5. Dissolve the pellet in 20% MeCN, 0.1% TFA in water, filter
with a 0.2 μm syringe filter, and purify with preparative
RP-HPLC, applying a linear gradient of a binary solvent system
of H 2 O–MeCN–TFA (A: 95:5:0.1; B: 5:95:0.1) at a flow rate
of 20 mL/min.
6. Lyophilize the purified sample to obtain white solids, which
can be stored long-term at À20
C until further use.
3.8 Boc/Bzl-SPPS
for the Insertion
of Amide-to Ester
Mutations
The insertion of an ester bond into a peptide is usually performed
by coupling the α-hydroxy acid to the N-terminus of the growing
peptide. Subsequently, an ester bond is formed with the following
α-amino acid. Another option to incorporate amide-to-ester substitution is the use of a preformed dipeptide building block
[Boc-amino acid-(CO)O-amino acid-OH] in standard Boc-SPPS.
These building blocks are produced by solution-phase synthesis.
The protocols below detail the synthesis of α-hydroxy acids.
3.8.1 Synthesis
of α-Hydroxy Acids
1. Treat 1 mmol Boc-protected amino acid with 3 mL TFA for
15 min to remove the Boc protection group.
2. Evaporate TFA and dissolve the residue in a 4 mL mixture of
1:1 (v/v) dioxane and water and cool it to 0
C in an ice bath.
Subsequently, add 2.0 mmol tert-butylnitrite and incubate at
20
C for 1 h, constantly stirring.
3. Upon completion, remove the solvent in vacuo and purify the
resulting residue by silica gel chromatography (DCM/MeOH
10/1 as a standard method).
3.8.2 Boc-SPPS
Coupling Cycle
Merrifield (chloromethyl) or PAM (phenylacetamidomethyl) resins
are the most commonly used in the standard Boc-SPPS protocol
and suitable for N-terminal fragments [31]. For thioester peptides,
Trt-S-Ac-L-Leu-PAM resin is used, which results in a functional
thioester after HF cleavage.
In the protocol below, we use HBTU and DIEA for coupling in
a 0.1 mmol scale. The stoichiometric ratio of the reagents is 4/4/8
(AA/HBTU/DIEA) throughout the whole protocol (see Note
24).
1. Weigh out the desired resin in an adequate amount and transfer
it to a fritted reaction vessel with a stopcock and a Teflon screw
cap. After closing the vessel with the stopcock, fill in DMF until
the resin is completely covered. Let the resin swell for at least
30 min. If the resin is precoupled with the first Boc-protected
amino acid, proceed to the deprotection step. If no
Boc-protection group is attached, proceed directly with the
coupling step (step 6).
2. Flow wash the resin with DMF for 30 s (see Note 25).
210
Christin Kossmann et al.
with a 0.2 μm syringe filter, and purify with preparative
RP-HPLC, applying a linear gradient of a binary solvent system
of H 2 O–MeCN–TFA (A: 95:5:0.1; B: 5:95:0.1) at a flow rate
of 20 mL/min.
6. Lyophilize the purified sample to obtain white solids, which
can be stored long-term at À20
C until further use.
3.8 Boc/Bzl-SPPS
for the Insertion
of Amide-to Ester
Mutations
The insertion of an ester bond into a peptide is usually performed
by coupling the α-hydroxy acid to the N-terminus of the growing
peptide. Subsequently, an ester bond is formed with the following
α-amino acid. Another option to incorporate amide-to-ester substitution is the use of a preformed dipeptide building block
[Boc-amino acid-(CO)O-amino acid-OH] in standard Boc-SPPS.
These building blocks are produced by solution-phase synthesis.
The protocols below detail the synthesis of α-hydroxy acids.
3.8.1 Synthesis
of α-Hydroxy Acids
1. Treat 1 mmol Boc-protected amino acid with 3 mL TFA for
15 min to remove the Boc protection group.
2. Evaporate TFA and dissolve the residue in a 4 mL mixture of
1:1 (v/v) dioxane and water and cool it to 0
C in an ice bath.
Subsequently, add 2.0 mmol tert-butylnitrite and incubate at
20
C for 1 h, constantly stirring.
3. Upon completion, remove the solvent in vacuo and purify the
resulting residue by silica gel chromatography (DCM/MeOH
10/1 as a standard method).
3.8.2 Boc-SPPS
Coupling Cycle
Merrifield (chloromethyl) or PAM (phenylacetamidomethyl) resins
are the most commonly used in the standard Boc-SPPS protocol
and suitable for N-terminal fragments [31]. For thioester peptides,
Trt-S-Ac-L-Leu-PAM resin is used, which results in a functional
thioester after HF cleavage.
In the protocol below, we use HBTU and DIEA for coupling in
a 0.1 mmol scale. The stoichiometric ratio of the reagents is 4/4/8
(AA/HBTU/DIEA) throughout the whole protocol (see Note
24).
1. Weigh out the desired resin in an adequate amount and transfer
it to a fritted reaction vessel with a stopcock and a Teflon screw
cap. After closing the vessel with the stopcock, fill in DMF until
the resin is completely covered. Let the resin swell for at least
30 min. If the resin is precoupled with the first Boc-protected
amino acid, proceed to the deprotection step. If no
Boc-protection group is attached, proceed directly with the
coupling step (step 6).
2. Flow wash the resin with DMF for 30 s (see Note 25).
210
Christin Kossmann et al.