Chemical synthesis of γ-secretase activating protein using pseudoglutamines as ligation sites.

Chemical synthesis of gamma-secretase activating protein using pseudo-glutamines as ligation sites. Paul W. R. Harrisa,b,c C. Squireb,c, Paul G. Youngc and Margaret A. Brimblea,b,c a

School of Chemical Sciences, The University of Auckland, 23 Symonds St, Auckland 1010, New Zealand; b Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1010, New Zealand; c School of Biological Sciences, The University of Auckland, 3A Symonds St, Auckland 1010, New Zealand. Correspondence should be addressed to: Dr Paul W. R. Harris or Prof. Margaret A. Brimble, School of Chemical Sciences, University of Auckland, 23 Symonds St, Auckland 1142, New Zealand. Email: [email protected]; [email protected]; Phone: +64 9238259; Fax +64 9 3737 422 Abstract The chemical synthesis of analog of a novel gamma-secretase activating protein, which may play a pivotal role in the formation of amyloid peptides, the precursor to Alzheimer’s disease, is described. The linear polypeptide sequence, consisting of 121 amino acids was assembled from four unprotected peptide building blocks using a convergent ligation-based synthesis. A strategic mutation of three glutamine residues to cysteine enabled the ligations, and the cysteines were subsequently converted to pseudo-glutamines, to mimic the native glutamine. The full length unfolded protein was obtained in milligram amounts and was demonstrated to be homogeneous by liquid chromatography and mass spectrometry. Keywords: Alzheimers; γ-Secretase; Ligation; pseudo-Glutamine. Introduction Alzheimer’s disease (AD) is a degenerative condition that affects 36 million people worldwide and for which there are currently no effective treatments.1 In the US alone the cost of care is estimated to be >US$220 billion per year. AD is thought to be associated with the formation of insoluble plaques in the brain predominantly by two 4 KDa amyloid beta peptides, Aβ40 and the more plaque-forming Aβ42 congener.2 These, in turn are secreted by the successive action of β- and γ-secretases on the amyloid precursor protein (APP), an integral membrane protein (Figure 1). Therefore, the inhibition of γ-secretase on the processing of APP may lead to a decrease in the production of the amyloid beta peptides; however γ-secretase is also integral to many other important biological functions therefore any potential inhibitors need to be highly selective.3 Recently, a 16 kDa protein, coined γ secretase activating protein (GSAP) was discovered and shown to increase amyloid production by interaction with γ-secretase and APP but importantly did not affect γsecretases’ normal processing functions.4 Moreover, it has been disclosed that Imatinib (Gleevac), currently used to treat chronic myeloid leukemia, selectively interacts with GSAP, This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/bip.22600 © 2014 Wiley Periodicals, Inc. This article is protected by copyright. All rights reserved.

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Biopolymers: Peptide Science

and prevents its binding to APP5 thus lowering amyloid beta production suggesting that GSAP may be a valid therapeutic target for the control of Alzheimer’s disease. However, it was reported that while reducing GSAP in cells decreased amyloid beta levels, overexpression of GSAP did not have any effect on amyloid production6. These findings and others7,8 have led to doubts over the exact role GSAP plays in modulation of amyloid beta peptide production. To clarify the role of GSAP and to more properly define its interaction with Imatinib which may lead to the development of other potential inhibitors, we describe herein a robust chemical synthesis of the 16 KDa GSAP polypeptide core using native chemical ligation of four peptide building blocks. Our convergent synthesis used non-native cysteines residues as ligation handles that were globally S-alkylated with iodoacetamide affording a pseudo glutamine at the three ligation points. Multi milligram quantities of the linear polypeptide were obtained which was characterised by mass spectrometry and liquid chromatography.

Figure 1. Enzymatic processing of the amyloid precursor protein (APP) by β-secretase and γsecretase leading to amyloid peptides. Experimental Methods Materials All solvents and reagents were used as supplied. O-(Benzotriazol-1-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate (HBTU), (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate) (HATU), and S-tritylmercaptopropionic acid were purchased from GL Biochem (Shanghai, China). Dimethylformamide (DMF) (AR grade) and acetonitrile (HPLC grade) were purchased from Scharlau (Barcelona, Spain). N,N’diisopropylethylamine (DIPEA), piperidine, 2,2′-(ethylenedioxy)diethanethiol (DODT), diisopropylcarbodiimide (DIC), and triisopropylsilane (TIPS), 4-mercaptophenylacetic acid (MPAA) were purchased from Aldrich (St Louis, MO) and N-methylpyrrolidine (NMP) was purchased from Fluka (Buchs, Switzerland). Guanidine hydrochloride (Gn.HCl) and tris(2carboxyethyl)phosphine hydrochloride (TCEP.HCl) were obtained from AK Scientific (Union City, CA). Trifluoroacetic acid (TFA) was purchased from Oakwood Chemical (River Edge, SC). 1-Hydroxybenzotriazole hydrate (HOBt.H2O) was purchased from Advanced Chemtech (Louisville, KY). Anhydrous hydrogen fluoride was obtained from Matheson Trigas (Basking Ridge, NJ). Aminomethyl polystyrene (AM-PS) resin was synthesised “in house” as described.9 Boc-Ala-PAM (PAM = phenylacetamidomethyl) linker was purchased from Polypeptides (Strasbourg, France). Fmoc-amino acids were purchased from GL Biochem with the following side chain protection: Fmoc-Arg(Pbf)-OH (Pbf = 2,2,4.6,7pentamethyldihydrobenzofuran-5-sulfonyl), Fmoc-Asn(Trt)-OH (Trt = triphenylmethyl), Fmoc-Asp(tBu)-OH (tBu = tert-butyl), Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)OH, Fmoc-His(Trt)-OH, Fmoc-Met(O)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-

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Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Trp(Boc)-OH. Boc-amino acids were purchased from Polypeptides with the following side chain protection: Boc-Arg(Tos)-OH (Tos = ptoluenesulfonyl), Boc-Asp(cHex)-OH (cHex = cyclohexyl), Boc-Cys(4-MeBn)-OH (Bn = benzyl), Boc-Asn(Xan)-OH (Xan = Xanthyl), Boc-Glu(cHex)-OH, Boc-His(DNP)-OH (DNP = dinitrophenyl), Boc-His(Tos)-OH.DCHA (DCHA = dicyclohexylamine), Boc-Met(O)-OH, BocLys(2-Cl-Z)-OH (Z = benzyloxycarbonyl), Boc-Ser(Bn)-OH, Boc-Thr(Bn)-OH, Boc-Tyr(2-Br-Z)OH, Boc-Trp(CHO)-OH. HPLC and LC-MS LC-MS was performed using an Agilent (Santa Clara, CA) 1100 Compact HPLC equipped with a single wavelength UV detector at 214 nm with an in-line Hewlett Packard (Palo Alto, CA) 1100MSD mass spectrometer using ESI in the positive mode. An Agilent Zorbax 300SB-C3 3.5µ; 3.0 x 150 mm) column was used using a linear gradient of 5% to 65%B over 21 mins at 40 °C at 0.3 ml/min. The solvent system used was A (0.1% formic acid in H2O) and B (0.1% formic acid in acetonitrile). Peptides were purified using a Dionex Ultimate 3000 system equipped with a Foxy Jr fraction collector using a Gemini C18 (5 μ; 10.0 x 250 mm) column (Phenomenex) at a flow rate of 5 mL/min or a X-Terra (19 x 300 mm) column (Waters) at a flow rate of 10mL/min and eluted on the slow gradient protocol10 based on the analytical HPLC profile. Fractions were collected, analysed by either HPLC or LC-MS, pooled and lyophilised. Synthesis of Peptide Fragments i): GSAP 734Leu-760Gly-COS-Ph-CH2CO2H 1: AM-PS resin (0.2 mmol, 0.2 g, loading 1 mmol/g) was shaken with Boc-Ala-PAM (0.4 mmol) and DIC (0.4 mmol) in dichloromethane (5 mL) for 1 h, washed with dichloromethane and dried. The Kaiser test was negative. The Boc group was removed with TFA (1 x 2 mins). DIPEA (0.4 ml) was added to S-tritylmercaptopropionic acid11 (0.48 g, 1.17 mmol) dissolved in 0.4 M HCTU (2.61 mL) and the solution added to the resin. After shaking for 20 mins, the resin was washed with DMF and the trityl group was removed with 95% TFA 2.5% TIPS, 2.5% water (v/v/v, 2 x 1 mins) and washed with DMF. Boc-Gly-OH (192 mg, 1.1 mmol), HCTU (435 mg, 1.05 mmol) were dissolved in DMF (2.3 mL). DIPEA (0.4 mL) was added and the solution added to the resin and shaken for 1 h. The peptide was then elongated manually by “in situ” neutralisation Boc SPPS12 using TFA (1 x 2 mins) for Boc removal and HCTU/DIPEA as coupling reagents. 700 mg of peptidyl resin was obtained which was cleaved using HF/p-cresol (20:1, v/v) for 1 h at 0 °C to afford 374 mg of crude 734Leu-760Gly-S-CH2CH2CO-Ala-OH ([M+H]3+ Calc. 1096.0, Found 1095.7 (see supporting information S1). 734Leu-760Gly-S-CH2CH2CO-Ala-OH (108 mg, 32.6 mmol) was dissolved in a buffer containing 6 M Gn.HCl, 0.2 Na2HPO4, 20 mM TCEP.HCl and 100 mM MPAA (15 mL) pH = 6.5 and the solution stood for 6 hours at room temperature. The solution was acidified with 5 M HCl to pH = 3, 0.1% TFA in water (30mL) was added, the residual MPAA was extracted with ether (3 x 100 mL) and the crude thiophenylester (96 mg)


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recovered by solid phase extraction. Purification by RP-HPLC on a X-Terra preparative column (19 x 300 mm, 10 ml/min) using a gradient of 5 to 30%B at 2%B/min then 30-60%B at 0.25%B/min afforded 734Leu-760Gly-COS-Ph-CH2CO2H 1 (24.1 mg, 22.5% ), [M+H]3+ Calc. 1092.9, Found 1092.7 (see supporting information S2). ii) GSAP 761Cys-764Cys(acm)-767Trp(CHO)-769His(DNP)-780His(DNP)-790Lys-COS-CH2CH2CO-AlaOH 2: Boc-Ala-PAM resin (0.3 mmol) was prepared as above and the Boc group was removed with TFA (1 x 2 mins). DIPEA (0.4 ml) was added to S-tritylmercaptopropionic acid (0.48 g, 1.17 mmol) dissolved in 0.4 M HATU (2.61 mL) and the solution added to the resin. After shaking for 5 mins, the resin was washed with DMF and the trityl group was removed with 95% TFA 2.5% TIPS, 2.5% water (v/v/v, 2 x 1 mins) and washed with DMF. Boc-Lys(2-ClZ)-OH (684 mg, 1.65 mmol), HATU (594 mg, 1.56 mmol) were dissolved in DMF (3.5 mL). DIPEA (0.6 mL) was added and the solution added to the resin and shaken for 1 h. The peptide was then elongated manually by “in situ” neutralisation Boc SPPS using TFA (1 x 2 mins) for Boc removal and HATU/DIPEA as coupling reagents. Boc-Met(O) was used for Met. At the completion of the sequence, 1.14 g of resin was obtained and 0.5 g of the peptide resin was treated with a solution of Bu4NI (554 mg, 1.5 mmol) and Me2S (120 µL, 1.5 mmol) in TFA (50 mL) (2 x 1 min) to effect on-resin conversion of Met(O) to Met.13 The resin was drained, washed with DMF, MeOH, dried under vacuum and the peptide cleaved using HF/p-cresol (20:1, v/v) for 1 h at °C to afford 287 mg of crude peptide. The procedure was repeated on the remaining resin (ca. 0.6 g) to afford a further 310 mg following HF cleavage. Purification by RP-HPLC using the one-step slow gradient chromatography procedure gave GSAP 761Cys-764Cys(acm)-767Trp(CHO)-769His(DNP)-780His(DNP)-790Lys-COS-CH2CH2CO-Ala-OH 2 (59 mg, 4.6% yield based on resin loading), [M+H]6+ Calc. 708.6, Found 708.6 (see supporting information S3). iii) GSAP 791Thz-823Asn-COS-CH2CH2CO-(Arg)6-Ala-OH 3: Boc-Ala-PAM resin (0.2 mmol) was prepared as above and elongated manually by “in situ” neutralisation Boc SPPS using TFA (1 x 2 mins) for Boc removal and HCTU/DIPEA as coupling reagents to incorporate the hexa arginine tag. The Boc group was removed with TFA (1 x 2 mins). DIPEA (0.4 ml) was added to S-tritylmercaptopropionic acid (0.48 g, 1.17 mmol) dissolved in 0.4 M HCTU (2.61 mL) and the solution added to the resin. After shaking for 5 mins, the resin was washed with DMF and the trityl group was removed with 95% TFA 2.5% TIPS, 2.5% water (v/v/v, 2 x 1 mins) and washed with DMF. Boc-Asn(Xan)-OH (453 mg, 1.1 mmol), HCTU (432 mg, 1.045 mmol) were dissolved in DMF (2.3 mL). DIPEA (0.4 mL) was added and the solution added to the resin and shaken for 1 h. The peptide was then elongated manually by “in situ” neutralisation Boc SPPS using TFA (1 x 2 mins) for Boc removal and HCTU/DIPEA as coupling reagents. Boc-Met(O) was used for Met. At the completion of the sequence, 1.08 g of resin was obtained which was treated with a solution of Bu4NI (554 mg, 1.5 mmol) and Me2S (120 µL, 1.5 mmol) in TFA (50 mL) (2 x 1 min) to effect on-resin conversion of Met(O) to Met. The resin was drained, washed with DMF, MeOH, dried under vacuum and the peptide cleaved using HF/p-cresol (20:1, v/v) for 1 h at °C to afford 456 mg of crude peptide. Purification by



RP-HPLC using the one-step slow gradient chromatography procedure gave GSAP 791Thz823 Asn-COS-CH2CH2CO-(Arg)6-Ala-OH 3 (37 mg, 3.75% yield based on resin loading), [M+H]7+ Calc. 703.5 , Found 703.3 (see supporting information S4). v) GSAP 824Cys-854Leu-OH 4: AM-PS resin (0.1 mmol, 0.1 g, loading 1 mmol/g) was shaken with Fmoc-Leu-HMPP (0.2 mmol) and DIC (0.2 mmol) in dichloromethane (2.5 mL) for 2 h, washed with dichloromethane and dried. The Kaiser test was negative. The peptide was elongated using a CEM Liberty 12 microwave peptide synthesiser using 5% (w/v) piperazine containing 0.1 M 6-Cl-HOBt as Fmoc deblocking reagent and HCTU/DIPEA as coupling reagents. The microwave settings and reaction times used have been described previously. 14 At the completion of the synthesis, 471 mg of resin was obtained which was cleaved with 94% TFA, 1% TIPS, 2.5% water, 2.5% DODT (10 mL, v/v/v/v) for 3 h and the peptide precipitated by dilution with cold diethyl ether and recovered by centrifugation to afford 265 mg of crude peptide. The peptide was dissolved in 50% aq. acetonitrile containing 0.1% formic acid (v/v) to give a final concentration of 3.5 mg/ml and shaken at 37 °C for 21 h to convert the methionine modified peptide15 to the native methionine. Purification by slow gradient RP-HPLC at 60 °C afforded GSAP 824Cys-854Leu-OH 4 (83 mg), [M+H]3+ Calc. 1135.9, Found 1135.7,( see supporting information S5). Chemical Ligation i) N-terminal fragment 734

Leu-760Gly-COS-Ph-CH2CO2H 1 (31 mg, 9.46 µmol) and GSAP 761Cys-764Cys(acm)767 Trp(CHO)-769His(DNP)-780His(DNP)-790Lys-COS-CH2CH2CO-Ala-OH 2 (60.5 mg, 14.1 µmol) were dissolved in a degassed aqueous buffer (9 mL) containing 0.2 M Na2HPO4 and 6 M Gn. HCl at pH 6.4. The solution was stood for 2 h, 10% (v/v) 2-mercaptoethanol was added and the mixture left for a further 1.5 h to remove the DNP protecting groups and effective transthioesterification. The ligation product 734Leu-764Cys(acm)-767Trp(CHO)-790Lys-COSCH2CH2OH (73 mg, >100% recovery) 5a was isolated by solid phase extraction. Purification by HPLC (C4 Jupiter 10 x 250 mm, 5 ml/min) using a gradient 5 to 20%B over 7 mins then 20 to 50%B over 300 mins afforded 734Leu-764Cys(acm)-767Trp(CHO)-790Lys-COS-CH2CH2OH 5a (15.6 mg, 24 %, based on the amount of 1 used). ESI-MS, [M+H]9+ Calc. 770.12, Found 769.9, [M+H]8+ Calc. 866.3, Found 866.1. ii) C-terminal fragment GSAP 791Thz-823Asn-COS-CH2CH2CO-(Arg)6-Ala-OH 3 (39.45 mg, 7.99 µmol) and GSAP 824Cys854 Leu-OH 4 (32.7 mg, 9.60 µmol) were dissolved in an aqueous buffer (8 mL) containing 0.2 M Na2HPO4, 6 M Gn.HCl, 100 mM MPAA and 20 mM TCEP.HCl at pH 6.9. After 1 h 0.2 M MeONH2.HCl (132.4 mg) was added, the pH adjusted to 4.0 with 5 M HCl and the mixture stored at room temperature for 3.5 h. Purification by HPLC (Diphenyl 10 x 250 mm, 5 ml/min, 60 °C) using a gradient 5 to 35%B over 16 mins then 35 to 60%B over 160 mins gave


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791

Cys-854Leu-OH 6 (9.2 mg, 16.1%). ESI-MS, [M+H]6+ Calc. 1200.4, Found 1200.6, [M+H]7+ Calc. 1029.0, Found 1029.3. iii) Ligation of N and C terminal fragments 734

Leu-764Cys(acm)-767Trp(CHO)-790Lys-COS-CH2CH2OH 5a (6.68 mg, 0.928 µmol) and 791Cys854 Leu-OH 6 (6.72 mg, 0.970 µmol) were dissolved in an aqueous buffer (1 mL) containing 0.2 M Na2HPO4, 6 M Gn. HCl, 178 mM MPAA and 20 mM TCEP.HCl at pH 6.8. The solution was stood at room temperature for 17 h and a degassed solution (1 ml) of 400 mM 2iodoacetamide in 0.2 M Na2HPO4, 6 M Gn. HCl was added After 15 mins, sodium 2sulfanylethanesulfonate (82 mg) was added, the reaction stood for 5 mins and then cooled to 0 °C. Piperidine (0.9 mL) and 2-mercaptoethanol (1.8 mL) were added and the solution stored for 1 h at 0 °C and the product (8.72 mg) was recovered by solid phase extraction. The crude polypeptide (8.7 mg, ca. 0.614 µmol) was dissolved in 50% aq. CH3CN containing 0.1% TFA (6 mL), silver acetate (30.7 mg, 300 eq, 0.183 mmol) was added, the mixture shaken at room temperature for 3 h and DTT (185 mg 1.2 mmol) was added. After 30 mins the peptide was recovered by centrifugation, the supernatant removed and replaced with 50% aq. CH3CN containing 0.1% TFA (8 mL) and the procedure repeated. The combined supernatants were purified by HPLC (Diphenyl, 10 x 250 mm, 5 ml/min, 50 °C) using a gradient 5 to 30%B over 15 mins then 30 to 60%B over 60 mins gave GSAP 734Leu-761ᴪGln791 ᴪGln-824ᴪGln-854Leu-OH 7 (4.15 mg, 30.5%), observed mass 14108.7± 1.4 Da., calculated mass 14112.3 Da. Results and Discussion GSAP is believed to be cleaved from the larger 98 kDa precursor protein that undergoes rapid proteolysis to afford the putative biologically-active C-terminal-derived 16 kDa protein. The primary amino acid sequence of this fragment, comprising residues Leu734Leu854 is depicted in Figure 1; the 121 Aa length is well within what can be achieved by modern chemical protein synthesis. Although this fragment has been expressed using recombinant technology, incorporating a C-terminal His6 purification tag, we sought to develop a reliable chemical synthesis principally using native chemical ligation (NCL).16,17 NCL, the chemoselective amide bond forming reaction between a peptide bearing a Cterminal thioester and one bearing an N-terminal cysteine, allows for greater flexibility in protein synthesis as site specific replacement with e.g. non-coded amino acids can be accomplished readily.

Figure 2. Amino acid sequence of the 16 KDa GSAP protein. Examination of the amino acid sequence (Figure 2) reveals that the sole cysteine residue (764Cys) was not an appropriate ligation site as the resultant C-terminal fragment (ca .90 residues) would be unable to be prepared satisfactorily by stepwise solid phase peptide



synthesis. We therefore devised a convergent synthesis using glutamine (Gln) as a ligation point as Gln regularly spans the sequence and conveniently results in more manageable polypeptide fragments consisting of ca. 30 residues. However, the required γ18 mercaptoglutamine building block that contains an appropriately placed thiol to facilitate NCL and suitably protected for SPPS is not commercially available and entails a multi-step (>10 steps) synthesis; a non-trivial post-ligation desulfurisation step(s) to regenerate the native Gln would also be required.19 Therefore we mutated the 761Gln, 791Gln and 824Gln residues to Cys to permit the NCL reaction; the resultant thiols can subsequently be globally capped with 2-iodoacetamide to afford the pseudo-Gln (ᴪ-Gln) as a glutamine surrogate.20 This strategy is outlined in Scheme 1. Thus it was envisaged that the N-terminal half of GSAP thioester (5 or 5a) could be accessed via a kinetically controlled ligation21 (KCL) between 734Leu-760Gly-thiophenylester (1) and the N-terminal cysteinyl peptide 761Cys-790Lys-thioalkylester (2) by exploiting the reactivity differences between thiophenylesters and alkyl thioesters.22 The resultant fragment 5 would still bear a C-terminal thioalkylester handle to allow the final NCL. The masking of the native 764Cys with an acetamidomethyl (Acm) group in 2 was two-fold; a) to circumvent potential side reactions during the KCL reaction which is carried out in the absence of thiols which serve to reverse unproductive thiolactone formation and b) avoid unwanted reaction with the alkylating reagent when capping the unprotected cysteines. The C-terminal region 6 can be prepared from 791Thz-823Asn thioalkylester (Thz = thialozidine) (3) and 824Cys-854Leu-OH (4) using an NCL reaction with an aryl thiol additive. In this case, the masked cysteine (Thz) can be revealed23 at the completion of the ligation to avoid oligomerisation or cyclisation during NCL. A final NCL reaction of 5 and 6, alkylation of the three cysteines by iodoacetamide and Acm removal from the 764Cys would furnish the full length GASP ᴪ-Gln analogue 7 for re-folding and biophysical characterisation.

Scheme 1. Convergent ligation strategy for the chemical synthesis of ᴪ-Gln-GSAP 7. Polypeptides 1, 2 and 3 containing the base sensitive C-terminal thioester were prepared by “in situ” neutralisation Boc SPPS on the HF labile PAM resin to circumvent problems encountered when preparing peptide thioesters using Fmoc SPPS.24,25 Thiophenylester 1, 734 Leu-760Gly-COS-Ph-CH2CO2H ([M+H]3+ Calc. 1092.9, Found 1092.7) was obtained by transthioesterification21 with 4-mercaptophenylacetic acid (MPAA)26 of the corresponding thioalkylester, 734Leu-760Gly-S-CH2CH2CO-Ala-OH which was easier to prepare and handle using Boc solid phase synthesis. Polypeptide 2, 761Cys-764Cys(acm)-767Trp(CHO)-769His(DNP)780 His(DNP)-790Lys-COS-CH2CH2CO-Ala-OH ([M+H]6+ Calc. 708.6, Found 708.6) contains two histidine residues that were protected during SPPS using the dinitrophenyl group (DNP). The DNP group is stable to HF and the presence of the C-terminal thioester in 2 precluded the removal of DNP using an on-resin procedure with PhSH prior to HF cleavage.


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Conveniently, though, the DNP group can be removed under NCL conditions. The alternative His protecting groups, namely, benzyloxymethyl (Bom) or p-toluenesulfonyl (Tos) both undergo side reactions during either chain assembly or HF cleavage.25 Peptide building block 3, 791Thz-823Asn-COS-CH2CH2CO-(Arg)6-Ala-OH, ([M+H]7+ Calc. 703.5 , Found 703.3) was prepared with a polyarginine solubilising tag to aid purification by HPLC. In the absence of the hexa-Arg tag,27 the resultant peptide, 791Thz-823Asn-COS-CH2CH2CO-Ala-OH 3a, was only recovered in very low yield (1.3 %) following HPLC purification and was problematic to use in NCL. Peptide 4, 824Cys-854Leu-OH ([M+H]3+ Calc. 1135.9, Found 1135.7), a non thioester was assembled by microwave Fmoc SPPS but using 5% piperazine/0.1 M Cl-HOBt in DMF as a milder Fmoc deblocking reagent28 to avoid undesired aspartimide formation of the Asp-Ala and Asp-Asn motifs.29 Additionally, during TFA mediated cleavage from the resin using DODT as a thiol scavenger, we observed a methionine modified by-product (+ 117 Da) which occurs via decomposition of DODT and subsequent alkylation of the thioether.15 This was able to be satisfactorily reversed by gentle heating of the crude polypeptide in dilute mild acid. With all four polypeptide fragments in hand, the convergent synthesis of GSAP was undertaken under standard KCL or NCL conditions. KCL of 1 (1 mM) and 2 (1.7 mM) was effected at pH = 6.3 in degassed 6M Gn. HCl/0.2 M phosphate in the absence of an auxiliary thiol to promote the ligation product 5. After 2h, LC-MS showed a complex product distribution, consisting of thioester 2, ligation product 5 and other unidentified peaks, presumably resulting from unwanted KCL-specific side reactions such as thiolactone formation and branched thioester formation.20 This mixture was suitably resolved by addition of 2-mercaptoethanol 10% (v/v) directly to the reaction mixture following KCL; conveniently the two DNP groups on 769His and 780His were smoothly removed under these reaction conditions. Furthermore, the C-terminal thioester moiety (–SCH2CH2-Ala-OH) was also quantitatively transthioesterified by 2-mercaptoethanol to afford the corresponding SCH2CH2-OH thioester with minimal hydrolysis.30 Purification by RP-HPLC afforded 5a in a reasonable yield of 24% at >95% purity (observed mass 6919.9 ± 0.91 Da., calculated mass 6922.1 Da.).

Figure 3. Kinetically controlled ligation of 734Leu-760Gly-COS-Ph-CH2CO2H 1 and GSAP 761Cys764 Cys(acm)-767Trp(CHO)-769His(DNP)-780His(DNP)-790Lys-COS-CH2CH2CO-Ala-OH 2 monitored by HPLC at 210 nm. A: after 2 h reaction; B: after 1 h treatment with 2-mercaptoethanol; C LC-MS of the purified product

NCL of 3 (1 mM) and 4 (1.2 mM) at pH = 6.9 using 100 mM MPAA and 20 mM TCEP.HCl in 6M Gn. HCl/0.2 M phosphate was essentially complete after 1 h affording the expected ligation product (observed mass 7205.4 ± 0.81 Da., calculated mass 7208.2 Da.).



Subsequent unmasking of the N-terminal thialozidine to the N-terminal cysteine with 0.2 M MeONH2.HCl at pH = 4 was quantitative after 4 h. Following purification by RP-HPLC at elevated temperature (60 °C) to ensure satisfactory recovery of the hydrophobic peptide, 6 was obtained in 16% yield (observed mass 7199.6 ± 0.81 Da., calculated mass 7196.1 Da.).

Figure 4. Native chemical ligation of GSAP 791Thz-823Asn-COS-CH2CH2CO-(Arg)6-Ala-OH and GSAP 824Cys-854Leu-OH monitored by LC-MS at 210 nm. A: after 1 h reaction; B: LC-MS of the purified product following Thz conversion to the N-terminal cysteine. Finally, the C terminal fragment 6 (1 mM) and N terminal fragment 5 (1 mM), were ligated under standard conditions (pH 6.8, 200 mM MPAA, 20 TCEP.HCl, 6M Gn. HCl/0.2 M phosphate) to give 8 and reaction was deemed complete after 17 h by LC-MS analysis (observed mass 14036.8 ± 1.4 Da., calculated mass 14040.1 Da). The three cysteines were subsequently capped by adding 2-iodoacetamide (200 mm) directly to the ligation mixture to afford the expected triple alkylation product (3 x 57 Da.) incorporating the three pseudoglutamines after only 15 mins (observed mass 14207.6± 1.3 Da., calculated mass 14211.3 Da). Excess superfluous iodoacetamide was then quenched by the addition of MesNa (250 mM). Subsequent formyl deprotection of 767Trp(CHO) was accomplished by addition of 20% (v/v) 2-mercaptoethanol and 40% piperidine (v/v) directly to the reaction mixture and was quantitative after 1 h at 0 °C by LC-MS analysis (observed mass 14179.1± 1.3 Da., calculated mass 14183.3 Da). The recovery by SPE of 734Leu-761ᴪGln-764Cys(acm)791ᴪGln-825ᴪGln854 Leu-OH 9 after execution of the 3 steps all carried out in the one pot (ligation, alkylation and formyl removal) was excellent (ca. 9 mg). Lastly, the acm group on 764Cys(acm) was cleanly removed by treatment with silver acetate in dilute acid at room temperature to afford the native cysteine residue. Purification by RP-HPLC afforded >4 mg of the GSAP ᴪGln analogue 7 in good recovery (>30% based on 5). Analysis of the purified 121 amino acid product by LC-MS revealed that the synthetic protein had the expected mass (observed mass 14108.7± 1.4 Da., calculated mass 14112.4.3 Da) and was obtained in high purity.

Figure 5. Native chemical ligation of 734Leu-764Cys(acm)-767Trp(CHO)-790Lys-COS-CH2CH2OH 5 and 791Cys-854Leu-OH 6 monitored by LC-MS at 210 nm. A: T = 0; B: T = 17 h; C: after treatment with I-CH2CONH2 and formyl deprotection of 767Trp; D: after acm removal from 764 Cys.

Figure 6. LC-MS of purified the 121 amino acid unfolded protein GSAP 734Leu-761ᴪGln791 ᴪGln-824ᴪGln-854Leu-OH 7. Conclusions


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Alzheimer’s disease is poised to become one of the biggest health care challenges in the coming decades and there is an ongoing need to develop effective therapies and diagnostics. We have outlined the chemical synthesis of an analogue of the putative GSAP protein that may play a role in the regulation of the levels of amyloidogenic peptides which progress to form the amyloid plaques that are associated with Alzheimer’s disease. We are currently exploring the re-folding of this synthetic protein with and without its reported inhibitor, Gleevac, under experimental conditions to enable probing of this interaction by structural studies using X-ray crystallography. Acknowledgments The authors with to thank the Maurice Wilkins Centre for Molecular Biodiscovery for financial assistance.

1. 2014 Alzheimer's Disease Facts and Figures; Alzheimers Association: Chicago, 2014. 2. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353-356. 3. Crump, C. J.; Johnson, D. S.; Li, Y. M. Biochemistry 2013, 52, 3197-3216. 4. He, G.; Luo, W. J.; Li, P.; Remmers, C.; Netzer, W. J.; Hendrick, J.; Bettayeb, K.; Flajolet, M.; Gorelick, F.; Wennogle, L. P.; Greengard, P. Nature 2010, 467, 95-U129. 5. Netzer, W. J.; Dou, F.; Cai, D. M.; Veach, D.; Jean, S.; Li, Y. M.; Bornamann, W. G.; Clarkson, B.; Xu, H. X.; Greengard, P. Proc Natl Acad Sci U S A 2003, 100, 12444-12449. 6. Hussain, I.; Fabregue, J.; Anderes, L.; Ousson, S.; Borlat, F.; Eligert, V.; Berger, S.; Dimitrov, M.; Alattia, J. R.; Fraering, P. C.; Beher, D. J Biol Chem 2013, 288, 2521-2531. 7. Olsson, B.; Legros, L.; Guilhot, F.; Strömberg, K.; Smith, J.; Livesey, F. J.; Wilson, D. H.; Zetterberg, H.; Blennow, K. Alzheimer's & Dementia: The Journal of the Alzheimer's Association 2014, 10, S374-S380. 8. Deatherage, C. L.; Hadziselimovic, A.; Sanders, C. R. Biochemistry 2012, 51, 5153-5159. 9. Harris, P. W. R.; Yang, S. H.; Brimble, M. A. Tetrahedron Lett 2011, 52, 6024-6026. 10. Harris, P. W. R.; Lee, D. J.; Brimble, M. A. J Pept Sci 2012, 18, 549-555. 11. Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Proc Natl Acad Sci U S A 1999, 96, 10068-10073. 12. Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H. Int J Pept Res Ther 2007, 13, 31-44. 13. Vilaseca, M.; Nicolas, E.; Capdevila, F.; Giralt, E. Tetrahedron 1998, 54, 15273-15286. 14. Harris, P. W. R.; Williams, G. M.; Shepherd, P.; Brimble, M. A. Int J Pept Res Ther 2008, 14, 387-392. 15. Harris, P. W. R.; Kowalczyk, R.; Yang, S.-H.; Williams, G. M.; Brimble, M. A. J Pept Sci 2014, 20, 186-190. 16. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776-779. 17. Kent, S. B. H. Chem Soc Rev 2009, 38, 338-351. 18. Siman, P.; Karthikeyan, S. V.; Brik, A. Org Lett 2012, 14, 1520-1523. 19. Wong, C. T. T.; Tung, C. L.; Li, X. Mol BioSyst 2013, 9, 826-833. 20. Torbeev, V. Y.; Kent, S. B. H. Angew Chem Int Ed Engl 2007, 46, 1667-1670. 21. Bang, D.; Pentelute, B. L.; Kent, S. B. H. Angew Chem Int Ed Engl 2006, 45, 3985-3988. 22. Lee, J.; Kwon, Y.; Pentelute, B. L.; Bang, D. Bioconjugate Chem 2011, 22, 1645-1649. 23. Villain, M.; Vizzavona, J.; Rose, K. Chem Biol 2001, 8, 673-679. 24. Mende, F.; Seitz, O. Angew Chem Int Ed Engl 2011, 50, 1232-1240. 25. Harris, P. W. R.; Brimble, M. A. Peptide Science 2013, 100, 356-365. 26. Johnson, E. C. B.; Kent, S. B. H. J Am Chem Soc 2006, 128, 6640-6646.



27. Melnyk, R. A.; Partridge, A. W.; Yip, J.; Wu, Y. Q.; Goto, N. K.; Deber, C. M. Biopolymers 2003, 71, 675-685. 28. Wade, J. D.; Mathieu, M. N.; Macris, M.; Tregear, G. W. Lett Pept Sci 2000, 7, 107-112. 29. Subirós-Funosas, R.; El-Faham, A.; Albericio, F. Tetrahedron 2011, 67, 8595-8606. 30. Gates, Z. P.; Stephan, J. R.; Lee, D. J.; Kent, S. B. H. Chem Commun (Cambridge, U K) 2013, 49, 786-788.


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Chemical Protein Synthesis with the KAHA Ligation.

Modern extensions of native chemical ligation for chemical protein synthesis.

Templated native chemical ligation: peptide chemistry beyond protein synthesis.

Chemical synthesis of a pore-forming antimicrobial protein, caenopore-5, by using native chemical ligation at a glu-cys site.

Solid phase chemical ligation employing a rink amide linker for the synthesis of histone H2B protein.

threonine ligation for the chemical synthesis of proteins.

Chemical synthesis of biologically active tat trans-activating protein of human immunodeficiency virus type 1.

Semisynthesis of peptoid-protein hybrids by chemical ligation at serine.

Chemical protein synthesis.

Chemical ligation of oligonucleotides using an electrophilic phosphorothioester.

Peptide o-aminoanilides as crypto-thioesters for protein chemical synthesis.

Total chemical synthesis of human interferon alpha-2b via native chemical ligation.

Recent extensions to native chemical ligation for the chemical synthesis of peptides and proteins.

A native chemical ligation handle that enables the synthesis of advanced activity-based probes: diubiquitin as a case study.

Chemical methods of protein synthesis and modification.

Tetrazine ligation for chemical proteomics.

Synthesis of cysteine-rich peptides by native chemical ligation without use of exogenous thiols.

One-pot native chemical ligation of peptide hydrazides enables total synthesis of modified histones.

Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis.

New methods for chemical protein synthesis.

Synthesis of thioester peptides for the incorporation of thioamides into proteins by native chemical ligation.

t-boc synthesis of huwentoxin-i through native chemical ligation incorporating a trifluoromethanesulfonic acid cleavage strategy.

Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide.

Synthesis and SAR Studies of Fused Oxadiazines as γ-Secretase Modulators for Treatment of Alzheimer's Disease.