-EM for large complexes) to capture structural changes at atomic detail.


Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe local dynamics and solvent accessibility.


Thermal shift assays (TSA) to assess global stability changes.



The target system will be a well-characterized, monomeric protein of moderate size (~20 kDa) with a known crystal structure. We will introduce small mutations (e.g., alanine scanning of surface residues) that are predicted to either preserve or disrupt the structural network.


3.1 Experimental Plan



Step Technique Purpose


1 Express and purify wild-type protein Obtain high-quality sample


2 Site-directed mutagenesis (alanine scanning) Introduce small, conservative mutations


3 Verify mutants by mass spectrometry Confirm correct sequence


4 CD spectroscopy at 25°C Assess secondary structure content


5 Differential scanning fluorimetry (DSF) Determine thermal stability ΔTm


6 DSF with varying buffer conditions (ionic strength, pH) Evaluate effect of electrostatics


7 Limited proteolysis (trypsin, chymotrypsin) over time course Monitor susceptibility to digestion


8 Mass spec mapping of cleavage sites Identify protected vs. exposed regions


9 Compare with control (wild-type) data


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4. Expected Outcomes & Interpretation



Observation Likely Explanation


ΔTm ≈ 0 and normal digestion pattern Protein remains unfolded; no protective effect of N‑terminal tail.


ΔTm > 5 °C (e.g., +10–15 °C) and delayed proteolysis Protective stabilisation from the N‑terminal tail; indicates that the unstructured segment is sufficient to shield the core, perhaps via transient interactions or steric hindrance.


ΔTm slightly positive (~1–3 °C) but normal digestion Tail may confer modest stability, not enough to prevent unfolding or proteolysis.


ΔTm negative (destabilisation) and faster proteolysis Tail might interfere with folding, exposing the core.



4.2 Interpretation






If significant stabilisation is observed, this supports the hypothesis that unstructured protein segments can function as "protective shells," offering an alternative to chaperone-mediated folding.


The magnitude of ΔTm provides a quantitative measure of the protective effect and can be correlated with tail length or amino acid composition (e.g., presence of hydrophobic residues, proline-rich motifs).


Observing no stabilisation suggests that the unstructured segment does not influence folding under the experimental conditions; perhaps the core is sufficiently stable or the tail fails to interact properly.







5. Critical Evaluation of Methodological Limitations and Mitigation Strategies



5.1. Sensitivity to Experimental Conditions




Buffer Composition: Ionic strength, pH, and presence of denaturants can dramatically alter protein stability and folding pathways.


Temperature Effects: Thermally induced unfolding may differ from chemical denaturation; choose appropriate temperature ranges.



Mitigation: Perform systematic buffer optimization studies; include controls at multiple temperatures.


5.2. Data Interpretation Challenges




Multiple Transition Events: Overlapping unfolding transitions can obscure clear melting points.


Non-equilibrium Effects: Rapid scans may not allow equilibrium to be reached, leading to kinetic artifacts.



Mitigation: Use slow scan rates; apply global fitting models that account for multiple states.


5.3. Instrumentation Limitations




Baseline Drift: Long measurements can introduce drift affecting data quality.


Detector Sensitivity: Low absorbance or fluorescence signals may be below detection thresholds.



Mitigation*: Regular instrument calibration; use more sensitive detectors or signal amplification strategies.





Conclusion


By systematically varying the temperature in a controlled manner and monitoring optical changes, one can obtain detailed thermodynamic information about protein folding transitions. The choice of spectroscopic technique (absorbance vs. fluorescence) depends on the specific molecular features accessible to each method. Careful experimental design—including selection of appropriate wavelengths, calibration procedures, and data acquisition protocols—ensures accurate determination of equilibrium constants and related parameters. Awareness of potential pitfalls in instrumentation and data analysis allows for robust interpretation of thermal denaturation experiments. This comprehensive approach is essential for elucidating the energetic landscape governing protein stability and folding dynamics.