Blog on Hiatus from December 21 to January 2

As the holidays approach, the weblog Philosophy to Chemistry to Elucidation (P2C2E) will be taking a short break. Posts will resume in the New Year.

Happy Holidays everyone.

Blog on Hiatus from December 21 to January 2

As the holidays approach, the weblog Philosophy to Chemistry to Elucidation (P2C2E) will be taking a short break. Posts will resume in the New Year.

Happy Holidays everyone.

How to Interpret an HSQC-COSY Experiment

Where a COSY or TOCSY spectrum can be a challenge for a structure with severe spectral overlap, collecting an HSQC-TOCSY spectrum can be a better choice. An HSQC-TOCSY experiment stands for Heteronuclear Single Quantum Coherence-Total Correlation Spectroscopy and other variants include HMQC-TOCSY, HSQC-COSY, etc. Depending on the mixing time, the hybrid experiment generally offers information on both short-range and long-range coupled nuclei.

For the diol fragment below, 1H-13C HSQC correlations are expected for C-H atoms labeled 4, 5 and 9.

On the 1H-13C IDR-HSQC-COSY spectrum below, the three HSQC signals are phased negative (blue). (Note: the acronym IDR stands for Inverted Direct Response.) If a rectangle is draw connecting two HSQC signals, the COSY signals (phased positive) are located at the opposite corners of the rectangle. The spectrum shows a COSY correlation between protons 4 and 5, and between protons 4 and 9.

 

How to Interpret an HSQC-COSY Experiment

Where a COSY or TOCSY spectrum can be a challenge for a structure with severe spectral overlap, collecting an HSQC-TOCSY spectrum can be a better choice. An HSQC-TOCSY experiment stands for Heteronuclear Single Quantum Coherence-Total Correlation Spectroscopy and other variants include HMQC-TOCSY, HSQC-COSY, etc. Depending on the mixing time, the hybrid experiment generally offers information on both short-range and long-range coupled nuclei.

For the diol fragment below, 1H-13C HSQC correlations are expected for C-H atoms labeled 4, 5 and 9.

On the 1H-13C IDR-HSQC-COSY spectrum below, the three HSQC signals are phased negative (blue). (Note: the acronym IDR stands for Inverted Direct Response.) If a rectangle is draw connecting two HSQC signals, the COSY signals (phased positive) are located at the opposite corners of the rectangle. The spectrum shows a COSY correlation between protons 4 and 5, and between protons 4 and 9.

 

Teaching and Learning by Spectral Data … Part 2

Part 1 of the series Teaching and Learning by Spectral Data explored the difference between presenting an NMR problem set to a student in the form of an alphanumerical text or as an actual NMR spectrum. Continuing on the same problem set, another issue arises. Is the information on the elements and the 1H NMR spectrum adequate for deducing the unknown?

From the following 1H NMR spectrum, the following fragments can be deduced:

1. the multiplet at 7.24-7.57 ppm (m, 5H) indicates a mono-substituted benzene ring system,

2. the pairing of the J values and the integral information indicates a CH3-CH2 and a CH=CH (trans) fragments (tilting is also evident),

3. the chemical shift for the CH2 at 4.45 ppm indicates an adjacent oxygen atom,

4. the chemical shifts for the CH=CH fragment, 6.49 and 7.83 ppm, indicate an adjacent oxygen atom and/or benzene ring.

The elucidation cannot be completed without additional analytical or spectral data such as elemental analysis, MS, IR, 13C NMR, 2D NMR, etc.

Teaching and Learning by Spectral Data … Part 2

Part 1 of the series Teaching and Learning by Spectral Data explored the difference between presenting an NMR problem set to a student in the form of an alphanumerical text or as an actual NMR spectrum. Continuing on the same problem set, another issue arises. Is the information on the elements and the 1H NMR spectrum adequate for deducing the unknown?

From the following 1H NMR spectrum, the following fragments can be deduced:

1. the multiplet at 7.24-7.57 ppm (m, 5H) indicates a mono-substituted benzene ring system,

2. the pairing of the J values and the integral information indicates a CH3-CH2 and a CH=CH (trans) fragments (tilting is also evident),

3. the chemical shift for the CH2 at 4.45 ppm indicates an adjacent oxygen atom,

4. the chemical shifts for the CH=CH fragment, 6.49 and 7.83 ppm, indicate an adjacent oxygen atom and/or benzene ring.

The elucidation cannot be completed without additional analytical or spectral data such as elemental analysis, MS, IR, 13C NMR, 2D NMR, etc.

Teaching and Learning by Spectral Example ... Part 1

There are many ways to teach the process of elucidating unknown structures. Offering a student a visual guide, such as seeing firsthand a spectral dataset, can enhance the learning process and better equip the student on future work.

Presented below is a typical elucidation question from a university test. The numerical values have been extracted from a 1H NMR spectrum and the student is basically left to focus on elucidating the unknown.

If a 1H NMR spectrum is presented with the question, as shown below, the student is faced with the tasks of interpreting the spectrum and elucidating the unknown.

Although both approaches can serve a purpose, the latter approach enhances the experience of learning how to elucidate an unknown.

Teaching and Learning by Spectral Example ... Part 1

There are many ways to teach the process of elucidating unknown structures. Offering a student a visual guide, such as seeing firsthand a spectral dataset, can enhance the learning process and better equip the student on future work.

Presented below is a typical elucidation question from a university test. The numerical values have been extracted from a 1H NMR spectrum and the student is basically left to focus on elucidating the unknown.

If a 1H NMR spectrum is presented with the question, as shown below, the student is faced with the tasks of interpreting the spectrum and elucidating the unknown.

Although both approaches can serve a purpose, the latter approach enhances the experience of learning how to elucidate an unknown.

Verifying, Confirming, Making Sure … Part 2

In the constant pursuit of new pharmaceutical drugs, process chemists (sometimes referred to as medicinal or synthetic chemists) must investigate all impurities detected in a new drug manufacturing process. The chemist’s procedure is simple: identify, elucidate and synthesize each and every single impurity.

The chromatogram (UV detector set at 254 nm) below shows three peaks. The large peak at 5.51 min. is the active pharmaceutical ingredient (API). The two small peaks on either side of the API are impurities from the manufacturing process. Depending on the dose and potency of the drug substance, typical regulatory requirements for impurities mandate that any peak with a threshold greater than 0.1% be identified, elucidated and synthesized.

Verifying, Confirming, Making Sure … Part 2

In the constant pursuit of new pharmaceutical drugs, process chemists (sometimes referred to as medicinal or synthetic chemists) must investigate all impurities detected in a new drug manufacturing process. The chemist’s procedure is simple: identify, elucidate and synthesize each and every single impurity.

The chromatogram (UV detector set at 254 nm) below shows three peaks. The large peak at 5.51 min. is the active pharmaceutical ingredient (API). The two small peaks on either side of the API are impurities from the manufacturing process. Depending on the dose and potency of the drug substance, typical regulatory requirements for impurities mandate that any peak with a threshold greater than 0.1% be identified, elucidated and synthesized.

Verifying, Confirming, Making Sure ... Part 1

After a long and arduous attempt at an elucidation, it is quite common to be left with more than one candidate structure. In some cases collecting more data is not an option and an exhaustive database/literature search turns up nothing useful, the alternative approach is to synthesize the proposed candidates and then compare the spectral data to the original unknown.

The example below shows three proposed candidate structure differing in the attachment of the hexopyranoside group. The high degree of similarity between the candidates and the high number of quaternary carbons (11 per structure) makes it very difficult to narrow the list to one candidate.

When data is limited and thus cannot assist in eliminating two out of the three candidates, then the only option left is to synthesize the candidates and compare the spectral data.  

Verifying, Confirming, Making Sure ... Part 1

After a long and arduous attempt at an elucidation, it is quite common to be left with more than one candidate structure. In some cases collecting more data is not an option and an exhaustive database/literature search turns up nothing useful, the alternative approach is to synthesize the proposed candidates and then compare the spectral data to the original unknown.

The example below shows three proposed candidate structure differing in the attachment of the hexopyranoside group. The high degree of similarity between the candidates and the high number of quaternary carbons (11 per structure) makes it very difficult to narrow the list to one candidate.

When data is limited and thus cannot assist in eliminating two out of the three candidates, then the only option left is to synthesize the candidates and compare the spectral data.  

Using a Smaller Structure to Elucidate a Larger Structure

Datasets for large unknown compounds tend to be complicated and typically require a lot of effort in distinguishing one signal from another. Having some background information about the unknown, such as a metabolite, a precursor, a derivative, etc. can be invaluable with the detail work needed to get through an elucidation.

The 13C NMR spectrum below is for an unknown compound with 38 carbons. Several carbons in the spectrum are overlapping with each other, thus, making the interpretation difficult.

To facilitate the process of elucidating the unknown, the 13C NMR spectrum for the unknown (drawn in black) is compared to the 13C NMR spectrum for a pure sample of cholesterol (drawn in red). The signals that line up are part of the same cholesterol scaffold; in all ~25 signals match up. The remaining 13 signals belong to the unknown part that requires further work.

 

The structures for the unknown and cholesterol are shown below.

Using a Smaller Structure to Elucidate a Larger Structure

Datasets for large unknown compounds tend to be complicated and typically require a lot of effort in distinguishing one signal from another. Having some background information about the unknown, such as a metabolite, a precursor, a derivative, etc. can be invaluable with the detail work needed to get through an elucidation.

The 13C NMR spectrum below is for an unknown compound with 38 carbons. Several carbons in the spectrum are overlapping with each other, thus, making the interpretation difficult.

To facilitate the process of elucidating the unknown, the 13C NMR spectrum for the unknown (drawn in black) is compared to the 13C NMR spectrum for a pure sample of cholesterol (drawn in red). The signals that line up are part of the same cholesterol scaffold; in all ~25 signals match up. The remaining 13 signals belong to the unknown part that requires further work.

 

The structures for the unknown and cholesterol are shown below.

Re-evaluating the data from MS and NMR … Part 4

With any type of data, there is an inherent risk of misinterpretation. My advice to elucidators is to consider multiple solutions and examine each one thoroughly. In the end, the answer to any problem set lies in tying together the bits of information in hopes of understanding the bigger picture.

Recap of the problem: The ESI+ MS shows a single [M+H]+ at m/z 102 allowing a maximum carbon count of 8. The 13C NMR shows there to be 12 carbons. How can the data from the MS and NMR present such different results for the same unknown?

The data from both the 13C NMR and DEPT-135 spectra are consistent with a mixture of two similar compounds at approximately a 1:1 ratio.

A mixture with an ESI+ MS exhibiting a single molecular ion indicates that the compounds in the mixture differ by a proton. For example, the mixture comprises of one compound with an R-NH2 group and the other with an R-NH3+ group. Some example amine/aminium mixtures are shown below.

Re-evaluating the data from MS and NMR … Part 4

With any type of data, there is an inherent risk of misinterpretation. My advice to elucidators is to consider multiple solutions and examine each one thoroughly. In the end, the answer to any problem set lies in tying together the bits of information in hopes of understanding the bigger picture.

Recap of the problem: The ESI+ MS shows a single [M+H]+ at m/z 102 allowing a maximum carbon count of 8. The 13C NMR shows there to be 12 carbons. How can the data from the MS and NMR present such different results for the same unknown?

The data from both the 13C NMR and DEPT-135 spectra are consistent with a mixture of two similar compounds at approximately a 1:1 ratio.

A mixture with an ESI+ MS exhibiting a single molecular ion indicates that the compounds in the mixture differ by a proton. For example, the mixture comprises of one compound with an R-NH2 group and the other with an R-NH3+ group. Some example amine/aminium mixtures are shown below.

Re-evaluating the data from MS and NMR … Part 3

Pattern recognition is an integral part of the process of structure elucidation. The quicker the elucidator can pick up on the pattern, the faster the elucidation can be accomplished and the less time wasted in elucidating the unknown.

Recap of the problem: The ESI+ MS shows a single [M+H]+ at m/z 102 allowing a maximum carbon count of 8. The 13C NMR shows there to be 12 carbons. How can the data from the MS and NMR present such different results for the same unknown?

The 13C NMR below shows a unique pattern that is not obvious at first. Each 13C signal seems to be paired to a nearby signal of similar peak intensity. The pattern is also noticeable in the 13C DEPT-135 NMR spectrum below.

 

This pairing pattern indicates that the unknown is a mixture at approximately a 1:1 ratio (note: the option of rotamers is also possible but is not being considered for this example). Therefore, instead of 12 carbons for a single unknown compound as perceived earlier, there are 6 carbons per unknown. This interpretation falls in line with what is being suggested by the MS data.

The last piece of the puzzle is to determine why the MS data shows only one molecular ion peak?

Re-evaluating the data from MS and NMR … Part 3

Pattern recognition is an integral part of the process of structure elucidation. The quicker the elucidator can pick up on the pattern, the faster the elucidation can be accomplished and the less time wasted in elucidating the unknown.

Recap of the problem: The ESI+ MS shows a single [M+H]+ at m/z 102 allowing a maximum carbon count of 8. The 13C NMR shows there to be 12 carbons. How can the data from the MS and NMR present such different results for the same unknown?

The 13C NMR below shows a unique pattern that is not obvious at first. Each 13C signal seems to be paired to a nearby signal of similar peak intensity. The pattern is also noticeable in the 13C DEPT-135 NMR spectrum below.

 

This pairing pattern indicates that the unknown is a mixture at approximately a 1:1 ratio (note: the option of rotamers is also possible but is not being considered for this example). Therefore, instead of 12 carbons for a single unknown compound as perceived earlier, there are 6 carbons per unknown. This interpretation falls in line with what is being suggested by the MS data.

The last piece of the puzzle is to determine why the MS data shows only one molecular ion peak?

Re-evaluating the data from MS and NMR … Part 2

Whenever data appear to contradict each other, an instinctive reaction to this problem is to collect more data. Collecting more data can help to understand the problem and/or complicate the matter. Remember the model for Elucidation Evolution? Maximize data extraction (MDE) while minimizing data collection (MDC).

Recap of the problem: The ESI+ MS shows a single [M+H]+ at m/z 102 allowing a maximum carbon count of 8. The 13C NMR shows there to be 12 carbons. How can the data from the MS and NMR present such different results for the same unknown?

The 1H NMR spectrum below is complicated due to significant peak overlap. As such, it does not offer any further insight into the problem.

Is there any data (or a different interpretation) that can assist in deciphering what the unknown is and thus explain why the MS and NMR data appear to contradict each other?

Re-evaluating the data from MS and NMR … Part 2

Whenever data appear to contradict each other, an instinctive reaction to this problem is to collect more data. Collecting more data can help to understand the problem and/or complicate the matter. Remember the model for Elucidation Evolution? Maximize data extraction (MDE) while minimizing data collection (MDC).

Recap of the problem: The ESI+ MS shows a single [M+H]+ at m/z 102 allowing a maximum carbon count of 8. The 13C NMR shows there to be 12 carbons. How can the data from the MS and NMR present such different results for the same unknown?

The 1H NMR spectrum below is complicated due to significant peak overlap. As such, it does not offer any further insight into the problem.

Is there any data (or a different interpretation) that can assist in deciphering what the unknown is and thus explain why the MS and NMR data appear to contradict each other?

Re-evaluating the data from MS and NMR … Part 1

Structure elucidators will routinely use data from multiple techniques such as MS and NMR to build a proposed structure(s). When dealing with data from multiple techniques, the issue may arise that the data seem to contradict each other. In these cases, it is best to step back and re-evaluate the data from a different angle.

The ESI+ MS data below shows a prominent [M+H]+ ion at m/z 102 and its sodiated adduct. The maximum number of carbons possible for the ion is 8 (= 102 / 12). The 13C NMR spectrum below shows 12 carbons signals, all aliphatic and no quaternary carbons.

 

 

Assuming no issues with the instruments, how the data was acquired or how the sample was prepared, the lingering issue is how can the data from the MS and NMR present such different results for the unknown?

Re-evaluating the data from MS and NMR … Part 1

Structure elucidators will routinely use data from multiple techniques such as MS and NMR to build a proposed structure(s). When dealing with data from multiple techniques, the issue may arise that the data seem to contradict each other. In these cases, it is best to step back and re-evaluate the data from a different angle.

The ESI+ MS data below shows a prominent [M+H]+ ion at m/z 102 and its sodiated adduct. The maximum number of carbons possible for the ion is 8 (= 102 / 12). The 13C NMR spectrum below shows 12 carbons signals, all aliphatic and no quaternary carbons.

 

 

Assuming no issues with the instruments, how the data was acquired or how the sample was prepared, the lingering issue is how can the data from the MS and NMR present such different results for the unknown?

Stereochemistry Information from NOESY/ROESY data … Part 2

NOESY, ROESY, COSY and TOCSY are all 2D NMR experiments that sound so similar but offer different pieces of information about the puzzle. When interpreting the NMR data, it is important to understand how the nuclei interact with each other. For example, the presence of a cross peak (a correlation off the diagonal) on a COSY dataset is a result of nuclei coupling through a bond(s) whereas a NOESY dataset measures NOE’s (Nuclear Overhauser Effect) through space regardless of the number of bonds separating the nuclei. An NOE is typically observed for nuclei that are separated no farther than 5 Å apart.

For the enantiomers example shown below, the NOESY and COSY experiments differ in the presence or absence of the cross peaks. A clear difference between the two experiments is the information provided on the diastereotopic protons of the CH2 group.

The NOESY spectrum, also outlined in Part 1, shows 2 correlations at (4.29,1.28) and (4.29,3.13) ppm. There are no NOE's to the proton signal at 2.68 ppm. The DQF-COSY below shows two-bond and three-bond correlations at (4.29,3.13), (4.29,1.28) and (3.13,2.68) ppm. There are no four-bond correlations present as the 4J coupling constants are close to zero.

  

Stereochemistry Information from NOESY/ROESY data … Part 2

NOESY, ROESY, COSY and TOCSY are all 2D NMR experiments that sound so similar but offer different pieces of information about the puzzle. When interpreting the NMR data, it is important to understand how the nuclei interact with each other. For example, the presence of a cross peak (a correlation off the diagonal) on a COSY dataset is a result of nuclei coupling through a bond(s) whereas a NOESY dataset measures NOE’s (Nuclear Overhauser Effect) through space regardless of the number of bonds separating the nuclei. An NOE is typically observed for nuclei that are separated no farther than 5 Å apart.

For the enantiomers example shown below, the NOESY and COSY experiments differ in the presence or absence of the cross peaks. A clear difference between the two experiments is the information provided on the diastereotopic protons of the CH2 group.

The NOESY spectrum, also outlined in Part 1, shows 2 correlations at (4.29,1.28) and (4.29,3.13) ppm. There are no NOE's to the proton signal at 2.68 ppm. The DQF-COSY below shows two-bond and three-bond correlations at (4.29,3.13), (4.29,1.28) and (3.13,2.68) ppm. There are no four-bond correlations present as the 4J coupling constants are close to zero.

  

Blog on Hiatus from September 21 to October 2

Due to some business travel, the blog P2C2E will be on hiatus for 2 weeks from September 21 to October 2. Posts will resume the following week.

Stay tuned for Part 2 of the series Stereochemistry Information from NOESY/ROESY data.

Blog on Hiatus from September 21 to October 2

Due to some business travel, the blog P2C2E will be on hiatus for 2 weeks from September 21 to October 2. Posts will resume the following week.

Stay tuned for Part 2 of the series Stereochemistry Information from NOESY/ROESY data.

Stereochemistry Information from NOESY/ROESY data … Part 1

Several NMR experiments offer tools to help determine the stereochemistry of a structure. Some typical experiments are 1D NOE (Nuclear Overhauser Effect), 2D NOESY (NOE Spectroscopy) and ROESY (Rotating-frame Overhauser Effect Spectroscopy). These experiments will produce signals for nuclei that are close to each other through space independent of the number of bonds separating the nuclei.

A simplified 1H-1H NOESY spectrum is shown below. The spectrum shows 2 correlations at (4.29,1.28) and (4.29,3.13) ppm. There is no correlation to the proton signal at 2.68 ppm.

Based on NOESY data, there are 2 possible conformations. The enantiomers (partially drawn) are shown below.

Stereochemistry Information from NOESY/ROESY data … Part 1

Several NMR experiments offer tools to help determine the stereochemistry of a structure. Some typical experiments are 1D NOE (Nuclear Overhauser Effect), 2D NOESY (NOE Spectroscopy) and ROESY (Rotating-frame Overhauser Effect Spectroscopy). These experiments will produce signals for nuclei that are close to each other through space independent of the number of bonds separating the nuclei.

A simplified 1H-1H NOESY spectrum is shown below. The spectrum shows 2 correlations at (4.29,1.28) and (4.29,3.13) ppm. There is no correlation to the proton signal at 2.68 ppm.

Based on NOESY data, there are 2 possible conformations. The enantiomers (partially drawn) are shown below.

Determining the Site of Modification … Part 3

Tandem mass spectrometry involves the process of selecting and separating a product ion(s) (or daughter ion(s)) and fragmenting it in a second mass analyzer. This is commonly referred to as MS/MS or MS2. Additional tandem processes can be applied to ions in the MS/MS data to create MS3 data, and so forth.

The metabolites A and B share the same exact mass, and as such, cannot be differentiated by the MS data alone (see Part 1 of this series). The MS/MS data, described in Part 2, offers fragment information that can assist in eliminating one of the candidates. Taking it a step further and thus verifying the candidate metabolite A, ESI+ MS3 data is presented herein.

The fragmentation scheme below shows both the Parent and Metabolite A with the fragment at 121 Da, in bold, fragmenting to create a fragment at 93 Da. This is supported by the nearly-identical MS3 data displayed below.

 

On the right-hand side of the fragmentation scheme, the fragments 106 and 108 Da are expected for the MS3 data of the Parent and Metabolite A, respectively.

Determining the Site of Modification … Part 3

Tandem mass spectrometry involves the process of selecting and separating a product ion(s) (or daughter ion(s)) and fragmenting it in a second mass analyzer. This is commonly referred to as MS/MS or MS2. Additional tandem processes can be applied to ions in the MS/MS data to create MS3 data, and so forth.

The metabolites A and B share the same exact mass, and as such, cannot be differentiated by the MS data alone (see Part 1 of this series). The MS/MS data, described in Part 2, offers fragment information that can assist in eliminating one of the candidates. Taking it a step further and thus verifying the candidate metabolite A, ESI+ MS3 data is presented herein.

The fragmentation scheme below shows both the Parent and Metabolite A with the fragment at 121 Da, in bold, fragmenting to create a fragment at 93 Da. This is supported by the nearly-identical MS3 data displayed below.

 

On the right-hand side of the fragmentation scheme, the fragments 106 and 108 Da are expected for the MS3 data of the Parent and Metabolite A, respectively.