The document describes a study examining the swelling behavior of peptide resins during solid phase peptide synthesis using a swellographic instrument. The instrument continuously recorded the displacement of a movable piston caused by resin volume changes. Excellent linear correlations between swelling volume changes (ΔV) and nominal molecular weight changes (ΔM) of the growing peptide chain were observed for most peptides, indicating regular solvation of the peptide chain without aggregation. Deviations from linearity occurred for some aggregation-prone peptides. The results provide insight into peptide-resin interactions and swelling dynamics during solid phase peptide synthesis.

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2. Russian research group
http://www.ibch.ru/structure/groups/depprotpeptide
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Rodionov, I.L.; Peshenko, I.A.; Baidakova, L.K.; Ivanov, V.T. Swellographic study of peptide resin swelling behavior
during solid phase peptide synthesis. Int. J. Pept. Res. Ther. 2007, 13, 161-171. 10.1007/s10989-006-9061-0

3. Introduction
• Tracing the swelling trend.
• Analyzing swelling volume changes (∆V).
• Nominal molecular weight change (∆M) of the pendant peptide chain.
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4. Swellographic instrument
Movable piston
Electric
displacement
meter
Real-time recording
of piston
displacements
caused by the resin
volume changes.
Continuous-flow reactor
Batchwise Continuous-flow
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5. Swellographic record
H-Ser(Bzl)-Asp(OcHx)-OMPA-Nle-NHCH2-resin:
Boc-Asp(OcHx)-OMPA-NHCH2-resin
1. DMF, 2. 50% TFA/DCM, 3. 10% DIEA/DCM, 4.
DCM, 5. 50% tert-butanol/THF
Boc-SPPS Fmoc-SPPS
Deprotected Rink-Amide resin
Fmoc-Ala-OPFP
1. 50% tert-butanol/THF, 2. DMF, 3. THF
OMPA: oxymethylphenylacetic
OPSP: Pentafluorophenyl esters 5

6. 0.96 mmol/g
SPPS
17-mer peptide
Un-cleaved
Peptide (PR)
1.556 g
0.43 g 0.41 mmol
Scale*m.wt
0.41*3636.8
1.501 g
PR
1.556 g
PR – native resin
1.556 - 0.43
1.13 g (peptide)
mass/density
1.13/1.0
1.13 mL (peptide)
Vol DMF??
Overall vol – Vol peptide
2.0 – 1.13
0.87 mL
Swelling vol/mass
0.87/1.13
0.77 mL/g
0.77 mL (DMF) per g (peptide)
9.82 mmol (DMF) per 0.275
mmol (peptide)
36 DMF molecules for 17-mer
Peptide
2 DMF molecules per AA
1 2
The space within the swollen
bead potentially accessible for
further chain growth.
Avogadro’s no.
Overall vol =
Swollen PR-Dry R
6
Peptide: Packing Density
Resin: Bulk
measurement

7. 0.96 mmol/g
SPPS
17-mer peptide
Un-cleaved
Peptide (PR)
1.556 g
0.43 g 0.41 mmol
Scale*m.wt
0.41*3636.8
1.501 g
PR
1.556 g
PR – native resin
1.556 - 0.43
1.13 g (peptide)
mass/density
1.13/1.0
1.13 mL (peptide)
Vol DMF??
Overall vol – Vol peptide
2.0 – 1.13
0.87 mL
Swelling vol/mass
0.87/1.13
0.77 mL/g
0.77 mL (DMF) per g (peptide)
9.82 mmol (DMF) per 0.275
mmol (peptide)
36 DMF molecules for 17-mer
Peptide
2 DMF molecules per AA
1 2
The space within the swollen
bead potentially accessible for
further chain growth.
Avogadro’s no.
Overall vol =
Swollen PR-Dry R
7
Peptide: Packing Density
Resin: Bulk
measurement

8. Linear relationship
Swelling volume of the peptide-resin
vs.
Mass increment due to peptide incorporation
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9. Merrifield tetra-peptide LAGV-OMPA
• Regression analysis, good linear correlation (r = 0.99) of total PR
swollen volumes in DMF and total PR mass increase.
• Volumes of the PRs swollen in CH2Cl2 (DCM) during the growing
of the peptide chain showed a less significant linear correlation (r =
0.89) with PR mass increase.
• Volumes and masses of dry PR demonstrated an excellent linear
relationship (r = 0.98).
Sarin, V.K.; Kent, S.B.H.; R. B, Merrifield. Properties of swollen polymer networks. Solvation and swelling of
peptide-containing resins in solid-phase peptide synthesis. J. Am. Chem. SOC. 1980, 102, 5463-5470. 9

10. Linearity plot: Plane polypeptide model
• Random polymerization; 19 times.
• Regression analysis, good linear
correlation; (r = 0.98) of total swollen
volumes in THF and total mass
increase.
• Regression analysis, good linear
correlation; (r = 0.99) of total swollen
volumes in DMF and total mass
increase.
• PR mass
• Increment / correction of initial mass of
Lys(Z)-NCA for quantities of carbon
dioxide evolved and Lys(Z)-NCA
unreacted.• Drying of the whole resin after
each step!!!
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N-benzyloxycarbonyllysine N-
carboxyanhydride

11. (Leu)18-Phe-OMPA
Linearity plot: Octadeca-Leu model
Volumes vs. Corresponding amino acid residue
• ∆m = (n)(scale)(m.wt)
• n: reaction step
• Reaction proceeds with
quantitative yield / scale is
constant / mechanical loss
ignored.
• ∆V to ∆M.
OMPA: oxymethylphenylacetic
• Considering AA residues is not
suitable for ∆v to ∆m estimation
(different m.wt’s).
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12. CEKDKFEWSNTDNNRRDE, linear relationship r > 0.99 KKYKYKYPYGYKYKY, linear relationship r > 0.99
Linearity plots, cont..
Boc-SPPS Fmoc-SPPS
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13. Linearity plots, cont..
Boc, ACP65-74 decapeptide, linear relationship r > 0.99
PR swelling volume drop to the initial
level at the final step of ACP synthesis??!!
Not only de-solvation of peptide chains,
but also as partial de-solvation of the
polystyrene matrix itself
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14. Theoretical interpretation
• Excellent liner ∆V vs. ∆M correlations:
• V remains nearly constant throughout chain assembly.
• The growing peptide chain is solvated in the same regular manner.
• Aggregation; loss of solvent molecule/s; no volume gain is observed.
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Slide 6 or 7

15. • Excellent linear ∆V vs. ∆M correlations (r > 0.97) throughout SPPS for
peptide resins swollen in DMF appeared to be typical of >60% of
analyzed peptides.
• Regression analysis, good linear correlation (r > 0.97) in Boc-
SPPS / 50% TFA/DCM, even ACP.
• Good linear correlation (r > 0.97) in Fmoc or Boc-SPPS / DMF (4th
residue). No aggregation, Only external solvation.
• Peptides with no tendency to aggregate (0.94 < r < 0.99) / DMF.
• (0.5 < r < 0.8), DCM.
• Aggregation prone peptides, deviation from the linear relationship;
DMF, DCM, THF.
Conclusion
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16. References
1. Rodionov, I.L.; Peshenko, I.A.; Baidakova, L.K.; Ivanov, V.T. Swellographic study of peptide resin swelling
behavior during solid phase peptide synthesis. Int. J. Pept. Res. Ther. 2007, 13, 161-171. 10.1007/s10989-
006-9061-0
2. Sarin, V.K.; Kent, S.B.H.; R. B, Merrifield. Properties of swollen polymer networks. Solvation and swelling of
peptide-containing resins in solid-phase peptide synthesis. J. Am. Chem. SOC. 1980, 102, 5463-5470.
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