Solution-phase peptide synthesis (LPPS) predates SPPS and remains essential for industrial-scale manufacture, very short peptides, difficult sequences, and convergent fragment assembly. Unlike SPPS, the growing peptide is not tethered to a resin; each coupling/deprotection step is followed by workup and purification in solution, enabling exceptional control over side reactions and stereochemical integrity.

When to Choose Solution-Phase

• Large-scale production (grams → kilograms) where solvent economy and crystallization-based purifications are advantageous.

• Fragment condensation strategies (e.g., coupling two protected 10–20-mer segments).

• Sequences prone to on-resin aggregation or side reactions (e.g., Asp–Gly, Met oxidation, Cys side chemistry).

• Need for ultra-high purity via repeated crystallization rather than chromatography.

Protecting-Group Logic

Orthogonality is critical because temporary and permanent protections must be removable without affecting others.

• N-terminus (temporary): typically Boc (acid-labile) or Cbz (hydrogenolysis).

• C-terminus: start from activated esters, acid chlorides, or protected amino acid derivatives; final deprotection yields the free acid or amide.

• Side chains (permanent during assembly):

  • Lys(Nε-Z or Boc), Arg(Pbf/Tos), His(Trt), Asp/Glu(OBzl/OtBu), Ser/Thr(TBDMS or Ac), Tyr(OBzl/OtBu), Cys(Acm/Trt/Bn), Trp(Boc/For).

• Orthogonal sets: e.g., Boc on the α-amine with Acm on Cys (stable to TFA, removable by Hg(II)/I₂ or Pd), or Alloc protections removable by Pd^0 under neutral conditions.

Activation and Coupling Strategies

1. Carbodiimide Couplings

   • DIC, EDC (water-soluble), often with additives to suppress racemization and O→N acyl migration.

   • Additives: Oxyma (ethyl cyano(hydroxyimino)acetate), HOAt/HOBt (historical; handle with strict safety), DMAP/PPY catalytic bases.

2. Active Ester Methods

   • p-Nitrophenyl (ONp), succinimidyl (OSu/NHS), pentafluorophenyl (OPfp) esters: pre-form the active ester, then couple with the amine partner.

3. Acid Halides / Mixed Anhydrides

   • Acid chlorides (via SOCl₂, Ghosez reagent) or mixed anhydrides (isobutyl chloroformate).

   • High reactivity—use low temperature and short times to limit racemization (especially at Cys, His, Trp and α-stereocenters next to carbonyls).

4. Uronium/Phosphonium Salts in Solution

   • HATU, HBTU, PyBOP effective off-resin; combine with DIPEA/Collidine in DMF/CH₂Cl₂/MeCN.

5. Segment (Fragment) Coupling

   • Convergent assembly: A_n–OH + H–B_m → A_n–B_m.

   • Employ C-terminal thioesters/oxoesters and N-terminal unprotected amines with chemoselective ligations.

Convergent Assembly & Ligation

• Fragment condensation: couple protected peptide segments; deprotect globally at the end.

• Native Chemical Ligation (NCL) (often classed as semisynthesis but routinely used with solution fragments):

  • C-terminal thioester + N-terminal Cys-peptide → native amide bond after S→N acyl shift in water at pH ~7.

  • Compatible with aqueous buffers, enables long sequence assembly (100+ residues) by iterative ligations.

• KAHA ligation, Ser/Thr ligation, oxime/hydrazone ligations: alternatives when Cys is unavailable.

Deprotection Workflows

• Boc strategy: periodic TFA for N-deprotection; side chains protected with acid-stable groups removed at the end (stronger acid or HF/TFA cocktails depending on groups).

• Cbz strategy: hydrogenolysis (H₂/Pd-C) for N-deprotection under neutral conditions (excellent for acid-sensitive sequences).

• Orthogonal side-chain releases: Acm (Cys) with I₂/Ag⁺/Hg²⁺, Alloc with Pd^0 + soft nucleophile, TBDMS with fluoride.

Purification and Isolation

• Crystallization/trituration is a major advantage at scale (choose counter-ions/solvent pairs to induce selective crystallization: e.g., EtOAc/hexanes, MTBE/MeOH).

• Liquid–liquid extraction to remove coupling byproducts (ureas from carbodiimides, N-acylureas, excess reagents).

• Silica gel chromatography for protected intermediates;

• Reverse-phase HPLC (C18) for final deprotected peptide; gradient water/acetonitrile + 0.1% TFA/formate.

• Lyophilization yields TFA or acetate salts (counter-ion selection affects solubility and bioassay).

Typical Process Map

1. Design orthogonal protection scheme and fragment boundaries (minimize epimerization hotspots; avoid Asp–Gly cleavage sites at junctions).

2. Assemble dipeptides/oligopeptides (protected) by DIC/Oxyma or HATU.

3. Couple fragments (active ester/NCL) under dilute conditions to avoid oligomerization.

4. Global deprotection in acid or hydrogenolysis, with scavengers (e.g., anisole, thioanisole, EDT) to capture carbocations.

5. Workup → polish by RP-HPLC → lyophilize.

6. Characterize (HR-MS/LC-MS, ^1H/^13C NMR for protected intermediates, amino acid analysis, analytical HPLC).

Key Side Reactions & Mitigation

• Racemization at C-terminal residue during activation

  • Use mild activators, low temperature, and additives (Oxyma/HOAt).

• N-acylurea formation (carbodiimides)

  • Convert O-acylisoureas to active esters rapidly; include additives.

• Aspartimide formation (Asp-X sequences in base)

  • Protect Asp with OBn/OtBu; minimize base exposure; use pH-neutral couplings.

• β-elimination at Cys/Ser/Thr under strong base

  • Avoid prolonged base; use neutral coupling buffers.

• Oxidation of Met/Trp/Cys

  • Deoxygenate solvents, add antioxidants (e.g., ascorbate), handle under inert atmosphere for sensitive steps.

Safety & Green Chemistry Notes

• HOBt/HOAt (especially anhydrous forms) can be energetic/explosive when dry—prefer Oxyma and keep wetted if used.

• Prioritize EDC (water-soluble) and phase-split aqueous workups to reduce halogenated solvent use.

• Recover and recycle solvents (MeOH, MeCN) at scale; choose greener alternatives where feasible.

Comparing LPPS vs SPPS

Quality Control & Release Testing

• Identity: HR-MS/ESI-MS, exact mass; optional MS/MS mapping.

• Purity: Analytical RP-HPLC (single dominant peak; report area %).

• Content: Amino acid analysis (hydrolysate) or quantitative NMR with internal standard.

• Counter-ion / residuals: Ion chromatography (TFA/acetate), residual solvents (GC), metals (ICP-MS if Pd/Hg used).

• Solid form: Water content (Karl Fischer), polymorph/amorphous checks where relevant.

Practical Tips

• Choose fragment junctions at Ala/Leu/Phe rather than Asp/Gly/Asn.

• Use slightly excess amine partner to drive couplings; quench promptly.

• Seeded crystallization of protected intermediates dramatically improves throughput.

• Document pH, temperature, and time rigorously—they are the main levers on epimerization.

Conclusion

Solution-phase synthesis remains a workhorse of peptide chemistry, particularly for scale, difficult sequences, and convergent strategies. In modern workflows, labs often combine LPPS (for fragments) with SPPS (for segments) and chemoselective ligations to deliver long, complex peptides at high purity with industrial practicality.