Materials

• Methanol (Beijing Chemical Works);
• CH₂Cl₂ (Dichloromethane, DCM, Beijing Chemical Works);
• DEP: 20% Piperidine in DMF (v/v) ;
• DIPEA: N, N-disopropylethylamine (Aladdin);
• DMF: N, N-dimethylamino (Beijing Chemical Works);
• Fmoc-Gly-OH  (GL Biochem (Shanghai) Corporation. Ltd.);
• Hexahydropyridine (Beijing Chemical Works);
• HOBt: 1-hydroxybenzotriazole (GL Biochem (Shanghai) Corporation. Ltd.);
• NMM: N-methylmorpholine (GL Biochem (Shanghai) Corporation. Ltd.);
• PyBOP: benzotrizaol-a-yloxytris (pyrrolidino) phosphonium hexafluorophosphate) (GL Biochem (Shanghai) Corporation. Ltd.);
• Silylating reagent:(CH₂Cl₂: SiMe₂Cl₂=4:1);

May 19^th

Pretreatment for synthesizing peptide using Fmoc Solid Phase Peptide Synthesis (SPPS)

• Silylating reagent was added to a reaction vessel, stood for 2 hours.
• Then the reaction vessel was dried at 140â„?for 1.5 hours.
• 0.13978g 2-Chlorotrityl Chloride Resin was swollen in DCM (10ml) for 2 hours and then replaced by DMF overnight.

May 20^th

Attachment of the first residue

• 0.09099g Fmoc-Gly-OH (3 eq.) was added to the reaction vessel at room temperature and DIPEA (6 eq.) was added.
• Sufficient DMF was added to cover resin with three-time bed volume.
• The reaction vessel was agitated at 25°C and 180 rpm/min for 2h.
• The complex was washed with DMF six times.
• The 4 steps mentioned above were repeated (0.09500g Fmoc-Gly-OH).
• 0.35mL DIPEA, 0.70mL MeOH and 5.95mL DMF was added to the reaction vessel and agitated for 25 minutes to block the resin.
• The complex was washed with DMF six times.
• The 2 steps mentioned above were repeated.

Deprotection of the first residue (Step 1)

• 10 mL DEP was added to the reaction vessel to cover the resin.
• The reaction vessel was agitated gently for 30 min. Reagent was drained off.
• The resin was washed with DMF six times.

Resin tests

The presence or absence of the free amino groups was tested by Kaiser test as followed.

• A few resin beads were transferred to a 1.5mL micro tube and 2 drops of 5% ninhydrin and 80% phenol was added to ethanol (w/v).
• The reagents were well mixed and reacted under 120â„?for 4-6 minutes.
• The presence of resin-bound free amine is indicated by blue resin beads.

Attachment (Step 2) and deprotection of the second residue

• 0.09057g Fmoc-Gly-OH, 0.04246g HOBt, 0.15621g PyBOP and 70μL NMM were added to the reaction vessel.
• 10mL DMF was added and the resin was agitated for 90 minutes. (180r/min)
• The complex was washed with DMF six times.
• Resin test: Yellow âˆ?
• DEP was added to remove the Fmoc protecting group for 30 minutes.
• The complex was washed with DMF six times.
• Resin test: Blue âˆ?

Attachment of the third residue

• 0.09281g Fmoc-Gly-OH, 0.04090g HOBt, 0.15632g PyBOP and 70μL NMM were added to the reaction vessel.
• The third residue was coupled as step 2.
• Resin test: Kind of blue ×.

Attachment and deprotection of the third residue

• 0.09133g Fmoc-Gly-OH, 0.04112g HOBt, 0.15690g PyBOP and 70μL NMM were added to the reaction vessel.
• The third residue was coupled as step 2 again.
• Resin test: Yellow âˆ?
• DEP was added to remove the Fmoc protecting group for 30 minutes.
• The complex was washed with DMF six times.
• Resin test: Blue âˆ?

May 21^st

Attachment of the fourth residue

• 0.09077g Fmoc-Gly-OH, 0.04089g HOBt, 0.15908g PyBOP and 70μL NMM were added to the reaction vessel.
• Step 2 was repeated and the fourth residue was coupled.
• Resin tests: ×.

Attachment and deprotection of the fourth residue

• 0.09187g Fmoc-Gly-OH, 0.04217g HOBt, 0.15906g PyBOP and 70μL NMM were added to the reaction vessel.
• Step 2 was repeated and the fourth residue was coupled again.
• Resin test: âˆ?

Attachment and deprotection of the fifth residue

• 0.09365g Fmoc-Gly-OH, 0.04123g HOBt, 0.15917g PyBOP and 70μL NMM was added to the reaction vessel.
• 0.09365g Fmoc-Gly-OH, 0.04123g HOBt, 0.15917g PyBOP and 70μL NMM was a to the reaction vessel.
• Step 2 was repeated and the fifth residue was coupled.
• Resin test: âˆ?

Attachment of the sixth residue

• 0.09453g Fmoc-Gly-OH, 0.04433g HOBt and 60μL NMM was added to the reaction vessle.
• Step 2 was repeated and the sixth residue was coupled.
• Resin test: âˆ?
• The resin was washed with DMF (six times), DCM (six times) and MeOH (six times).
• The resin was dried under vacuum overnight.

May 23^rd

Cleavage of the linear precursor (Step 3)

• 10 mL cleavage reagent (9.75mL TFA and 250μL water TFA/TIS/water/DCM 1: 2.5: 2.5: 94) was added to the reaction vessel.
• The resin was agitated under room temperature for 30 min.
• The resin was removed by filtration under reduced pressure through the reaction vessel and was washed twice with DCM.
• The filtrates were dropped into cold diethyl ether. The supernatant solvent was decanted and the solid was washed with cold diethyl ether twice.
• The raw product was dissolved in little methanol and 30ml water was added.
• The mixture was freeze-dried in vacuum. The white solid product was used to next step without further purification.

May 31^st-June 1^st

Cyclizing of the linear peptide Gly₆

• The linear precursor (0.02764g, 76.78μmol) was dissolved in 90mL DMF. The solution was cooled to 0 â„?in an ice bath.
• 119.83mg PyBOP (230.34μmol)was dissolved in 30mL DMF; The liquid was cooled to 0 â„?and was slowly added to the solution mixture while stirring at â„?
• 79.44μL DIPEA was added to the mixture slowly.
• The reaction mixture was left to be stirred for 6 hours at 0 â„?and then at room temperature for 2 days.
• DMF was removed by vacuum distillation at 55â„?
• The crude product was purified by HPLC on C18 reversed-phase silica gel (20% to 90% acetonitrile with 0.1% TFS in 45min).

June 12^th

Vacuum distillation

• DMF was removed by vacuum distillation at 55â„?

Experiment

• Cyclo-(L-Asp-D-Ala)₃ Synthesis

Materials

• CH₂Cl₂ (Dichloromethane, DCM, Beijing Chemical Works);
• DEP: 20% Piperidine in DMF (v/v) ;
• DIPEA: N, N-disopropylethylamine (Aladdin);
• DMF: N, N-dimethylamino (Beijing Chemical Works);
• Fmoc-D-Ala-OH GL (Biochemistry (Shanghai) LTD);
• Fmoc-L-Asp(-OtBu)-OH;
• Hexahydropyridine (Beijing Chemical Works);
• HOBt: 1-hydroxybenzotriazole (GL Biochem (Shanghai) Corporation. Ltd.);
• Ninhydrin; (A: 5% (ω/V) 0.5 10mL Ninhydrin-ethanol solution: B: 80% (ω/V) phenol-ethanol solution);
• NMM: N-methylmorpholine (GL Biochem (Shanghai) Corporation. Ltd.);
• PyBOP: Benzotrizaol-a-yloxytris (pyrrolidino) phosphonium (Lot NO: 150106-00804) ;
• Silylating reagent: (CH₂Cl₂: SiMe₂Cl₂=4: 1);

July 18^th

Pretreatment

• 1.40040g 2-Chlorotrityl Chloride Resin was added.
• Dichloromethane was added to the resin and stood overnight.
• Silylating reagent was added to the reaction vessel and the reaction vessel stood overnight.
• The complex was dried at 140â„?for 1.5 hours.

July 19^th

Attachment of the first residue (D-Ala) (Twice)

• The resin was added to the reaction vessel.
• 0.96g Fmoc-D-Ala-OH, dichloromethane and 330μL DIPEA was added.
• The reaction vessel was agitated for 10 minutes.
• 660μL DIPEA was added and the reaction vessel was agitated for 2 hours.
• A blocking agent was prepared with 5mL DIPEA, 10mL MeOH and 85mL DCM.
• The reaction vessel was washed with dichloromethane six times.
• The six steps mentioned above were repeated.
• The reaction vessel was washed with dichloromethane six times.
• 11mL blocking agent was added and the reaction vessel was agitated for 40 minutes (160r/min).
• The liquid was discarded and 10.5mL blocking agent was added to the reaction vessel.
• The reaction vessel was agitated for 30 minutes.

Deprotection of the first residue (D-Ala)

• DEP was added to remove the Fmoc protecting group for 30 minutes.
• Resin test: Blue âˆ?

July 20^th

Attachment and deprotection of the second residue (L-Asp)

• 1.26g Fmoc-L-Asp(-OtBu)-OH, 0.42609g HOBt, 1.56956g PyBOP and 700μL NMM was added to the reaction vessel.
• 10mL DMF was added and the resin was agitated for 10 minutes artificially and then agitated for 90 minutes (180r/min).
• The resin was washed with DMF six times.
• Resin test: Yellow âˆ?
• DEP was added to remove the Fmoc protecting group for 30 minutes (180r/min).
• Resin test: Blue âˆ?

Attachment and deprotection of the third residue (D-Ala)

• 0.96520g Fmoc-D-Ala-OH, 0.42793g HOBt, 1.56673g PyBOP and 700μL NMM was added to the reaction vessel.
• The third residue was coupled as step 2.
• Resin test: Yellow âˆ?
• DEP was added to remove the Fmoc protecting group for 30 minutes.
• Resin test: Blue âˆ?

Attachment and deprotection of the fourth residue (L-Asp)

• 1.28301g Fmoc-L-Asp(-OtBu)-OH 0.42142g HOBt, 1.56052g PyBOP and 700μL NMM was added to the reaction vessel.
• The fourth residue was coupled as step 2.
• Resin test: Yellow âˆ?
• DEP was added to remove the Fmoc protecting group for 30 minutes.
• Resin test: Blue âˆ?

Attachment and deprotection of the fifth residue (D-Ala)

• 0.96848g Fmoc-D-Ala-OH, 0.42521g HOBt, 1.56310g PyBOP and 700μL NMM was added to the reaction vessel.
• The fifth residue was coupled as step 2.
• Resin test: Yellow âˆ?
• DEP was added to remove the Fmoc protecting group for 30 minutes.
• Resin test: Blue âˆ?

Attachment and deprotection of the sixth residue (L-Asp)

• 1.28647g Fmoc-L-Asp(-OtBu)-OH, 0.42283g HOBt, 1.56g PyBOP and 700μL NMM was added to the reaction vessel.
• The sixth residue was coupled as step 2.
• The resin was tested with ninhydrin reaction: Yellow.
• Resin test: Yellow âˆ?
• DEP was added to remove the Fmoc protecting group for 30 minutes.
• Resin test: Blue âˆ?

Cleavage of the linear precursor

• The resin was washed with MeOH six times and dried under vacuum.
• A small amount of resin was added to a flask and 1% TFA (Solvent: CH₂Cl₂) was added.
• The resin and solution were added to cold ethylether.
• The mixture was centrifuged under 6000r/min at 4â„?for 5 minutes.

MS Analysis

July 21^st

• 0.05043g Resin-Cyclo-(L-Asp-D-Ala)₃ was added to be cleaved with 0.75mL cleaving reagent (TFA/TIS/water/DCM 1: 2.5: 2.5: 94) as a condition test.
• The cyclic peptide was cleaved for 45 minutes under room temperature.

July 24^th

Characterization of cyclo-(DG)₃+ ethylenediamine

August 2^nd

Cyclo-(Asp-Gly)₃ assembling and purification

1. Hexamethylendiamine as the linker (-COOH:-NH₂=1:10)
• 0.5mg Cyclo-(Asp-Gly)₃, 2mg hexamethylendiamine, 0.0312g PyBOP, 4.029μL triethylamine and 5mL DMF were added to a flask.
• The flask was sealed and the liquid was stirred for one day.
• Dialysis: One day.
2. Hexamethylendiamine as the linker (-COOH:-NH₂=1:50)
• 0.5mg Cyclo-(Asp-Gly)₃, 8.5mg hexamethylendiamine, 0.1544g PyBOP, 4.029μL triethylamine and 5mL DMF were added to a flask.
• The flask was sealed and the liquid was stirred for one day.
• Dialysis: One day.

August 3^rd

• 1.23686g Resin-Cyclo-(L-Asp-D-Ala)₃ was added to be cleaved with 17.50mL cleaving reagent (Prepared with 4mL TFE, 16mL DCM).
• The mixture was stirred for 45 minutes.

Cyclo-(Asp-Pro)₃ assembling and purification

1. ethylenediamine as the linker (-COOH:-NH₂=1:50)
• 3.27μL triethylamine, 0.5mg Cyclo-(Asp-Pro)₃, 3.543mg ethylenediamine, 122.71mg PyBOP and 3.841 mL DMF were added to a flask.
• The flask was sealed and the liquid was stirred for 2 days.
• Dialysis: 2 days.
2. Hexamethylendiamine as the linker (-COOH:-NH₂=1:50)
• 3.27μL triethylamine, 0.5mg Cyclo-(Asp-Pro)₃, 6.852mg hexamethylendiamine, 122.71mg PyBOP and 3.841 mL DMF were added to a flask.
• The flask was sealed and the liquid was stirred for 2 days.
• Dialysis: 2 days.

August 7^th

Cyclo-(Asp-Pro)₃ Synthesis

• Cyclo-(Asp-Pro)₃ was purchased from SynPeptide Co. Ltd.(Shanghai, China)

HPLC analysis of Cyclo-(Asp-Pro)₃

• Pump A: 0.1% Trifluoroacetic in 100% Water
• Pump B: 0.1% Trifluoroacetic in 100% Acetonirtrile
• Total Flow: 1ml/min
• Wavelength: 214nm
• Analytial column type: SHIMADZU Inertsil ODS-SP (4.6*250mm*5um)

MS analysis of Cyclo-(Asp-Pro)₃

August 8^th

Cyclo-(Arg-Pro)₆ assembling and purification

1. Adipic acid as the linker (-CN₃H₄:-COOH=1:10)
• 0.5mg cyclo-(Arg-Pro)₆, 1.443mg adipic acid and 1.646mL DMF were added to a flask.
• The flask was sealed and the liquid was stirred for 2 days.
2. Adipic acid as the linker (-CN₃H₄:-COOH=1:50)
• 0.5mg cyclo-(Arg-Pro)₆, 7.216mg adipic acid and 1.646mL DMF were added to a flask.
• The flask was sealed and the liquid was stirred for 2 days.

August 13^th

Cyclo-(Arg-Pro)₃ Synthesis

• Cyclo-(Arg-Pro)₃ was purchased from ChinaPeptides Co.,Ltd.(Shanghai, China) (Received: August 29^th)

HPLC analysis of Cyclo-(Arg-Pro)₆

• Measurement: Peak Area
• Run Time: 11min
• Calculation Type: Percent
• Wavelength: 220nm
• Flow Rate: 1ml/min
• Inj.Vol: 10μL
• Buffer A: 0.1% TFA in water
• Buffer B: 0.1% TFA in Acetonitrile
• Column: Kromasil 100-5C18, 4.6mm×250mm, 5 micron
• Column Temp: 35â„?
• Gradient(linear): 10%-41% buffer B in 11min

 

MS analysis of Cyclo-(Arg-Pro)₃

• Expected MS: 759.93 Mass Spectrometer: API 150EX
• Ion Source: ESI B.conc: 80%ACN/20%H2O
• NEB: 10.00 CUR: 12.00 IS: +4500 TEM: 0.00
• Flow Rate: 0.2ml/min Run Time: 1min

August 14^th

Cyclo-(Arg-Arg) Synthesis

• Cyclo-(Arg-Arg) was purchased from ChinaPeptides Co.,Ltd.(Shanghai, China) (Received: August 29^th)

HPLC analysis of Cyclo-(Arg-Arg)

• Measurement: Peak Area Run Time: 13min
• Calculation Type: Percent Wavelength : 220nm
• Flow Rate: 1ml/min Inj.Vol: 10uL
• Buffer A: 0.1% TFA in water Buffer B: 0.1%TFA in Acetonitrile
• Column: Atlantis C18,4.6mmX250mm,5 micron Column Temp: 35â„?
• Gradient(linear): 0%-20% buffer B in 13min

MS analysis of Cyclo-(Arg-Arg)

• Expected MS: 312.39 Mass Spectrometer: API 150EX
• Ion Source: ESI B.conc: 75%ACN/24.5%H2O/0.5%Ac
• NEB: 10.00 CUR: 12.00 IS: +4500 TEM: 0.00
• Flow Rate: 0.2ml/min Run Time: 1min

August 17^th

Characterization of Cyclo-(Asp-Pro)₃+ ethylenediamine

Cyclo-(Asp-Pro)₃ assembling and purification

1. Hexamethylendiamine as the linker (-COOH:-NH₂=1:10)
• 0.5mg Cyclo-(Asp-Pro)₃, 1.3704mg hexamethylendiamine, 24.542mg PyBOP, 3.27μL triethylamine and 3.841 mL DMF were added to a flask.
• The flask was sealed and the liquid was stirred for 2 days.
• Dialysis: 2 days.
2. Hexamethylendiamine as the linker (-COOH:-NH₂=1:50)
• 0.5mg Cyclo-(Asp-Pro)₃, 6.852mg hexamethylendiamine, 122.71mg PyBOP, 3.27μL triethylamine and 0.8mL DMF were added to a flask.
• The flask was sealed and the liquid was stirred for 2 days.
• Dialysis: 2 days.
3. Cyclo-(Arg-Arg) as the linker (-COOH:-CN₃H₄ =1:10)
• 0.5mg Cyclo-(Asp-Pro)₃, 3.68mg cyclo-(Arg-Arg) and 0.8mL DMF were added to a flask.
• The flask was sealed and the liquid was stirred for 2 days.
• The assembly was not dialyzed.
4. Cyclo-(Arg-Arg) as the linker (-COOH:-CN₃H₄ =1:50)
• 0.5mg Cyclo-(Asp-Pro)₃, 0.736mg cyclo-(Arg-Arg) and 0.8mL DMF were added to a flask.
• The flask was sealed and the liquid was stirred for 2 days.
• The assembly was not dialyzed.

August 19^th

Cyclizing the linear peptide (D-Ala-L-Asp)3

• 3.72mg (D-Ala-L-Asp(-OtBu))₃ and 1.461mg NaCl were dissolved in 1mL DMF.
• 3.1224mg PyBOP and 1.4μL Et₃N were added.

August 24^th

• Silylating.
• The reaction vessel was dried at 140â„?for 1.5 hours.
• 1.4023g resin was added to the reaction vessel.
• Dichloromethane was added to the resin and stood overnight.

 

• The resin and 1.4g Fmoc-Lys(-Boc)-OH were added to the reaction vessel.
• Dichloromethane and 33μL DIPEA were added.
• The reaction vessel was agitated for 10 minutes.
• 66μL DIPEA was added and the reaction vessel was agitated for 2 hours.

 

• The reaction vessel was washed with dichloromethane six times.
• 11mL blocking agent was added and the reaction vessel was agitated for 25 minutes.

 

• The reaction vessel was washed with dichloromethane six times.
• DEP was added to remove the Fmoc protecting group for 30 minutes.
• Resin test: Blue âˆ?

 

• 1.14g Fmoc-Ser(-tBu)-OH was added to the reaction vessel.
• DMF, 0.4260g HOBt, 1.56956g PyBOP and 700μL NMM were added.
• The reaction vessel was agitated.

August 25^th

• The reaction vessel was washed with DMF six times.
• Resin test: Yellow âˆ?
• DEP was added and the second residue was deprotected for 30 minutes.
• Resin test: A bit blue.
• Ninhydrin was prepared.

August 26^th

• The reaction vessel was silylated and was dried at 140â„?
• 0.28g 2-Chlorotrityl Chloride Resin was added and swollen in dichloromethane. .

 

• The resin was added to the reaction vessel.
• 0.1401g Fmoc-Lys(-Boc)-OH was added to the reaction vessel.
• Dichloromethane and 33μL DIPEA were added.
• The reaction vessel was agitated for 10 minutes.
• 66μL DIPEA was added and the reaction vessel was agitated for 2 hours.

August 27^th

• A small reaction vessel was silylated and dried at 140â„?for 1.5 hours.
• 0.28g Fmoc-Lys(-Boc)-OH was added to the reaction vessel.
• Dichloromethane and 66μL DIPEA were added.
• The reaction vessel was agitated for 10 minutes.
• 132μL DIPEA was added and the reaction vessel was agitated for 2 hours.

 

• The reaction vessel was washed with DCM six times.
• 0.28g Fmoc-Lys(-Boc)-OH was added to the reaction vessel.
• Dichloromethane and 66μL DIPEA were added.
• The reaction vessel was agitated for 10 minutes.
• 132μL DIPEA was added and the reaction vessel was agitated for 2 hours.

 

• The reaction vessel was washed with DCM six times and the resin was blocked.
• The reaction vessel was washed with DMF six times.
• DEP was added and the amino residue was deprotected for 30 minutes.
• DEP was added to remove the Fmoc protecting group for 30 minutes.
• Resin test: Blue âˆ?

 

• 0.23g Fmoc-Ser(-tBu)-OH was added to the reaction vessel.
• DMF, 0.4260g HOBt, 0.312g PyBOP and 600μL NMM was added and the reaction vessel were agitated.
• The reaction vessel was washed with DMF six times.
• Resin test: Yellow.

August 28^th

• DEP was added to remove the Fmoc protecting group for 30 minutes.
• Resin test: Blue âˆ?

 

• 0.28g Fmoc-Lys(-Boc)-OH was added to the reaction vessel.
• DMF, 0.085g HOBt, 0.312g PyBOP and 140μL NMM were added.
• The reaction vessel was washed with DMF six times after 90 minutes.
• Resin test: Yellow âˆ?
• DEP was added to remove the Fmoc protecting group for 30 minutes.
• Resin test: Blue âˆ?

 

• 0.23g Fmoc-Ser(-tBu)-OH was added to the reaction vessel.
• DMF, 0.085g HOBt, 0.312g PyBOP and 140μL NMM were added and the reaction vessel was agitated.
• The reaction vessel was washed with DMF six times.
• Resin test: Yellow âˆ?
• DEP was added to remove the Fmoc protecting group for 30 minutes.
• Resin test: Blue âˆ?

August 29^th

• Assembling: [(DP)₃+ hexamethylendiamine (-COOH:-NH₂=1:10 & -COOH:-NH₂=1:50)]assembly was dialyzed against water.

 

• 0.28g Fmoc-Lys(-Boc)-OH was added to the reaction vessel.
• DMF, 0.085g HOBt, 0.312g PyBOP and 140μL NMM was added.
• The reaction vessel was washed with DMF six times after 90 minutes.

August 30^th

• Resin test: Yellow âˆ?
• DEP was added to remove the Fmoc protecting group for 30 minutes.

 

• 0.37g Fmoc-His(-Trt)-OH was added to the reaction vessel.
• DMF, 0.085g HOBt, 0.312g PyBOP and 140μL NMM was added.
• Resin test: Yellow âˆ?

August 31^st

• Assembling

• DEP was added and the residue was deprotected for 30 minutes.
• The reaction vessel was washed with DMF six times and the resin was tested with ninhydrin reaction: Red.
• The residue was deprotected again.

September 2^nd

Characterization of Cyclo-(Asp-Pro)₃+ Cyclo-RR

Characterization of Cyclo-(Asp-Pro)₃+ hexamethylendiamine

September 7^th

• Assembling

Electrophoresis

October 15^th

Transfection Result

DLS Result

c(PD)₃ assembly (cyclo-(Asp-Pro)₃ assembly, hexamethylendiamine as linker, 50:1, dialyzed in water)

Zeta Potential

 

Size distribution