Side Reactions in Peptide Synthesis and mitigation MCQs With Answer

Introduction: Side Reactions in Peptide Synthesis and mitigation MCQs With Answer is a focused quiz collection designed for M.Pharm students studying advanced peptide chemistry. This set highlights common side reactions encountered during both solution- and solid-phase peptide synthesis—such as racemization, aspartimide formation, diketopiperazine cyclization, O→N acyl shifts, and cysteine oxidation—and emphasizes practical mitigation strategies. Each question links mechanism to preventive measures: choice of coupling reagents, protecting-group strategies, additives (HOBt/HOAt), backbone protection, temperature control, and scavengers during cleavage. The objective is to deepen conceptual understanding and improve problem-solving skills required for designing robust peptide synthesis protocols in research and pharmaceutical development.

Q1. What is the primary mechanistic cause of racemization at the activated C-terminal amino acid during peptide coupling?

• Oxidation of the α-carbon by atmospheric oxygen
• Formation of an oxazolone intermediate or enolization of the activated species
• Base-catalyzed N-acyl transfer from backbone amide
• Intramolecular cyclization to diketopiperazine

Correct Answer: Formation of an oxazolone intermediate or enolization of the activated species

Q2. Which additive is most commonly used to suppress racemization during carbodiimide-mediated couplings?

• Triisopropylsilane (TIS)
• Hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azabenzotriazole (HOAt)
• Piperidine
• Diphenyl ether

Correct Answer: Hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azabenzotriazole (HOAt)

Q3. Which side reaction commonly occurs with sequences containing Asp-Gly or Asp-Ser under basic Fmoc deprotection conditions?

• Formation of diketopiperazine (DKP)
• Aspartimide formation leading to succinimide and subsequent rearrangements
• Oxidation of methionine to sulfoxide
• O-Acylation of serine side chain to form esters stable to cleavage

Correct Answer: Aspartimide formation leading to succinimide and subsequent rearrangements

Q4. What is an effective mitigation to prevent aspartimide formation during solid-phase peptide synthesis?

• Using stronger base than piperidine for Fmoc removal
• Temporary side-chain protection of Asp using bulky esters or use of pseudoproline/Backbone protecting groups
• Prolonged exposure to TFA during cleavage
• Replacing Asp with glutamic acid

Correct Answer: Temporary side-chain protection of Asp using bulky esters or use of pseudoproline/Backbone protecting groups

Q5. Diketopiperazine (DKP) formation is a common side reaction—what sequence context most favors its formation during SPPS?

• N-terminal Pro immediately followed by Ala
• Free N-terminal dipeptide (usually Xaa–Pro or Xaa–Ala) on the resin during elongation
• Long hydrophobic stretches of residues
• Presence of aspartic acid at the C-terminus

Correct Answer: Free N-terminal dipeptide (usually Xaa–Pro or Xaa–Ala) on the resin during elongation

Q6. Which strategy reduces DKP formation during manual elongation steps in SPPS?

• Delay capping after coupling to allow side reactions
• Use of pre-activation with short-lived activated species and rapid coupling; minimize time that N-terminal dipeptide is free
• Lowering the resin loading does not affect DKP
• Replacing Fmoc with Boc chemistry without other changes

Correct Answer: Use of pre-activation with short-lived activated species and rapid coupling; minimize time that N-terminal dipeptide is free

Q7. Carbodiimide couplings (e.g., DIC, EDC) can produce N-acylurea byproducts. What procedural change minimizes N-acylurea formation?

• Omitting any additive and using high temperature
• Adding HOBt/HOAt to intercept O-acylisourea and form less reactive active esters
• Prolonging the reaction time to full consumption of reagents
• Replacing carbodiimide with elemental sulfur

Correct Answer: Adding HOBt/HOAt to intercept O-acylisourea and form less reactive active esters

Q8. During Fmoc deprotection, prolonged exposure to 20% piperidine can cause which of the following side reactions?

• Epimerization only at the C-terminal residue
• Asparagine dehydration to form succinimide
• β-Elimination of serine/threonine side chains and base-catalyzed aspartimide formation
• Sulfoxide formation of methionine

Correct Answer: β-Elimination of serine/threonine side chains and base-catalyzed aspartimide formation

Q9. What protective group strategy can be employed to prevent O-acyl migration from serine or threonine side chains?

• Use of temporary allyl or trityl protection on the hydroxyl side chain
• Leaving the hydroxyl unprotected and performing strong base deprotection
• Using Boc chemistry with neat TFA for Fmoc replacement
• Oxidizing the side chain prior to coupling

Correct Answer: Use of temporary allyl or trityl protection on the hydroxyl side chain

Q10. Cysteine residues are prone to oxidation during peptide synthesis and handling. Which reagent is typically used to prevent oxidation on-resin or in crude peptides?

• Hydrogen peroxide
• Tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) as reducing agents; and thiol scavengers (EDT) during cleavage
• Sodium chlorite
• Sodium azide

Correct Answer: Tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) as reducing agents; and thiol scavengers (EDT) during cleavage

Q11. Nucleophilic aromatic substitution of activated side chains is rare, but which protecting group on lysine is most suitable to avoid undesired N-acylation during harsh activation?

• Alloc (allyloxycarbonyl) for orthogonal removal
• Boc without additional precautions
• Unprotected lysine
• Trimethylsilyl (TMS) protection

Correct Answer: Alloc (allyloxycarbonyl) for orthogonal removal

Q12. What is the main advantage of using uronium-type coupling reagents (e.g., HATU, HBTU) over simple carbodiimides for difficult couplings?

• They always eliminate the need for any additive
• They form more reactive and less racemization-prone activated species, improving yields and reducing byproducts
• They convert peptides to peptidomimetics spontaneously
• They are inert to nucleophilic side chains, so no protection is needed

Correct Answer: They form more reactive and less racemization-prone activated species, improving yields and reducing byproducts

Q13. Which cleavage cocktail additive helps scavenge carbocations formed during TFA cleavage to prevent side-chain alkylation?

• Triisopropylsilane (TIS) or ethanedithiol (EDT) as scavengers
• Sodium hydroxide
• Hydrogen peroxide
• Piperidine

Correct Answer: Triisopropylsilane (TIS) or ethanedithiol (EDT) as scavengers

Q14. O-to-N acyl shift (O-acylation of serine/threonine followed by migration) can produce sequence errors. Which practice reduces this risk?

• Using very long coupling times with excess base
• Protecting the hydroxyl group during coupling and using mild activation methods
• Performing couplings at elevated temperatures to speed reaction
• Adding oxidants to the coupling mixture

Correct Answer: Protecting the hydroxyl group during coupling and using mild activation methods

Q15. During solution-phase peptide coupling, pre-activation of the carboxyl component for extended periods can increase what side reaction?

• Racemization via formation of oxazolone or activated enolates
• Complete protection of all side chains
• Formation of stable amide bonds without side products
• Selective N-methylation of the amide nitrogen

Correct Answer: Racemization via formation of oxazolone or activated enolates

Q16. Which backbone-protecting strategy is used to prevent aggregation and mitigate difficult couplings or aspartimide formation?

• Use of pseudoproline dipeptides or backbone amide protection (e.g., Boc-ψPro)
• Leaving the backbone fully unprotected to enhance solubility
• Performing all couplings at pH > 12
• Using metallic salts to cross-link the resin

Correct Answer: Use of pseudoproline dipeptides or backbone amide protection (e.g., Boc-ψPro)

Q17. Formation of peptide-resin cleavage artifacts (e.g., t-butylated side chains) occurs when inadequate scavengers are present. Which change in cleavage cocktail reduces such artifacts?

• Increasing the TFA concentration to 99.9% without scavengers
• Inclusion of appropriate scavengers like TIS, water, anisole, or EDT in the cleavage cocktail
• Using strong bases instead of acid for cleavage
• Extending cleavage time to 72 hours at room temperature

Correct Answer: Inclusion of appropriate scavengers like TIS, water, anisole, or EDT in the cleavage cocktail

Q18. When using EDC in aqueous peptide couplings (e.g., for conjugation), what side reaction is a particular concern and how is it prevented?

• Formation of N-acylurea in water; prevented by adding NHS or sulfo-NHS to form stable active esters
• Oxidation of cysteine; prevented by oxygen bubbling
• Complete hydrolysis of peptide bonds; prevented by raising pH to 12
• Formation of azo compounds; prevented by adding sodium nitrite

Correct Answer: Formation of N-acylurea in water; prevented by adding NHS or sulfo-NHS to form stable active esters

Q19. Which analytical sign in crude peptide HPLC/MS suggests aspartimide formation has occurred?

• Single peak with expected mass only
• Additional peaks with masses corresponding to −18 Da (dehydration) or mass shifts consistent with succinimide intermediates
• Appearance of a single broad peak at very low retention time always
• Loss of UV absorbance at 214 nm only

Correct Answer: Additional peaks with masses corresponding to −18 Da (dehydration) or mass shifts consistent with succinimide intermediates

Q20. To minimize epimerization of His, Cys, and other sensitive residues, which combined approach is most effective?

• Use strong bases and long activation times to ensure full coupling
• Cold preactivation, rapid coupling, use of HOAt/HOBt or uronium reagents, and minimizing exposure to base
• Replace all sensitive residues with alanine
• Always perform couplings in protic solvents without additives

Correct Answer: Cold preactivation, rapid coupling, use of HOAt/HOBt or uronium reagents, and minimizing exposure to base

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