Peptide Chemistry: coupling reactions and strategies MCQs With Answer

Introduction

Peptide chemistry is central to M.Pharm training, especially for designing and synthesizing therapeutic peptides. This quiz collection focuses on coupling reactions and practical strategies used in both solid-phase and solution-phase peptide synthesis. Topics include coupling reagents (carbodiimides, uronium/phosphonium reagents), additives (HOBt, HOAt, Oxyma), mechanisms that cause racemization, protecting group strategies, and troubleshooting difficult sequences. These MCQs are designed to test conceptual understanding and practical decision-making required in laboratory peptide assembly. Each question highlights common side reactions, reagent selection, and methods to improve yield and stereochemical integrity—essential knowledge for advanced organic and medicinal chemistry in peptide drug development.

Q1. Which reagent is a carbodiimide commonly used for peptide bond formation by activating carboxyl groups?

• EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)
• HATU (O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate)
• PyBOP (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate)
• Oxyma (ethyl cyano(hydroxyimino)acetate)

Correct Answer: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)

Q2. Which additive is commonly used with carbodiimides to suppress O-acylurea formation and reduce racemization?

• HOBt (1-hydroxybenzotriazole)
• DMF (dimethylformamide)
• THF (tetrahydrofuran)
• TFA (trifluoroacetic acid)

Correct Answer: HOBt (1-hydroxybenzotriazole)

Q3. Which coupling reagent is preferred to minimize racemization and often used in difficult sequences due to fast activation and low epimerization?

• DCC (dicyclohexylcarbodiimide)
• HATU
• EDCI alone
• Acetic anhydride

Correct Answer: HATU

Q4. In solid-phase peptide synthesis using Fmoc strategy, which base is commonly used to remove the Fmoc protecting group?

• Piperidine
• Hydrazine
• Sodium hydroxide
• Trifluoroacetic acid

Correct Answer: Piperidine

Q5. Which side reaction results from intramolecular cyclization between the N-terminal amine and a C-terminal activated ester, leading to loss of product in short peptides?

• Diketopiperazine formation
• Aspartimide formation
• O-acylurea formation
• Epimerization

Correct Answer: Diketopiperazine formation

Q6. Which reagent pair is commonly used in peptide coupling that generates a highly reactive OAt ester to reduce racemization compared to HOBt?

• HBTU / HOBt
• HATU / HOAt
• EDC / NHS
• DCC / DMAP

Correct Answer: HATU / HOAt

Q7. In carbodiimide-mediated coupling, what is the key intermediate that can rearrange to an inert byproduct (O-acylurea) causing reduced yield?

• O-acylurea intermediate
• Acyl chloride intermediate
• Peptidyl thioester
• Isocyanate intermediate

Correct Answer: O-acylurea intermediate

Q8. Which of the following coupling additives is considered safer and often replaces HOBt due to explosion risk in its crystalline form?

• HOAt
• Oxyma Pure
• HBTU
• DIC

Correct Answer: Oxyma Pure

Q9. For on-resin coupling of sterically hindered residues such as Val–Pro, which strategy often improves coupling efficiency?

• Use of double coupling with stronger activator like HATU
• Reducing reaction time to avoid side reactions
• Using only carbodiimide without additive
• Perform coupling at 0 °C

Correct Answer: Use of double coupling with stronger activator like HATU

Q10. Native chemical ligation (NCL) is a method to join unprotected peptide segments. What functional group is required at the C-terminus of one peptide fragment?

• Thioester
• Methyl ester
• Tert-butyl ester
• Acid chloride

Correct Answer: Thioester

Q11. Which protecting group strategy is orthogonal and commonly used for temporary N-terminus protection in SPPS allowing mild base removal?

• Boc (tert-butyloxycarbonyl)
• Cbz (benzyloxycarbonyl)
• Fmoc (9-fluorenylmethoxycarbonyl)
• Trt (trityl)

Correct Answer: Fmoc (9-fluorenylmethoxycarbonyl)

Q12. Which side chain sequence is particularly prone to aspartimide formation during base-mediated deprotection steps?

• Sequences containing Asp-Gly or Asp-Ser
• Sequences containing Ala-Leu
• Sequences containing Val-Pro
• Sequences containing Lys-Arg

Correct Answer: Sequences containing Asp-Gly or Asp-Ser

Q13. Which reagent is a phosphonium-based coupling reagent known for efficient peptide couplings with low racemization?

• PyBOP
• DCC
• EDC
• HOBt

Correct Answer: PyBOP

Q14. Which solvent is most commonly used for Fmoc-SPPS due to good solubility of reagents and resin swelling?

• DMF (dimethylformamide)
• Water
• Hexane
• Acetone

Correct Answer: DMF (dimethylformamide)

Q15. What is the principal cause of racemization during peptide coupling at the activated carboxylate?

• Formation of an oxazolone intermediate
• Hydrolysis by water
• Cleavage of side-chain protecting groups
• Peptide aggregation on resin

Correct Answer: Formation of an oxazolone intermediate

Q16. Which approach reduces epimerization for coupling of sensitive amino acids like Cys and His?

• Using pre-activated esters with HOAt or Oxyma and rapid coupling
• Performing coupling at high temperature for longer time
• Excluding additives to avoid side reactions
• Using DCC without additive

Correct Answer: Using pre-activated esters with HOAt or Oxyma and rapid coupling

Q17. Which reagent combination forms an active ester in situ by carbodiimide activation followed by N-hydroxysuccinimide capture, commonly used in solution-phase peptide coupling?

• EDC / NHS
• HATU / HOAt
• PyBOP / HOBt
• DCC / HBTU

Correct Answer: EDC / NHS

Q18. In “preactivation” coupling strategy, what is typically done before adding the nucleophilic amino component?

• Carboxyl component is activated with coupling reagent to form an active ester
• Product peptide is precipitated
• Resin swelling is reversed
• Base is neutralized completely

Correct Answer: Carboxyl component is activated with coupling reagent to form an active ester

Q19. Which protective group for cysteine is commonly used to prevent disulfide formation during chain assembly and is removable under mild acidic conditions?

• Trt (trityl)
• Acetamidomethyl (Acm)
• Methyl (Me)
• Benzyl (Bn)

Correct Answer: Trt (trityl)

Q20. Which tactic is effective to minimize aggregation and improve coupling yield for long hydrophobic peptide sequences during SPPS?

• Use of pseudoproline dipeptides or backbone-protecting strategies
• Reducing solvent volume drastically
• Omitting agitation during coupling
• Using only the weakest activators to slow reaction

Correct Answer: Use of pseudoproline dipeptides or backbone-protecting strategies

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