Swellographic technique for SPPS

1. 1

2. Russian research group
http://www.ibch.ru/structure/groups/depprotpeptide
2
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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1 2
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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