Solution Phase Peptide Strategies: segmental and sequential synthesis MCQs With Answer

Introduction:

Solution-phase peptide strategies are essential for assembling medium-to-large peptides and small proteins in M.Pharm practice. This quiz focuses on two main solution-based approaches: sequential (linear, stepwise) synthesis and segmental (fragment-based/convergent) synthesis. You will be tested on practical aspects such as coupling reagents, protecting-group strategies, chemoselective ligation methods (e.g., native chemical ligation, KAHA, sortase), methods to generate thioesters, handling solubility and aggregation, and strategies to minimize racemization and side reactions. These MCQs are designed to deepen your understanding of advanced peptide assembly techniques, troubleshooting, and selection of appropriate methods for complex therapeutic peptides and peptide-based research.

Q1. What best defines segmental (fragment-based) peptide synthesis?

• Repeating single-residue couplings in a stepwise manner
• Joining pre-synthesized peptide fragments to assemble larger peptides
• Enzymatic polymerization of amino acids
• Synthesis of peptides only on solid supports

Correct Answer: Joining pre-synthesized peptide fragments to assemble larger peptides

Q2. Which statement accurately describes sequential solution-phase peptide synthesis?

• Parallel synthesis of many peptide fragments simultaneously
• Assembly of peptides by stepwise addition of amino acids to a growing chain in solution
• Formation of peptides by fragment condensation only
• Cyclization of peptides using enzymatic ligation

Correct Answer: Assembly of peptides by stepwise addition of amino acids to a growing chain in solution

Q3. A principal advantage of segmental synthesis over long linear sequential synthesis is:

• It avoids the need for protecting groups entirely
• Improved overall yields for long sequences by assembling larger fragments
• It eliminates racemization during couplings
• It always uses enzymatic ligation exclusively

Correct Answer: Improved overall yields for long sequences by assembling larger fragments

Q4. What are the essential functional groups required for native chemical ligation (NCL)?

• N-terminal serine and C-terminal methyl ester
• N-terminal cysteine and C-terminal peptide thioester
• Two free N-terminal amines
• C-terminal carboxylic acid and N-terminal proline

Correct Answer: N-terminal cysteine and C-terminal peptide thioester

Q5. Which method is commonly used in solution-phase chemistry to prepare peptide C-terminal thioesters for NCL?

• Direct coupling with DCC to give a stable thioester
• Conversion of peptide hydrazides to thioesters (Dawson method)
• Direct oxidative formation from C-terminal alcohols
• Use of Fmoc deprotection to form thioesters

Correct Answer: Conversion of peptide hydrazides to thioesters (Dawson method)

Q6. What is the major drawback of long linear sequential solution-phase peptide synthesis?

• It always requires enzymes
• Cumulative loss of yield with each coupling leading to low overall yield for long peptides
• It prevents incorporation of modified amino acids
• It cannot use protecting groups

Correct Answer: Cumulative loss of yield with each coupling leading to low overall yield for long peptides

Q7. Which coupling reagent combination is preferred to minimize racemization in solution-phase peptide couplings?

• DCC alone
• EDC/HOAt
• Excess HCl
• Sodium hydroxide activation

Correct Answer: EDC/HOAt

Q8. Why are solubility-enhancing tags (e.g., PEGylated handles, temporary charged tags) used in fragment-based peptide synthesis?

• To permanently modify the final therapeutic peptide
• To increase sequence hydrophobicity for membrane insertion
• To prevent aggregation and facilitate purification and handling of large fragments
• To avoid the use of protecting groups

Correct Answer: To prevent aggregation and facilitate purification and handling of large fragments

Q9. In convergent (segmental) peptide synthesis versus purely linear assembly, a principal synthetic benefit is:

• Fewer purification steps overall because small molecules are eliminated
• Ability to test fragment folding independently and improve global yield by coupling larger, purified segments
• Requirement for no activating reagents
• Avoiding any risk of racemization

Correct Answer: Ability to test fragment folding independently and improve global yield by coupling larger, purified segments

Q10. What chemical sequence most commonly leads to aspartimide formation during peptide synthesis?

• Activation of Asp-containing sequences under basic or strongly activating conditions causing cyclization to a succinimide
• Acidic cleavage of t-Bu side chains only
• Use of EDC in presence of HOAt
• FTP coupling of proline residues

Correct Answer: Activation of Asp-containing sequences under basic or strongly activating conditions causing cyclization to a succinimide

Q11. Which pair of protecting groups is commonly used as orthogonal thiol protections for cysteine in solution-phase strategies?

• Boc and Fmoc
• Acm (acetamidomethyl) and Trt (trityl)
• Methyl and ethyl ethers
• t-Bu and benzyl on the backbone amide

Correct Answer: Acm (acetamidomethyl) and Trt (trityl)

Q12. What solvent system is typically chosen for native chemical ligation of peptide segments in solution-phase synthesis?

• Pure hexane
• Aqueous buffer containing chaotropic agent (e.g., 6 M guanidine·HCl) to maintain solubility
• Concentrated sulfuric acid
• Neat DMF at high temperature

Correct Answer: Aqueous buffer containing chaotropic agent (e.g., 6 M guanidine·HCl) to maintain solubility

Q13. The most reliable approach to form multiple disulfide bonds in a defined pattern in solution-phase peptide assembly is:

• Random air oxidation of all cysteines at once
• Stepwise deprotection of orthogonally protected cysteines followed by controlled oxidation
• Exposure to strong base to form disulfides
• Use of cyanogen bromide to cleave at methionine

Correct Answer: Stepwise deprotection of orthogonally protected cysteines followed by controlled oxidation

Q14. KAHA ligation (alpha-ketoacid-hydroxylamine) requires which pair of reactive termini on the fragments?

• C-terminal carboxamide and N-terminal proline
• C-terminal alpha-ketoacid and N-terminal hydroxylamine (or aminooxy) derivative
• Two free thiols only
• C-terminal methyl ester and N-terminal serine

Correct Answer: C-terminal alpha-ketoacid and N-terminal hydroxylamine (or aminooxy) derivative

Q15. What is the main synthetic benefit of incorporating pseudoproline dipeptides during long peptide assembly?

• They permanently cap the peptide chain
• They act as strong oxidants for disulfide formation
• They disrupt aggregation and secondary structure, improving coupling efficiency
• They eliminate the need for deprotection

Correct Answer: They disrupt aggregation and secondary structure, improving coupling efficiency

Q16. To minimize racemization during fragment condensation of sensitive residues (e.g., Cys, His), the recommended practice is to:

• Use strong bases and high temperature
• Employ HOAt/Oxyma-containing activation systems, minimize preactivation time, and keep reactions cold
• Always pre-oxidize cysteines before coupling
• Use excess acid during coupling

Correct Answer: Employ HOAt/Oxyma-containing activation systems, minimize preactivation time, and keep reactions cold

Q17. Segmental isotopic labeling in solution-phase peptide assembly is mainly used to:

• Increase peptide hydrophobicity for membrane studies
• Simplify NMR and MS analysis by introducing isotopes into specific segments of a large protein
• Prevent racemization during couplings
• Speed up enzymatic digestion reactions

Correct Answer: Simplify NMR and MS analysis by introducing isotopes into specific segments of a large protein

Q18. Sortase-mediated ligation requires which recognition motif on the donor peptide?

• LPXTG motif at the C-terminus of the donor and an N-terminal glycine on the acceptor
• A single proline residue anywhere in the sequence
• A C-terminal thioester and an N-terminal cysteine
• An N-terminal alpha-ketoacid only

Correct Answer: LPXTG motif at the C-terminus of the donor and an N-terminal glycine on the acceptor

Q19. A frequent practical limitation encountered during solution-phase fragment condensation is:

• The impossibility of using protecting groups
• Solubility and purification difficulties of large fragments leading to low isolated yields
• Complete elimination of side reactions
• Inability to use coupling reagents like EDC or HATU

Correct Answer: Solubility and purification difficulties of large fragments leading to low isolated yields

Q20. Which strategy is often most effective for head-to-tail cyclization of peptides in solution-phase to favor intramolecular reaction?

• High-concentration coupling with excess reagent
• Thioester-mediated intramolecular native chemical ligation under high-dilution conditions
• Oxidative cleavage of side chains to induce cyclization
• Adding metal catalysts to condense multiple molecules together

Correct Answer: Thioester-mediated intramolecular native chemical ligation under high-dilution conditions

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