Modulation of the antagonistic properties of an insulin mimetic peptide by disulfide bridge modifications

Insulin is a peptide responsible for regulating the metabolic homeostasis of the organism; it elicits its effects through binding to the transmembrane insulin receptor (IR). Insulin mimetics with agonistic or antagonistic effects toward the receptor are an exciting field of research and could find applications in treating diabetes or malignant diseases. We prepared five variants of a previously reported 20‐amino acid insulin‐mimicking peptide. These peptides differ from each other by the structure of the covalent bridge connecting positions 11 and 18. In addition to the peptide with a disulfide bridge, a derivative with a dicarba bridge and three derivatives with a 1,2,3‐triazole differing from each other by the presence of sulfur or oxygen in their staples were prepared. The strongest binding to IR was exhibited by the peptide with a disulfide bridge. All other derivatives only weakly bound to IR, and a relationship between increasing bridge length and lower binding affinity can be inferred. Despite their nanomolar affinities, none of the prepared peptide mimetics was able to activate the insulin receptor even at high concentrations, but all mimetics were able to inhibit insulin‐induced receptor activation. However, the receptor remained approximately 30% active even at the highest concentration of the agents; thus, the agents behave as partial antagonists. An interesting observation is that these mimetic peptides do not antagonize insulin action in proportion to their binding affinities. The compounds characterized in this study show that it is possible to modulate the functional properties of insulin receptor peptide ligands using disulfide mimetics.


Table of contents:
List of abbreviations Detailed structures of peptides 1-5 (Figure S1)
Melting points were determined on a Boetius block and are uncorrected.
Optical rotation values were measured in 100 mm cell on Perkin Elmer 241 MC under Na lamp radiation.1 H and 13 C NMR spectra were acquired on Bruker AVANCE-600 spectrometer ( 1 H at 600.13 MHz, 13 C at 150.9 MHz) in CDCl3 or DMSO-d6 at 300 K.The 2D-H,H-COSY, 2D-H,C-HSQC and 2D-H,C-HMBC spectra were recorded and used for the structural assignment of proton and carbon signals.
IR spectra were recorded on a Bruker IFS 55 Equinox apparatus.HRMS data were obtained on a FTMS mass spectrometer LTQ-orbitrap XL (Thermo Fisher, Bremen, Germany) in electrospray ionization mode.
The cooling bath was removed, and the reaction mixture was allowed to react overnight at rt. Next, the mixture was again ice-cooled, 100 ml water was added and then the pH value was adjusted approximately to 3 with 10 % citric acid.A brown solution was extracted 3 x with 100 ml ethyl acetate; the combined organic layers were washed 2 x with 100 ml brine, dried with Na2SO4 and concentrated under reduced pressure.The resulting oil (8 g) was subjected to flash chromatography on silica, using a linear gradient of ethyl acetate in toluene to afford 4.85 g (S)-2-[(tert.butoxycarbonyl)amino]-3-(prop-2-yn-1-yloxy)propanoic acid 6 (yellow oil).The purity of 6 was checked by TLC chromatography; Rf = 0.52 (ethyl acetate : MeOH : acetone : water = 4 : 1 : 1 : 1, plate was exposed before elution by gaseous ammonia).
Intermediate 6 (4.85 g; 19.95 mmol) was dissolved in 5 ml of DCM, and 10 ml of TFA was added during ice-cooling.The reaction mixture was stirred for another 2 hours at rt and the complete removal of Boc group was checked by TLC analysis (ethyl acetate : MeOH : acetone water = 6 : 1 : 1 : 0.5).Volatile solvents were evaporated and the oil brown residue was dissolved in 50 ml of saturated aqueous solution of NaHCO3, ice-cooled, and Fmoc-OSu (6.7 g; 19.95 mmol) in 50 ml dioxane was added dropwise.The reaction mixture was stirred for 1 hour at 0 °C and then overnight at rt.The flask was cooled again in an ice bath and 1M HCl was added until pH ~ 1 was achieved.The reaction mixture was extracted 3 x with 100 ml ethyl acetate; the combined organic phases were washed with 100 ml water, 100 ml brine, dried on Na2SO4 and concentrated in vacuo to give a crude solid.Double crystallization from a mixture of ethyl acetate and hexane afforded the compound 7.  L-Cystine (4.8 g; 20 mmol) was loaded into a 500 ml Erlenmeyer flask, fitted with a Claisen adapter, a dry ice cooler, a glass-coated magnetic rod, and potassium hydroxide tubing at the inlet and outlet.The apparatus was first flushed with a stream of dry ammonia.When approximately 200 ml of liquid ammonia was condensed, the gas intake was closed, the inlet tube was removed and replaced with a stopper.Then, metallic sodium (1.93 g; 84 mmol) was added in several small portions during 1 h until the dark blue color persisted for at least half an hour.The excess of free sodium was quenched by adding a small portion of ammonium chloride, which resulted in full decolorization of the solution.
Subsequently, 80 % propargyl bromide (4.7 ml; 40 mmol) was added, stirring and cooling was stopped, and ammonia was allowed to evaporate spontaneously overnight.The resulting dark solid was dissolved in 100 ml water and 1 M HCl was added during ice-cooling and stirring, until pH ~ 5 -6 was reached.
Massively precipitating crystals were filtered off, washed with 50 ml cold water and dried with P2O5.
Intermediate 8 (4.4 g; 28 mmol) was dissolved in 100 ml of saturated NaHCO3, ice-cooled and Fmoc-Osu (9.4 g; 28 mmol) in 100 ml dioxane was added dropwise.The reaction mixture was stirred for 1 hour at 0 °C and then overnight at rt.Then 200 ml of water was added, the flask was cooled again in an ice bath and 1M HCl was added until pH ~ 1 was reached.The reaction mixture was extracted 3 x with 100 ml ethyl acetate; the combined organic phases were washed with 100 ml water, 100 ml brine, dried with Na2SO4 and concentrated in vacuo to give a crude oily material, which was subjected to flash chromatography on silica, using a linear gradient of 1 % AcOH/ethyl acetate in toluene.The product (10 g, light yellow oil) was dissolved during heating (60 °C) in a minimal amount of toluene, and the flask was placed at -20 °C overnight.Next, 50 ml of hexane was added to the gelatinous material, the mixture was sonicated for 10 minutes in an ice-cooled bath and then again placed at -20 °C overnight.The compound was prepared according to the earlier described Method 2 (Ref. 2 ) by the reaction of L-cysteine (5 g; 41.3 mmol) and 80 % propargyl bromide (6.9 ml; 62 mmol).Yield 3.6 g (56 %).Compound 11 was prepared using the slightly modified protocol published earlier 3 (including work-up and isolation previously employed for 9) from acid 10 (3.8 g; 23.9 mmol) and Fmoc-OSu (8.1 g ; 23.9 mmol).Yield 8.1 g (89 %).
Alternatively, Fmoc-L-Cys-OH 4 (5.6 g; 16.4 mmol) and 80 % propargyl bromide (2.2 ml; 19.7 mmol) were dissolved in 100 ml of argon-bubbled ethyl acetate and the deaerated aqueous solution (100 ml) of NaHCO3 (5.5 g; 65.6 mmol) and TBAB (0.52 g; 1.6 mmol) were added in one step.The reaction mixture was vigorously stirred in argon atmosphere at rt for 4 days.Afterwards, 1 M HCl was slowly added dropwise until pH ~ 1 was reached.The organic layer was separated and the aqueous phase was extracted 2 x with 100 ml ethyl acetate.The combined organic phases were washed with 100 ml water, 2 x 100 ml brine, dried on sodium sulfate and evaporated under reduced pressure to afford 8.7 g of oily residue.Flash chromatography and crystallization of the product were performed by the same method as for compound 9. O-tert-Butyl-N,N´-diisopropyl isourea (9.7 g; 48.7 mmol) in 250 ml DCM was added to a stirred cloudy solution of Boc-L-Ser-OH (10 g; 48.7 mmol) and the reaction mixture was allowed to react at rt overnight.Then, 4 portions of isourea (1.9 g; 9.7 mmol) were added in 12-hour periods, until TLC analysis revealed the almost complete disappearance of the starting compound.The volatile material was evaporated under reduced pressure, the semi-solid residue was dissolved in 100 ml ethyl acetate and placed at -20 °C.After several hours, the precipitated crystals (urea) were filtered off and washed with chilled ethyl acetate (100 ml).The filtrate phase was evaporated in vacuo to afford a crude yellow oil (14 g), which was subjected to flash chromatography on silica using a linear gradient ethyl acetate in toluene.Yield 9.2 g (72 %).Clear viscous oil.The physical-chemical characteristics were in a full agreement with those published earlier 5 .
Alternatively, 18 (9.1 g ; 34.3 mmol) and O-tert-butyl-N,N´-diisopropyl isourea (6.9 g ; 34.3 mmol) in 100 ml of DCM were allowed to react at rt overnight.Then, 5 portions of isourea (1.4 g; 6.9 mmol) were added in 12-hour periods, until TLC analysis (DCM : MeOH : NH4OH conc.= 75 : 22 : 3) revealed almost complete consumption of the starting compound 18.The volatile material was evaporated under reduced pressure, the residue was dissolved in 100 ml ethyl acetate and placed at -20 °C.After several hours, the precipitated DIU was filtered off and washed with 100 ml of chilled ethyl acetate.The filtrate was evaporated in vacuo to afford a crude yellow oil (6.9 g), which was subjected to flash chromatography on silica using a linear gradient of ethyl acetate in toluene.Yield 6.1 g (55 %).Ester 14 (7.8 g; 24.3 mmol) and CBr4 (8.9 g ; 26.7 mmol) were dissolved in 100 ml of DCM.
The flask was ice-cooled and PPh3 (7 g; 26.7 mmol) in 50 ml of DCM was slowly added dropwise during stirring.Stirring was continued for 1 hour at 0 °C and then at rt overnight.The volatile material was removed on the evaporator under reduced pressure and the resulting yellow oil was dissolved (sonication) in a mixture of diethyl ether (500 ml) and petroleum ether (2 000 ml).The solution was placed at -20 °C overnight.The precipitate of triphenylphosphine oxide was filtered off and washed with a mixture of 500 ml chilled diethyl ether and petroleum ether (1 : 4).The filtrate was evaporated under reduced pressure to give 11.5 g of bright yellow oil, which was subjected to flash chromatography on silica using a linear gradient ethyl acetate in toluene.Yield Ester 14 (6.1 g ; 19 mmol) was dissolved in 100 ml of DCM with TEA (4 ml; 28.5 mmol).The reaction mixture was ice-cooled and MsCl (1.6 ml; 20.9 mmol) was added dropwise.After 1 hour of stirring at 0 °C, TLC analysis (ethyl acetate : toluene 1 : 1) revealed complete consumption of the starting compound 14.Excess of TEA was removed by addition of 10 % citric acid, the organic phase was separated, washed with 50 ml water and 2 x 50 ml brine and dried on Na2SO4.The filtrate was evaporated under reduced pressure to give 7.7 g of clear oil.The crude mesylated-intermediate was dissolved in 70 ml of anhydrous DMSO with NaN3 (5 g; 76 mmol) and heated at 70 °C overnight.After cooling, 150 ml water was added, and the solution was extracted with 4 x 100 ml ethyl acetate.The combined organic phases were washed successively with 1 x 100 ml water and 2 x 100 ml brine and dried over Na2SO4.
The filtrate was evaporated under reduced pressure to give 8 g of yellow oil, which was subjected to flash chromatography on silica using a linear gradient of ethyl acetate in toluene.Yield 5.3 g (80 %, for 2 steps).
Alternatively, 15 (5.8 g; 15.1 mmol) and NaN3 (2 g; 70.2 mmol) were heated at 70 °C in 40 ml of anhydrous DMSO overnight.After cooling, 100 ml water was added and the solution was extracted with 3 x 100 ml ethyl acetate.The combined organic phases were washed with 50 ml water and 2 x 50 ml brine and dried on Na2SO4.The filtrate was evaporated under reduced pressure to give 5.5 g of yellow oil, which was subjected to flash chromatography on silica using a linear gradient of ethyl acetate in

2-(R)-(tert-Butoxycarbonylamino)-3-[(2-hydroxyethyl)sulfanyl]propanoic acid 18
L-Cysteine (9.7 g; 80 mmol) in 250 ml of deaerated methanol was placed in a 500 ml roundbottom flask, equipped with a magnetic rod, and surrounded by an ice-bath.Stirring was started under the protective atmosphere of argon, and small portions of sodium (3.9 g; 168 mmol) were added carefully during a 1 h period.When the slurry became clear (approx.after 0.5 hour), 2-bromoethanol (10 g; 80 mmol) was added dropwise and the reaction mixture was allowed to react at rt overnight.
Methanol was evaporated under reduced pressure, the residue was dissolved in 50 ml water and the solution was applied on a column of Dowex in H + form (160 g; 5.2 ± 0.3 meq/g), which was then washed with 750 ml of a mixture of methanol-water (1 : 9).The product was liberated from the ion-exchange resin by washing with 1.5 l of mixture of conc.NH4OH : MeOH : water (1 : 1 : 3).The collected filtrate was evaporated under reduced pressure on an evaporator; the resulting slurry of crystals was coevaporated with 2 x 500 ml of toluene, and dried under deep vacuum overnight.The solid material was dissolved at 80 °C in 30 ml water and then 120 ml of 96 % ethanol was added.The clear solution was allowed to stand overnight at rt.The precipitate was filtered off and the crystals (6.4 g) were washed with 50 ml of chilled ethanol.The mother liquor was concentrated on the evaporator, and crystallization from water (10 ml) and ethanol (80 ml) produced the second portion of crystals (4.8 g).TLC analysis (IPA : conc.NH4OH : water = 7 : 1 : 2) revealed identical profiles for both samples: major spot (Rf = 0.50) of 2-(R)-3-[(2-hydroxyethyl)sulfanyl]propanoic acid 17 and minor spot (Rf = 0.32) of unknown impurity.The material was used for further reaction without additional purification.
Compound 17 (5 g; 30.3 mmol) and Na2CO3 (6.4 g ; 60.6 mmol) were dissolved in 150 ml water and stirred.Then Boc anhydride (6.6 g ; 30.3 mmol) in 100 ml of dioxane was added dropwise at 0 °C during a period of 0.5 hour.The cooling bath was removed, and the reaction mixture was allowed to react overnight.Thereafter, 500 ml water was added, the solution was saturated with sodium chloride and acidified to pH ~ 3 with 10 % citric acid while being efficiently cooled with ice.The reaction mixture was extracted 3 x with 150 ml of ethyl acetate; the combined organic phases were washed 3 x with 100 ml brine and dried on Na2SO4, filtered and the solvent was removed under reduced pressure.The oily residue was subjected to flash chromatography on silica using linear gradient of ethyl acetate : methanol : acetone ( 4 : 3 : 1) in ethyl acetate.Yield 6.1 g (76 %).Colorless and viscous oil.Rf = 0.24 (DCM : Potassium hydroxide (4.1 g; 73.4 mmol) in 50 ml methanol was added dropwise under argon atmosphere to a stirred slurry of L-cysteine (4.4 g; 36.7 mmol) in 100 ml of deaerated methanol.After 10 minutes, the suspension turned into a completely clear solution.Then, the protected bromide 24 (8.2 g; 36.7 mmol) in 50 ml of THF was added in one portion and the reaction was allowed to react overnight at rt.The solid citric acid was slowly added until pH ~ 6 was reached and methanol was evaporated under reduced pressure.Thereafter, the resulting bulky precipitate was dissolved in 150 ml water; the pH value of the turbid solution was adjusted to 8 with NaHCO3 and Fmoc-OSu (12.4 g; 36.7 mmol) in 150 ml of dioxane was added dropwise during stirring and ice-cooling.After 12 hours of stirring at rt, the reaction mixture was acidified to pH ~ 3 with 1 M citric acid.Next, 250 ml water was added, and the reaction mixture was extracted with 3 x 150 ml of ethyl acetate.The combined organic phases were washed with 1 x 150 ml water, 2 x 150 ml brine and dried on Na2SO4.The filtrate was evaporated under reduced pressure and the yellow oil was subjected to flash chromatography on silica using linear gradient of ethyl acetate in toluene to afford 12.2 g 2-(R)-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[(2-tert butoxycarbonylaminoethyl)sulfanyl]propanoic acid 22. HPLC analysis (see Figure S7) revealed satisfactory purity (92 % rel.) of 22, which enabled a diazo-transfer reaction to be performed.
12.2 g of 22 was treated with an acidic cocktail, consisting of 40 ml of DCM, 60 ml of TFA and 3 ml of TIPS.When the dramatic release of gas slowed down, the reaction mixture was allowed to react for another 2 hours.Volatile materials were evaporated under reduced pressure and the resulting yellow oil was dissolved in a mixture of 200 ml methanol and 200 ml saturated NaHCO3.Then, a solution of freshly prepared triflic azide in 100 ml of DCM (prepared according to previously published protocol 4 from 50.2 mmol of triflic anhydride and 251 mmol of NaN3) was added dropwise and the reaction mixture was allowed to react overnight at rt.The pH was adjusted to ~ 6 by adding 10 % citric acid and volatile solvents (MeOH, DCM) were evaporated under reduced pressure.Water (200 ml) was added to the resulting dense slurry and the mixture was acidified with 1 M HCl.The reaction mixture was extracted with 3 x 150 ml of ethyl acetate, and the combined organic layers were washed with 150 ml water, 2 x 100 ml brine and dried on Na2SO4.Sodium sulfate was filtered off and the filtrate evaporated.
The solution was placed at -20 °C overnight.The resulting precipitate of triphenylphosphine oxide was filtered off and washed with a mixture of 500 ml of chilled mixture diethyl ether and petroleum ether (1 : 4).The filtrate was evaporated under reduced pressure to give 30 g of bright yellow oil, which was subjected to flash chromatography on silica using a linear gradient ethyl acetate in toluene.Yield 12 g (69 %).Bright yellowish oil.Rf = 0.44 (toluene : ethyl acetate 90 : 10).methylhept-6-enoic acid) were attached manually, as described earlier 6 .
Generally, two couplings for 1h were done for each position of the peptides.The first coupling was done with 4 eq. of Fmoc-amino acid and 4.1 eq. of DIC.The second coupling was done with 4 eq.
of Fmoc-amino acid, 3.8 eq. of HBTU and 7.6 eq. of DIPEA.The Fmoc group was cleaved with 20 % 4-methylpiperidine in DMF for 2 and 20 min.After the couplings and Fmoc group deprotection, the resin was washed 5 x 2 min with 2 ml of DMF.
Peptides were cleaved from the resin by treatment with an acidic cocktail (TFA : TIPS : H2O = 95 : 2.5 : 2.5 v/v) for 2 hours.Crude peptides were precipitated with chilled ether, dried, dissolved in a mixture of CAN and water and lyophilized.Peptides were purified by a Waters HPLC system (Waters 600 with 2487 Dual λ Absorbance Detector), using a Nucleosil 100-7 C8 column (250 x 10 mm, 7 µm, Macherey-Nagel) at a flow rate of 4 ml/min and the following gradient: t = 0 min/10 % B, t = 30 min/100 % B, t = 31 min/10 % B. Solvent A is 0.1 % TFA in water and solvent B is 80 % ACN in A (v/v).
Compounds were detected at 218 and 254 nm.The purity of the final peptides was checked by using HPLC on a Watrex HPLC system (Watrex DeltaChrom™ P200 binary Pump and Wufeng LC-100 UV Detector), using a Nucleosil 120-5 C8 column (250 x 4.6 mm, 5 µm, Macherey-Nagel) at a flow rate of 1 ml/min, with the same gradient and solvents as described for the preparative HPLC.

Formation of disulfide bridge in peptide 1 (Scheme S1)
Peptide 1 with Acm protection of cysteine thiol groups was dissolved at a concentration 10 -3 M in 40 % AcOH.Iodine (25 eq.) in AcOH was added.The resulting solution was stirred at RT for 20 min and then 1 M ascorbic acid in water was added until the dark iodine color disappeared.Peptide 1 was purified as described above.

Staple formation by ring-closing olefin metathesis (RCM) in peptide 2 (Scheme S2)
RCM was performed on the resin-bound peptide with the N-terminal amino acid protected with Fmoc.RCM was carried out using freshly prepared 6 mM Grubbs catalyst 1st generation (20 mol% regarding the resin substitution) in 1,2-dichloroethane (DCE).The resin was agitated under nitrogen at rt for 2 h protected from light.The reaction was repeated once more and a test cleavage with MS analysis was performed.If the RCM reaction was incomplete, it was repeated until completion.Thereafter, the Fmoc group was removed, and the peptide cleaved from resin and purified as described above.Synthetic scheme S10 shows the preparation of peptide 2.

Figure S1 .
Figure S1.Structures of peptides 1-5.The parts by which the peptides differ are in blue.
Figure S2.HPLC profile of purified compound 7 using a gradient from Method 1.

Figure S3 .
Figure S3.HPLC profile of purified compound 9 using a gradient from Method 1.

Figure S4 .
Figure S4.HPLC profile of purified compound 11 (prepared from compound 10) using a gradient from

Figure S6 .
Figure S6.HPLC profile of purified compound 19 using a gradient from Method 1.

Figure S7 .
Figure S7.HPLC profile of crude compound 22 using a gradient from Method 1.

Figure S8 . 5
Figure S8.RP-HPLC profile of purified compound 23 using a gradient from Method 1.

CuAAC
Scheme S3. Preparation peptide 3. o denotes C α atom of non-standard amino acids.
Scheme S4. Preparation peptide 4. o denotes C α atom of non-standard amino acids.

Figure S19 .
Figure S19.Representative Western blots for the abilities of peptides to stimulate IR-A phosphorylation