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Do the phosphoester and glycosidic bonds of dNTPs increase thermal stability?
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+ Biochemistry
+ Chemistry
+ Stability
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Marcos Esteban
Do the phosphoester and glycosidic bonds of dNTPs increase thermal stability?
How wrong is my approach to model the stability of the whole dNTP by the stability of its pieces?
There are two classes of errors that such an approach introduces. First, it is possible that the pieces have different reactivity in the context of the complete molecule. Second, this approach does not consider breaking the molecule at the connections.
Do the connecting bonds stabilize the pieces?
The glycosidic bond prevents the ribose ring from opening via the usual linear/hemiacetal pathway. In the linear form, the aldehyde could be oxidized to a carboxylic acid or reduced to an alcohol. In the ring form, this is not possible. The triphosphate group on its own could lose the alpha or the gamma phosphate in the same way (it is a symmetric species with anhydride linkages on both ends). Once it forms an ester, this would change the reactivity of the anhydride linkage on the alpha carbon side. Once the nucleobase is connected to the sugar, the linking nitrogen no longer has a protonated and deprotonated form. This might also change some degradation reactions.
Are the connecting bonds part of degradation mechanism?
So, any mechanism that results in a product that (1) interferes with hydrogen bonding associations of the dNTP with its template or (2) prevents subsequent phosphodiester bond formation is fair game.
There are two types of degradation products that should be considered; those that the polymerase no longer incorporates into the growing strand (having those accumulate would make the syntheses a bit slower) and those that inhibit polymerization (even small concentrations of the latter could inhibit polymerization strongly).
What is the organic chemistry explanation (resonance?) for why the intact molecule would be more (or less) stable than its least stable constituent in a neutral-buffered aqueous solution at 95 °C?
The explanation would depend on the specifics (conditions during the PCR - pH, presence or absence of divalent cations, other additives, which nucleotide are we talking about). Resonance is not the core explanation of the effects because the linkages are pretty much single bonds. If we are just substituting -OH with -OR or -NH with -N-C..., there is no drastic effect on the aromatic systems and double bonds in the constituents.
How wrong is my approach to model the stability of the whole dNTP by the stability of its pieces?
There are two classes of errors that such an approach introduces. First, it is possible that the pieces have different reactivity in the context of the complete molecule. Second, this approach does not consider breaking the molecule at the connections.
Do the connecting bonds stabilize the pieces?
The glycosidic bond prevents the ribose ring from opening via the usual linear/hemiacetal pathway. In the linear form, the aldehyde could be oxidized to a carboxylic acid or reduced to an alcohol. In the ring form, this is not possible. The triphosphate group on its own could lose the alpha or the gamma phosphate in the same way (it is a symmetric species with anhydride linkages on both ends). Once it forms an ester, this would change the reactivity of the anhydride linkage on the alpha carbon side. Once the nucleobase is connected to the sugar, the linking nitrogen no longer has a protonated and deprotonated form. This might also change some degradation reactions.
Are the connecting bonds part of degradation mechanism?
So, any mechanism that results in a product that (1) interferes with hydrogen bonding associations of the dNTP with its template or (2) prevents subsequent phosphodiester bond formation is fair game.
There are two types of degradation products that should be considered; those that the polymerase no longer incorporates into the growing strand (having those accumulate would make the syntheses a bit slower) and those that inhibit polymerization (even small concentrations of the latter could inhibit polymerization strongly).
What is the organic chemistry explanation (resonance?) for why the intact molecule would be more (or less) stable than its least stable constituent in a neutral-buffered aqueous solution at 95 °C?
The explanation would depend on the specifics (conditions during the PCR - pH, presence or absence of divalent cations, other additives, which nucleotide are we talking about). Resonance is not the core explanation of the effects because the linkages are pretty much single bonds. If we are just substituting -OH with -OR or -NH with -N-C..., there is no drastic effect on the aromatic systems and double bonds in the constituents.
There are two classes of errors that such an approach introduces. First, it is possible that the pieces have different reactivity in the context of the complete molecule. Second, this approach does not consider breaking the molecule at the connections.
Do the connecting bonds stabilize the pieces?
The glycosidic bond prevents the ribose ring from opening via the usual linear/hemiacetal pathway. In the linear form, the aldehyde could be oxidized to a carboxylic acid or reduced to an alcohol. In the ring form, this is not possible. The triphosphate group on its own could lose the alpha or the gamma phosphate in the same way (it is a symmetric species with anhydride linkages on both ends). Once it forms an ester, this would change the reactivity of the anhydride linkage on the alpha carbon side. Once the nucleobase is connected to the sugar, the linking nitrogen no longer has a protonated and deprotonated form. This might also change some degradation reactions.
Are the connecting bonds part of degradation mechanism?
The glycosidic bond is subject to hydrolysis (see e.g. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC523769/pdf/nar00047-0143.pdf). Once the glycosidic bond is gone, the sugar will potentially also be degraded. The phosphoester bond is cleaved in vivo by enzymes; non-enzymatic hydrolysis is possible under harsh conditions (see e.g. http://www.tud.ttu.ee/im/Tonu.Reintamm/shabarova/4.5.html).
There are two types of degradation products that should be considered; those that the polymerase no longer incorporates into the growing strand (having those accumulate would make the syntheses a bit slower) and those that inhibit polymerization (even small concentrations of the latter could inhibit polymerization strongly).
The explanation would depend on the specifics (conditions during the PCR - pH, presence or absence of divalent cations, other additives, which nucleotide are we talking about). Resonance is not the core explanation of the effects because the linkages are pretty much single bonds. If we are just substituting -OH with -OR or -NH with -N-C..., there is no drastic effect on the aromatic systems and double bonds in the constituents.
There are two classes of errors that such an approach introduces. First, it is possible that the pieces have different reactivity in the context of the complete molecule. Second, this approach does not consider breaking the molecule at the connections.
Do the connecting bonds stabilize the pieces?
The glycosidic bond prevents the ribose ring from opening via the usual linear/hemiacetal pathway. In the linear form, the aldehyde could be oxidized to a carboxylic acid or reduced to an alcohol. In the ring form, this is not possible. The triphosphate group on its own could lose the alpha or the gamma phosphate in the same way (it is a symmetric species with anhydride linkages on both ends). Once it forms an ester, this would change the reactivity of the anhydride linkage on the alpha carbon side. Once the nucleobase is connected to the sugar, the linking nitrogen no longer has a protonated and deprotonated form. This might also change some degradation reactions.
Are the connecting bonds part of degradation mechanism?
The glycosidic bond is subject to hydrolysis (see e.g. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC523769/pdf/nar00047-0143.pdf). Once the glycosidic bond is gone, the sugar will potentially also be degraded. The phosphoester bond is cleaved in vivo by enzymes; non-enzymatic hydrolysis is possible under harsh conditions (see e.g. http://www.tud.ttu.ee/im/Tonu.Reintamm/shabarova/4.5.html).
There are two types of degradation products that should be considered; those that the polymerase no longer incorporates into the growing strand (having those accumulate would make the syntheses a bit slower) and those that inhibit polymerization (even small concentrations of the latter could inhibit polymerization strongly).
The explanation would depend on the specifics (conditions during the PCR - pH, presence or absence of divalent cations, other additives, which nucleotide are we talking about). Resonance is not the core explanation of the effects because the linkages are pretty much single bonds. If we are just substituting -OH with -OR or -NH with -N-C..., there is no drastic effect on the aromatic systems and double bonds in the constituents.
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