Ligations of O -acyl threonine units to give native peptides via 5, 8, 9-and 10-membered cyclic transition states

N -Acyl threonine isopeptides undergo acyl transfer in chemical ligations via 5, 8, 9-and 10-membered cyclic transition states to yield natural peptides, representing the first examples of successful isopeptide ligations from N-acyl threonine units


Introduction
Synthetic methods for peptides are of great interest: native chemical ligation (NCL), first reported by Wieland 1 and developed by Kent 2,3 is a chemo-and regio-selective reaction of a peptide-thioester with a N-terminal Cys-peptide that produces a long chain polypeptide with a native amide bond at the ligation site through rapid S-to N-acyl transfer within the initial thioester.
The chemical basis of such ligations is a regiospecific coupling of a C-terminal electrophile of one peptide with a N-terminal nucleophile of a second peptide, without any protection or activation step.The peptide segments can be synthetic [4][5][6][7] or biosynthetic in origin. 8,9Many organic reactions have been enabled by ligations via a variety of chemical linkages, including amide, 10 thioester, 11 thiazolidine, 12 oxaproline, 13 oxime, 14 hydrazone, 15 and thioether moieties. 16CL development as a synthetic tool for building peptides depends on additives to increase both ligation rates (from hours to days) and yields.8][19][20][21][22] In addition, the low abundance of cysteine (1.7% of the residues in protein sequences) is a major drawback of this methodology since NCL is restricted to Cysteine residues.
To address this limitation, efficient synthetic approaches have been developed 20,[23][24][25][26][27][28][29][30][31] including our recent report on classic O-to N-acyl shifts via a 8 and 11-membered transition states in O-acyl serine 32 and 12-to 19-membered cyclic transition states in O-acyl tyrosinepeptides. 33Thus, "traceless" chemical ligation involving O-to N-acyl shift (at Ser and Tyr site) involving neither Cys nor an auxiliary group at the ligation site is possible. 34ur focus has been on threonines, each possessing the 1,2-hydroxylamine bifunctionality 34 (corresponding to the SH/NH 2 in cysteine) and thus affording chemoselective ligation by O-to N-acyl transfer without the need of cysteine residues.We now report migrations of acyl groups from O-acylated threonine isopeptides via 5-8-, 9-and 10-membered cyclic transition states to give natural peptides.

Results and Discussion
We synthesized the intermediate mono-isodipeptide 4 to study the O-acyl migration from the oxygen to the N-terminal group of threonine amino acid sequence via a 5-membered transition state and also to serve as starting material to study the possibility of O-to N-acyl migration via 8-, 9-and 10-membered cyclic transition states.Compound 4 on coupling with α-, βor γ-amino acids gave the starting mono-isotripeptides (8a-c) needed for the ligation studies.To avoid steric problems and enhance migration rates, we used a glycine unit at the N-terminus of monoisotripeptide 8a and βand γ-amino acid units in mono-isotripeptides 8b and 8c respectively.

Study of ON acyl migration via a 5-membered cyclic transition state
Chemical ligation via a 5-membered cyclic transition state was investigated by subjecting monoisodipeptide 4 to microwave irradiation at 50 °C, 50 W for 3 h using aqueous conditions (pH 7.3, 1 M buffer strength) as well as basic condition (DMF-piperidine).(Scheme 2) The reaction was allowed to cool to room temperature and acidified with 2 N HCl to pH = 1.The mixture was extracted with ethyl acetate (3 x 20 mL), the combined organic extracts were dried over MgSO 4 and solvent was removed under reduced pressure.The ligation mixture was weighed and then a solution in methanol (1 mg mL -1 ) was analyzed by HPLC-MS.HPLC-MS (ESI) analysis of the ligated mixtures showed both in aqueous buffer as well as DMF-piperidine the expected migration product 5 (rt 38.08, m⁄z 325.0) together with intermolecular bis-acylation product 6 (rt 60.58, m⁄z 530.1) in.HPLC-HRMS, via (+) ESI-MS, confirmed that the ligated product 5 (rt 38.08, m⁄z 325.0) and starting mono-isohexapeptide 4 (rt 34.61, m⁄z 325.0) produced different MS patterns.The result indicates the formation of ligated product 5 with 89% and 86% relative abundance in aqueous buffer and DMF-piperidine respectively, which infers that O-to N-acyl group migration via a 5-membered transition state is preferred over intermolecular acylation.Thus, acyl migration via a 5-membered cyclic transition state is feasible and may afford a promising approach for the synthesis of native peptide analog containing threonine.

Preparation of mono-isotripeptide 9a-c
Unprotected mono-isodipeptide 4, on coupling with benzotriazolide of Boc protected α-, βor γamino acids 7a-c at room temperature in acetonitrile in the presence of 1.5 equiv. of triethylamine gave mono-isotripetides 8a-c in good yields.Compounds 8a-c on deprotection with dioxane-HCl solution afforded unprotected mono-isotripeptides 9a-c in good yields, which were fully characterized by 1 H, 13 C NMR and HRMS analysis (Scheme 3).Scheme 3. Synthesis of mono-isotripeptides 9a-c.

Study of ON acyl migration via a 8-, 9-and 10-membered cyclic transition state
When treated under aqueous conditions, (pH 7.3, 1 M buffer strength, MW 50 o C, 50 W, 3 h), 9a-c did not form the desired ligated products 10a-c or bis-acylated products 11a-c (Scheme 4, Table 1).Microwave irradiation of 9b in piperidine-DMF at 50 °C, 50 W for 3 h gave 10b (57%) and 11b (36%) as observed by HPLC-MS.We also observed bis-acylated product 11c in case of 9c.The retention times and fragmentation patterns of 9b and 10b were and HPLC-MS, via (−)ESI-MS/MS, confirmed that 9b and 10b, had different fragmentation patterns, thus proving the formation of intramolecular ligated product 10b via a 9-membered TS (see Supplementary Material).Scheme 4. of ON acyl migrations via a 8-, 9-and 10-membered cyclic transition states.

Conclusions
Threonine isopeptides with α-, β-, or γ-amino acid units were synthesized and acyl migration form threonine oxygen to terminal NH 2 was studied under microwave irradiation.From the experiments two conclusion cab be drawn: (i) intramolecular acyl transfer through 5-and 9membered transition states was favoured over 8-and 11-membered transition state and (ii) intramolecular acyl migration occur more readily in basic non-aqueous media relative to aqueous buffered conditions.

Experimental Section
General.All commercial materials (Aldrich, Fluka) were used without further purification.All solvents were reagent grade or HPLC grade (Fisher Solvents were dried using standard protocols kept under a dry atmosphere of nitrogen.Melting points were determined on capillary point apparatus equipped with a digital thermometer and are uncorrected. 1H NMR and 13 C NMR spectra were recorded in CDCl 3 , DMSO-d 6 or CD 3 OD using a 300 MHz spectrometer (with TMS as an internal standard) at ambient temperature unless otherwise stated.All microwave assisted reactions were carried out with a single mode cavity Discover Microwave Synthesizer (CEM Corporation, NC).The reaction mixtures were transferred into a 10 mL glass pressure microwave tube equipped with a magnetic stirrer bar.The tube was closed with a silicon septum and the reaction mixture was subjected to microwave irradiation (Discover mode; run time: 60 sec.; PowerMax-cooling mode).HPLC-MS analyses were performed on reverse phase gradient Phenomenex Synergi Hydro-RP (2.1×150 mm; 5 um) + guard column (2×4 mm) or Thermoscientific Hypurity C8 (5um; 2.1×100 mm + guard column) using 0.2% acetic acid in H 2 O/methanol as mobile phases; wavelength = 254 nm; and mass spectrometry was done with electro spray ionization (ESI).

N-(4-Aminobutanoyl)-O-(((benzyloxy)carbonyl)-L-alanyl)-L-threonine hydrochloride (9c).
Off white sticky solid; Yield: 96%; General procedure for the synthesis of ligated products 5 and 6 in buffer.Isotetrapeptide (4) (0.20 mmol) were each suspended in deoxygenated phosphate buffer (NaH 2 PO 4 /Na 2 HPO 4 ) (1 M, pH = 7.4, 8 mL) and irradiated with microwave (50 °C, 50 W, 3 h).Each reaction mixture was allowed to cool to room temperature, acidified with 2 N HCl to pH = 1, and extracted with ethyl acetate (3×10 mL).The combined organic extracts were dried over Na 2 SO 4 and the solvent was removed under reduced pressure.Each ligation mixture was weighed and a solution in methanol (1 mg mL -1 ) was analysed by HPLC-MS.General procedure for synthesis of ligated products 5, 6, 10b and 11b in DMF-piperidine.Isotripeptides (4 & 9) (0.20 mmol) were each dissolved in a mixture of DMF-piperidine (5mL/1.5mL)and the mixture was irradiated with microwave (50 °C, 50 W, 3 h) in a microwave tube.After cooling to room temperature the reaction mixtures were acidified with 2 N HCl to pH = 1.Each mixture was extracted with ethyl acetate (3×10 mL), the combined organic extracts were dried over sodium sulfate and the solvent removed under reduced pressure.Each ligation mixture was weighed and then a solution in methanol (1 mg mL -1 ) was analysed by HPLC-MS.