2.2 Results
29
added to make pH value of the mixture stay at 2–3. The reaction was extracted
with ethyl acetate for three times. The organic phase was dried with anhydrous
magnesium sulfate. The final product S 6 was obtained after the purification of flash
chromatography (Hexane: EA = 5:1) (5.2 g, yield: 41%)
2.2.2 Solid Phase Peptide Synthesis and Thiol-Ene
Photoreactions
Peptides were synthesized on MBHA resin (loading capacity: 0.37 mmol/g) by standard Fmoc-based SPPS (Scheme 2.3). At first, the resin was swelled in NMP for
30 min. Then 50% (vol/vol) morpholine in NMP was used to deprotect the Fmoc
group on amine group for 30 min × 2. Next the resin was washed with DCM and
NMP alternatively for three time (3×1 min). In coupling process, for natural amino
acids, the Fmoc-protected amino acids (5.0 equiv), HCTU (4.9 equiv), DIPEA (10.0
equiv) were dissolved in NMP and mixed with resin for 2 h, followed by washing
with DCM and NMP for three times (3×1 min). For unnatural amino acids, Fmocprotected acids (2.5 equiv), HCTU (2.4 equiv), and DIEA (5.0 equiv) were dissolved
in NMP and mixed with resin for 4 h, followed by washing with DCM and NMP for
several times. After we completed the synthesis of designed peptide, the intramolecular thiol-ene reaction was performed. The resins were drained and transferred to a
suitable flask, mixed with 1.2 eq MAP/MNP (1:1) catalyst in DMF and reacted at
ultraviolet light (365 nm) for 2 h. Final the resins were treated with a mixture of
TFA:H 2 O:TIS (95:2.5:2.5 by volume) for 2 h and dried by blowing nitrogen. Then
the peptides were precipitated with Hexane:Et 2 O (1:1 in volume) at 4°C, isolated by
centrifugation and dissolved in 40% (vol/vol) acetonitrile/water, purified by HPLC
with UV absorbance at 220 nm or 280 nm and later analyzed by LC-MS.
2.2.3 Circular Dichroism Study of Secondary Structures
To eliminate possible amino acid residue perturbations, a single turn pentapeptide
system was employed as a model system based on previous literature [13]. In order
to access the linear peptide and both cyclic peptide diastereomers in one synthesis,
the unnatural amino acid epimers were used. The structure-activity relationship of
the tether ring sizes and chiral center positions are summarized in Scheme 2.4 and
Fig. 2.2 and the optimal tether is shown in Fig. 2.3. These results suggested that
a seven-atom tether with a C-terminal γ-position chiral center induces the highest
helical content of peptides. Cyclic peptides 1a and 1b were synthesized from the
linear peptide 1, cyclo-Ac-CAAAS 5 (2-Me)-NH 2 (S 5 : (S)-pentenylglycine, 2-Me:
the methyl group located at the β position to the α carbon of amino acid), via a thiolene reaction as shown in Fig. 2.3a. Peptides 1a and 1b were readily separable by
29
added to make pH value of the mixture stay at 2–3. The reaction was extracted
with ethyl acetate for three times. The organic phase was dried with anhydrous
magnesium sulfate. The final product S 6 was obtained after the purification of flash
chromatography (Hexane: EA = 5:1) (5.2 g, yield: 41%)
2.2.2 Solid Phase Peptide Synthesis and Thiol-Ene
Photoreactions
Peptides were synthesized on MBHA resin (loading capacity: 0.37 mmol/g) by standard Fmoc-based SPPS (Scheme 2.3). At first, the resin was swelled in NMP for
30 min. Then 50% (vol/vol) morpholine in NMP was used to deprotect the Fmoc
group on amine group for 30 min × 2. Next the resin was washed with DCM and
NMP alternatively for three time (3×1 min). In coupling process, for natural amino
acids, the Fmoc-protected amino acids (5.0 equiv), HCTU (4.9 equiv), DIPEA (10.0
equiv) were dissolved in NMP and mixed with resin for 2 h, followed by washing
with DCM and NMP for three times (3×1 min). For unnatural amino acids, Fmocprotected acids (2.5 equiv), HCTU (2.4 equiv), and DIEA (5.0 equiv) were dissolved
in NMP and mixed with resin for 4 h, followed by washing with DCM and NMP for
several times. After we completed the synthesis of designed peptide, the intramolecular thiol-ene reaction was performed. The resins were drained and transferred to a
suitable flask, mixed with 1.2 eq MAP/MNP (1:1) catalyst in DMF and reacted at
ultraviolet light (365 nm) for 2 h. Final the resins were treated with a mixture of
TFA:H 2 O:TIS (95:2.5:2.5 by volume) for 2 h and dried by blowing nitrogen. Then
the peptides were precipitated with Hexane:Et 2 O (1:1 in volume) at 4°C, isolated by
centrifugation and dissolved in 40% (vol/vol) acetonitrile/water, purified by HPLC
with UV absorbance at 220 nm or 280 nm and later analyzed by LC-MS.
2.2.3 Circular Dichroism Study of Secondary Structures
To eliminate possible amino acid residue perturbations, a single turn pentapeptide
system was employed as a model system based on previous literature [13]. In order
to access the linear peptide and both cyclic peptide diastereomers in one synthesis,
the unnatural amino acid epimers were used. The structure-activity relationship of
the tether ring sizes and chiral center positions are summarized in Scheme 2.4 and
Fig. 2.2 and the optimal tether is shown in Fig. 2.3. These results suggested that
a seven-atom tether with a C-terminal γ-position chiral center induces the highest
helical content of peptides. Cyclic peptides 1a and 1b were synthesized from the
linear peptide 1, cyclo-Ac-CAAAS 5 (2-Me)-NH 2 (S 5 : (S)-pentenylglycine, 2-Me:
the methyl group located at the β position to the α carbon of amino acid), via a thiolene reaction as shown in Fig. 2.3a. Peptides 1a and 1b were readily separable by