Solid Phase Synthesis of HyNic‐Peptides 1. N‐terminal HyNic incorporation using 6‐Boc‐HyNic
Method 1‐1: Incorporation of HyNic to the N‐terminus of a 10‐mer peptide
1) Dissolve 6‐Boc‐HyNic (5 eq), HBTU (5 eq) in a minimum amount of DMF.
2) Add DiPEA (15 eq) to mixture and add immediately to Fmoc‐deblocked peptide resin.
3) Incubate for 1‐2 h.
4) Check completeness of reaction with Kaiser or TNBS test.
5) Wash resin and repeat reaction cycle if required.
Application 1‐1: Synthesis of HyNic‐10‐mer peptide
The peptide was assembled on a Rainin Symphony Peptide Synthesizer using
Rink amide resin. All acylation reactions were carried out using a 5‐fold excess of Fmoc‐amino acid activated with HBTU/DiPEA. A coupling time of 1 hour was used for each cycle. The HyNic‐peptidyl resin was cleaved using TFA/TIS/acetone/water (92.5/2.5/2.5/2.5) for 2 h. The crude peptide was analyzed by HPLC and ES‐MS with an m/e of 1314.6 as expected for the desired peptide (Figure 6). The addition of acetone is critical as the
in situ formation of the acetone hydrazone of the deprotected peptide blocks the formation of the trifluoroacetamide.13, 14 The product was isolated by RP‐HPLC using a gradient of 90% A (0.1% TFA/water) to 100% B (0.1% TFA/80% ACN/20% water) in 15 min.
Figure 1: RP‐HPLC (left) and mass spectrum (right) of crude C‐terminus‐HyNic 14mer peptide.
2. C‐terminal HyNic incorporation using Fmoc‐Lysine‐ε‐(6‐Boc‐HyNic) OH
Fmoc‐Lysine‐ε
‐(6‐Boc‐HyNic)OHMethod 2‐1: Incorporation of HyNic on the C‐terminus of a peptide
1) Dissolve Fmoc‐Lysine‐ε‐(6‐Boc‐HyNic)OH (5 eq), HBTU (5 eq) in a minimum amount of DMF.
2) Add DiPEA (15 eq) to mixture and add immediately to Fmoc‐deblocked peptide resin.
3) Incubate for 1‐2 h.
4) Check completeness of reaction with Kaiser of TNBS test.
5) Wash resin and repeat reaction if required.
Application 2‐1: Synthesis of C‐terminal HyNic‐14‐mer peptide
The peptide was assembled on a Rainin Symphony Peptide Synthesizer using
Rink amide resin. All acylation reactions were carried out using a 5‐fold excess of Fmoc‐amino acid activated with HBTU/DiPEA(5 eq). A coupling time of 1 hour was used for each cycle. The HyNic‐peptidyl resin was cleaved using
TFA/TIS/acetone/water (92.5/2.5/2.5/2.5) for 2 h. The crude peptide was analyzed by HPLC and ES‐MS with an m/e 1612 as expected for the desired peptide (Figure 7). The addition of acetone is critical as the
in situ formation of the acetone hydrazone of the deprotected peptide blocks the formation of thetrifluoroacetamide.13, 14 The product was isolated by RP‐HPLC using a gradient of 90% A (0.1% TFA/water) to 100% B (0.1% TFA/80% ACN/20% water) in 15 min.
Figure2: RP‐HPLC (left) and mass spectrum (right) of crude N‐terminus‐ HyNic‐10mer peptide.
3. N‐Terminal Biotin incorporation using ChromaLink Biotin
Method 3‐1: Incorporation of ChromaLink Biotin‐COOH on N‐terminus of a peptide
1) Dissolve ChromaLink Biotin‐COOH (5 eq), HBTU (5 eq) in a minimum amount of DMF.
2) Add DiPEA (15 eq) to mixture and add immediately to Fmoc‐deblocked peptide resin.
3) Incubate for 1‐2 h.
4) Check completeness of reaction with Kaiser of TNBS test.
5) Wash resin and repeat reaction if required.
Application 3‐1: Synthesis of N‐terminal biotinylated 10‐mer peptide
The peptide was assembled on a Rainin Symphony Peptide Synthesizer using
Rink amide resin. All
acylation reactions were carried out using a 5‐fold excess of ChromaLink Biotin‐COOH with
HBTU/DiPEA (15 equiv.). A coupling time of 1 hour was used for each cycle. The ChromaLink Biotinpeptidyl
resin was cleaved using TFA/TIS/water (95/2.5/2.5) for 2 h. The crude peptide was analyzed by
ES‐MS and had m/e 1869 as expected for the desired peptide. The product was isolated by RP‐HPLC using
a gradient of 90% A (0.1% TFA/water) to 100% B (0.1% TFA/80% ACN/20% water) over 15 min. RP‐HPLC of
the crude reaction product is illustrated in Figure 3.
Figure 3: RP‐HPLC (left) and mass spectrum (right) of crude 10mer ChromaLink Biotin peptide.
