Full Solution-Phase Synthesis of Acetyl Hexapeptide-3 by Fragments

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Full solution-phase synthesis of acetyl hexapeptide-3 by fragments coupling strategy Teng Zhang, Wei Song, Jin li Zhao, and Jian-Li Liu Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b03299 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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Industrial & Engineering Chemistry Research

Full solution-phase synthesis of acetyl hexapeptide-3 by fragments coupling strategy Teng Zhang,a Wei Song,b Jinli Zhaob and Jianli Liu*,c a

College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, P. R. China; b

Department of Polypeptide Engineering, Active Protein & Polypeptide Engineering Center of Xi'an Hua Ao Li Kang, Xi’an, 710054, P. R. China; c

College of Life Science, Northwest University, Xi’an, 710069, P.R. China.

Keywords: acetyl Hexapeptide-3, solution-phase synthesis, in water, peptide fragments Abstract: Acetyl hexapeptide-3, a competitive SNAP-25 inhibitor that mimics the action of botulinum neurotoxins but is non-toxic, was successively synthesized in a solution by peptide chemistry with a “3+2+1” fragment coupling strategy. The difficulties encountered in convergent route, an optimization on scalable process and the controls on potential epimerization were systematically discussed. Final purification led to the target product overall 36.5% yield with 98.3% purity and excellent optical purity. 1. INTRODUCTION Biological peptides have attracted an enormous amount of attention for decades from synthetic chemists due to both their stereochemically complex structure and the wide range of clinically relevant applications.[1] However, such compounds are not available in quantity usually from naturally occurring sources, rendering analogue synthesis and drug development efforts extremely resource-intensive and time-consuming. In this respect, solid-phase peptide synthesis (SPPS) has opened an avenue in the development of modern peptide chemistry.[2]

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Especially, with the recent progress and the collection of available reagents,[3] new and improved strategies lead towards an efficient synthesis of complex peptide targets, and even preparations on the industrial scale.[4] While great progress has been made in the development of new methods to address the synthetically challenging of complex peptide chain, as a result, in the development of more highly efficient syntheses, that combines high efficiency of atom economy with high levels of environmental-friendly and scalability has remained elusive. Inspired by this, we intend to explore a facile and cost-effective approach for the practical synthesis of peptides by a novel convergent strategy. Acetyl hexapeptide-3 (known as Argireline)[5] is a topically simulating peptide that has garnered significant interest from synthetic chemists due primarily to its promising applications on desirable cosmetic enhancements[6] and a treatment of blepharospasm.[7] Although it has a mechanism of action similar to botulinum neurotoxin (BoNT),[8] inhibiting neurotransmitter release at the neuromuscular junction, its potency is about 4000 times lower than that of BoNTs, so it does not exhibit in vivo oral toxicity or primary irritation at high doses.[9] As shown in Fig. 1, Acetyl hexapeptide-3 is a stereochemically complex molecule that contains four amino acids and six stereocenters with the sequence Ac-Glu12-Glu13-Met14-Gln15-Arg16-Arg17-NH2. Besides N-terminal amino and C-terminal carboxyl groups closed with amide groups, other groups are all free with distinct reactivity. Twin-arginine is also known as “difficult sequence” because of their poor solubility and strong basic of guanidino groups.[10] This synthetically challenging play an outsized role in asymmetric peptide chemistry. In this arena, SPPS seems the first way to access significant quantities of the objective hexapeptide.[11] Advantages of SPPS are numerous including possible automation and parallel library synthesis. However, the excessive amounts of reagents often used in a heterogeneous system to forced the coupling completion, that could drive up the cost and increase the pollution in batch production. In fact, in vivo tests have shown that to reduce the depth of skin wrinkles by 30%, a 10% (w/v) concentration of acetyl hexapeptide-3 is


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required.[5] The use of such a high concentration has significantly increased the demand for this product far outpacing conventional production capabilities. In addition, the peptide chain constructed by solidphase synthesis is not easy to monitor the epimerization unless cleavage cocktail treatment and HPLC purification at each step. Therefore, the development of more effective and stepcontrollable syntheses of acetyl hexapeptide-3 is still required. Alternatively, a solution approach would allow for the specific benefits of good batch reproducibility and possibly low cost due to its considerable freedom for synthetic scheme and completely coupling in homogeneous system.[4b] Those properties suggest that it is a feasible alternative to the solidphase routes to construct hexapeptide and possible to obtain significant amounts of this structurally challenging molecule. In this communication, we present our work on the first full solution-phase synthesis of acetyl hexapeptide-3 including a construction of the stereochemically complex structure and an examination of its epimerization in the synthetic process. Additionally, the scalability and practicability of the route have been demonstrated by performing on a meaningful scale. This work has achieved not only the development of a practical synthesis for bioactive peptides, but also serve a good reference in the field of sustainable chemistry and modern process research.

Figure 1.

Structure of acetyl hexapeptide-3.

2. RESULTS AND DISCUSSION Originally, we had planned to prepare the hexapeptide by the stepwise construction of the peptide chain starting from the glutamate moiety. However, as the peptide chain grew in complexity and length, the solubility of the peptides decreased, making additional synthesis


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steps difficult. Moreover, impurities gradually accumulated and were removed with great difficulty. Thus, we decided to perform the synthesis by the condensation of different fragments. In our preceding communication,[12] the protected tripeptide fragments, Ac-Glu(OtBu)Glu(OtBu)-Met-OH

and H-Gln(Trt)-Arg-Arg-NH2,

had been

successfully

synthesized.

However, the desired product was failed to obtained by a “3+3” coupling in solution, because the C-terminal fragment was insoluble and reacted unsatisfactorily. Further analyses found that arginine-containing peptides are sparingly soluble in most organic solvents because of preferential intramolecular salt formations. Therefore, it was necessary to further separate the twin-arginine residues in the C-terminal fragment.

Figure 2.

Synthetic analysis of acetyl hexapeptide-3.

As described above, the peptide chain was finally divided into three different sequences: AcGlu(OtBu)-Glu(OtBu)-Met-OH, H-Gln(Trt)-Arg-OH and H-Arg-NH2 (Fig. 2). The intermediates were obtained as follows.[3c] The α-amino group of glutamic acid12 was blocked by acetylation, the γ-carboxyl group12,13 was protected with -OtBu group. The amide group of glutamine15 was converted into a -Trt, and its α-amino group was protected by the formation of Fmoc-NH. Additionally, the carboxyl group of the C-terminal arginine17 was closed with an amide group. Other amino acids13,14,15,16 were used as free bases in which the carboxyl groups were converted into their salt forms. The peptide bonds were synthesized in good yield by using active ester mediated solution phase synthesis.[13] The protected tripeptide Ac-Glu(OtBu)-Glu(OtBu)-Met-OH (A-3) was prepared by the stepwise addition of amino acid moieties as our previous report (Scheme 1).[12] The dipeptide Fmoc-Gln(Trt)-Arg-OH (B-1) was obtained by the condensation of Fmoc-Gln(Trt)-OSu with free arginine (Scheme 2). After removed the ACS Paragon Plus Environment

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Scheme 1.

Solution-phase synthesis of fragment A.

Scheme 2.

Solution-phase synthesis of fragment B.

Scheme 3. “3+2+1” fragments coupling of acetyl hexapeptide-3.


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Fmoc- protecting group with 10% DEA in DCM (v/v), the free dipeptide (B-2) was coupled with activated tripeptide (A-4) to produce the fragment AB (Scheme 3). The fragment C was prepared by ammonolysis of Arg-OMe·HCl. The final step consisted of a condensationbetween the arginine (AB) and H-Arg-NH2(C) lead to the protected hexapeptide with satisfied yield (ABC, 67.7%). After treatment with 50% TFA in DCM (v/v) to remove the side chain protecting groups on ABC and subsequent ion exchange chromatography (IEC) purification, the desired product was finally obtained with 36.5% overall yield and 98.3% purity (see the ESI for details). “AB + C” condensation is crucial for the synthetic preparation of the protected hexapeptide (ABC). We initially attempted to prepare hexapeptide (ABC) by activation of pentapeptide (AB) followed by subsequent coupling with the fragment C. However, there were difficulties with the activation reaction. First, the protected pentapeptide was sparingly soluble in a single solvent. Second, even after a prolonged period of time, no activated derivative was formed, and only reaction by-products were detected (according to TLC). We assumed that the formation of the activated ester was hindered by the ionisation of the carboxyl group of the pentapeptide and the formation of its inner salt with the arginine residues (Fig. 3a). Alternatively, Arg derivatives tend to be worse activated, mainly because of the formation of the δ-lactam from the activated species (Fig. 3b). In an effort to overcome this difficulty, many experiments were carried out in our research by varying the number of equivalents of coupling

Figure 3.

Mechanism of inner salt and δ-lactam formation.


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reagent, bases (nucleophilic assistance) and the reaction time as well as the different types of mixed solvents. Under optimal conditions, the best result was obtained by adding 1.2 equivalents of EDC·HCl as a coupling reagent and HOBt as an auxiliary nucleophile with the presence of a tertiary amine in onepot reaction. We speculated that the one-pot condensation prevented the formation of the inner salt and δ-lactam and appropriately improved the stability of the activated compound, leading to a rapid formation of the protected hexapeptide (ABC).[14] Other than the condensing agent and the additives, both solvent system and additional base have a significant effect on the synthetic yield of the product. Due to the poor solubility of the pentapeptide (AB), only in specific homogeneous systems do both fragments AB and C show satisfactory solubilities where the desired coupling reaction is more rapid than the decomposition of the active esters. In general (see ESI Table S1), due to the lack of a strong polar solvent, when a single solvent was used in the reaction system, the expected yield was not obtained, but the corresponding yield of product was increased when using mixed solvents. For example, the synthetic yield in varying the volume ratio of THF and DMF significantly increased (39.7% - 46.2%) when the proportion was changed from 2 : 1 to 1 : 1. A similar result was observed between THF and DMSO (36.5%- 40.0%). This may contribute to the fact that the carbonyls of the solvents could easily form hydrogen bonds with the N-H groups of the peptide chain, which reduces the aggregation and increases the solubility of the peptide fragments.[15] Thus, the optimal solvent system for “AB + C” condensation was an isocratic solution of DMF and DMSO. Under the same conditions, “AB + C” condensation was performed in the presence of an additional base, different bases may have different effect on the synthetic yield of the product (see ESI Figure S1). The yield of non-nucleophilic tertiary amine was generally higher than those of inorganic bases. This could be because the organic bases were liquid and easily dispersable in the solvents system to promote aminolysis. More specifically, NaHCO3 was much more reactive than Na2CO3, likely because the latter was difficult to solubilise and hindered the reaction equilibrium from peptide formation. While the ACS Paragon Plus Environment

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reaction efficiency with DIEA was enhanced and the yield was increased (comparable with that of TEA) because of it’s increasing electron-donating ability. Additionally, all base-promoted synthetic yields eventually reached maximum yields and even reduced when the reaction time exceeded an optimal point. To our satisfaction, better yield was observed when performing the coupling with 4 equivalents of NMM as the associated promotor at room temperature reaction for about 30 h. Therefore, NMM is our top pick in this one-pot coupling process. Additionally, the scalability and practicability of the route have been demonstrated by performing all of the steps on a meaningful scale (>500g). One of important process was the post-treatment involved in the multi-step synthesis with possibly large-scale production. According to the different physiochemical characteristics, the aforementioned intermediates could be separated from liquid mixtures by specialized methods. As we described in previous work,[16] for example, the full protected amino acids usually produce a hydrophobic peptide products in two phase mixture (H2O/THF), after removed the organic phase by reduced pressure distillation, the product could be precipitated and filtrated by addition of 20% aqueous solution of citric acid (m/m), while excess free amino acids, base and hydrophilic byproducts remain soluble in the aqueous phase. If necessary, an ultrasonic-assisted extraction or recrystallisation was used to further purification. In addition, most lipophilic impurities are soluble in iced diethyl ether and readily separated from the insoluble final product. With such a protocol, a series of peptide intermediates were quickly prepared at high yields with excellent purity. At present, the pilot scale of operation provides each fragment in 1kg batches. As for the time, all fragments were synthesized within 7 days, the next from fragments assembly and then deprotection to completion of scale purification is about half a month, so the entire operation time for batch production is less than 23 days. The fragment yields are steady from 45% to 85% with purity of 93-96%. The efficiency of fragment coupling is approximately 65-68%, and the overall yield of pure product is about 36.5%. Due to a full solution phase process that the reaction was carried out in an aqueous mixture of an environmentally benign


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solvent, it reduced the potential of organic contamination and the solvents can also be recycled and reused. After achieving the synthesis, we were interested in examining the potential epimerization in this full solution phase route. It is generally known that racemization is usually occurred during peptide activation and then promote the generation of isomers in the peptide coupling. In this process, active ester acted both as activating and leaving group, eliminating frequent use of protection and subsequent deprotection, it indicated that an appropriate active ester with a steady and faster coupling rate than hydrolysis would suppressed the epimerization in solution. Following this basic rationale, we have performed control experiments to screen the optimal combination of additive and condensing reagent to reduce the produce of epimers in each coupling step.[3b,3e] Under the optimal conditions, HOSu/DCC was used to synthesize the fragments A and B, because it forms a relatively stable activated ester to avoid both epimerization and hydrolysis in solution phase synthesis. HONp/DCC is more prone to peptide bond formation and contribute to the coupling of fragment A and B due to its fast coupling rate. While both of them are less reactive than HOBt/EDC, which is suit for condensation between fragment AB and C in one-pot reaction, where the -OBt ester was directly prepared in situ as an intermediate and subsequently react with fragment C quickly to form the protected hexapeptide (ABC). All the fragments were detected by comparing the chiral chromatogram of the desired peptide with the epimerized peptide (see the ESI for details). In order to further ensure the configuration of the acetyl hexapeptide-3, three isopeptides, incoluding Ac-Glu-Glu-D-Met-Gln-Arg-Arg-NH2, Ac-Glu-Glu-Met-Gln-D-Arg-Arg-NH2 and Ac-Glu-Glu-Met-Gln-Arg-D-Arg-NH2, in which L-amino acid at the C-terminus of each fragment was replaced with D-amino acid, were conveniently obtained by solid-phase peptide synthesis. Under the same conditions, the synthesized acetyl hexapeptide-3 was completely separated with three isopeptides by HPLC (Fig. 4), its absolute configuration accounted for 98.4% of the total peak area with trace detectable epimerization (≈ 1.6%). As a control, the comparison of the major product chromatography of four different batches also revealed that about 96.0% ee were maintained. These


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Figure 4.

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Chiral HPLC analysis of epimerization of acetyl hexapeptide-3.

results show that acetyl hexapeptide-3 synthesized with the full solution phase chemistry exhibited minimal epimers and it is an excellent method for successfully obtaining hexapeptide with high optical purity. 3. EXPERIMENTAL SECTION The homogeneity of the intermediates was checked by TLC on pre-coated silica gel plates (GF254) in different chromatographic systems. Spots were detected by irradiation with UV light or by spraying with 0.05g/mL ninhydrin in ethanol or potassium iodide reagent. Mass spectra were recorded on LTQXL with direct sample injection. 1H-NMR spectra were recorded on a Bruker Avance-400 MHz spectrometer. Optical rotations were measured using an Autopol II automatic polarimeter. HPLC analysis was carried out on a Hitachi L2000 instrument using a Kromasil C18 column (4.6 × 250mm) with a linear gradient from 10% to 90% of aqueous acetonitrile (0.1% trifluoroacetic acid) over 30 min at a flow rate of 1.0 mL/min. The crude product was purified by cation exchange chromatography using a SP sepharose fast flow column (GE Healthcare), and desalted by reversed phase liquid chromatography on a SBC MCI GEI column (Sci-Bio-Chem Co., Ltd.), and then lyophilized using a ACS Paragon Plus Environment

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Biocool lyophilizer. The determination of enantiomeric excess was performed via chiral phase HPLC analysis using Thermo U-3000 HPLC workstation with an AS-H column or OD-H column. The LAmino acids (protected or free) were obtained from GL Biochem Ltd. (Shanghai, China). Other reagents were provided by Sigma-Aldrich. Important notes: It should be noted that, in order to minimize potentially racemization and reduce the formation of by-products, several positive parameters have been applied in experimental procedure. A key issue is the use of appropriate additive and condensing reagent in peptide-coupling reaction. A further issue is that L-amino acids were exactingly used and material ratio was strictly controlled, the organic solvents must be dried before use. In addition, the activating reaction must be carried out in icesalt bath, while the coupling reactions can be perform at room temperature. The reaction mixture was evaporated in vacuo below 37°C, the solvents can be recycled and reused. The prepared compounds are dried under reduced pressure at room temperature and the product was obtained by freeze-drying, etc. Abbreviations: ACN, acetonitrile; AcOH, Acetic acid; BoNT, botulinum neurotoxin; DCC, N,N'dicyclohexylcarbodiimide; DCM, Dichloromethane; DCU, N, N'-dicyclohexylurea; DEA, Diethylamine; DIEA, Diisopropylethylamine; DMF, Mimethylformamide; DMSO, Dimethylsulfoxide; EDC·HCl, 1(3-dimethyl-aminopropyl)-3-ethylcarbodiimide

hydrochloride;

EtOAc,

Ethyl

acetate;

Fmoc-,

Fluorenylmethoxycarbonyl; HEX, n-hexanol; HOBt, 1-Hydroxylbenzotrizole; HONp, p-Nitrophenol; HOSu, N-Hydroxysuccinimide; IEC, ion exchange chromatography; IPA, Isopropanol; MeOH, Methyl alcohol; Na2CO3, Sodium carbonate; NaHCO3, Sodium bicarbonate; Nin, Ninhydrin; NMM, NMethylmorpholine; PE, petroleum ether; RT, room temperature; SOCl2, Thionyl chloride; SPPS, solidphase peptide synthesis; -tBu, tert-Butyl; TEA, Triethylamine; TFA, Trifluoroacetic acid; THF, Tetrahydrofuran; Tis, Trisisopropylsilane; Trt, Trityl. Ac-Glu(OtBu)-OH (A-1)


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Acetyl chloride (32 mL, 0.45 mol) was slowly added to the reaction mixture of HOSu (46 g, 0.4 mol) and DIEA (79.5 mL, 0.48 mol) in dry THF (400 mL) under ice cooling. After stirring for 5 h, the reaction mixture was used as is.[13] H-Glu(OtBu)-OH (89.3 g, 0.44 mol) was dissolved in H2O (300 mL) and NaHCO3 (67.2 g, 0.8 mol) was added while stirring. The resultant solution was dropwise-added to the solution of Ac-OSu (obtained from the previous step). The mixture was stirred at room temperature and the solution became clear. After the Ac-OSu completely reacted as indicated by TLC, the mixture was concentrated to a small volume and adjusted to pH 2-3 with 20% citric acid aqueous solution. A white solid precipitate out, then filtered and washed with water to provide Ac-Glu(OtBu)-OH (86 g, yield 87.7%) as a white squamous solid. m.p.173.5-174.6°C. TLC: Rf=0.3, EtOAc:HEX:AcOH= 10:10:1. HPLC: A (0.1% TFA/ACN), B (0.1% TFA/H2O), 30 min 10-90%, 12.88 min, purity 93.11%. ESI-MS for [M] calcd: 245.12, found: 268.0 ([M+Na]+, 100%); 211.9 ([M-34], 70%); 513 (40%). 1H NMR (400 MHz, MeOD) δ = 4.42 (dd, J=9.1, 5.0, 1H), 2.35 (t, J=7.5, 2H), 2.21 – 2.09 (m, 1H), 2.01 (s, 3H), 1.96 – 1.83 (m, 1H), 1.50 – 1.42 (m, 9H). Ac-Glu(OtBu)-Glu(OtBu)-OH (A-2) To a stirred solution of Ac-Glu(OtBu)-OH (85 g, 0.347 mol) and HOSu (47.9 g, 0.416 mol) in dry THF (850 mL), a solution of DCC (93 g, 0.451 mol) in dry THF (100 mL) was added under ice cooling. After 2 h, the reaction was allowed to warm to room temperature and stirred overnight. The DCU precipitate was removed and the filtrate was used without further purification.[13] H-Glu(OtBu)-OH (77.5 g, 0.382 mol) and NaHCO3 (43.7 g, 0.52 mol) were dissolved in H2O (300 mL). The mixture was slowly added to the solution of Ac-Glu(OtBu)-OSu (obtained from the previous step) and stirred at room temperature for 8 h. The solution was concentrated and adjusted with 20% citric acid aqueous solution to pH 2-3, then extracted with ethyl acetate (300 mL and 100 mL). The organic phase were washed with water (200 mL, 3×), saline (200 mL, 2×) and dried with magnesium sulfate. Activated carbon (10 g) was added in organic phase with stirring at 40-45°C for 20 min, After


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filtered, the filtrate was evaporated to yield a transparent syrup (approximately 140 g). After crystallisation from EtOAc and PE (1.3 : 1), the pure crystalline was obtained (88 g, yield 59.0%). m.p. 113.7-117.1°C. TLC: Rf=0.2, EtOAc:HEX:AcOH=15:5:1. HPLC: A (0.1% TFA/ACN), B(0.1% TFA/H2O), 30 min 10-90%, 17.63 min, purity 88.39%. ESI-MS for [M] calcd: 430.5, found: 430.3 ([M], 90%); 859.4 ([2M]+, 100%). 1H NMR (400 MHz, MeOD) δ = 4.41 (ddd, J=18.9, 8.8, 5.4, 2H), 2.41 – 2.28 (m, 4H), 2.18 (d, J=7.4, 1H), 2.12 – 2.03 (m, 1H), 2.01 (d, J=6.6, 3H), 1.91 (ddd, J=18.0, 11.7, 7.5, 2H), 1.47 (s, 18H). Ac-Glu(OtBu)-Glu(OtBu)-Met-OH (A-3) Ac-Glu(OtBu)-Glu(OtBu)-OH (86g, 0.2mol) and HOSu (27.6g, 0.24mol) were dissolved in dry THF (500 mL) and the resulting solution was cooled under ice bath. The solution of DCC (53.5g, 0.26mol) in THF (100 mL) was then slowly added. After stirring for 2 h, the reaction was allowed to warm to room temperature overnight. The resulting precipitate (DCU) was filtered and the filtrate was used for the next step.[13] H-Met-OH (26.8g, 0.24mol) and NaHCO3 (25.2g, 0.3mol) were dissolved in H2O (300 mL). The solution of Ac-Glu(OtBu)-Glu(OtBu)-OSu (obtained from the previous step) was gradually added. The mixture was stirred at room temperature until the active ester disappeared as indicated by TLC. The solution was evaporated and adjusted to pH 2-3 with 20% citric acid aqueous solution, then extracted with EtOAc (200 mL, 3×). The organic layers were washed with water (200 mL, 2×) and saline (200 mL, 2×), dried with magnesium sulfate, and evaporated to give white solid Ac-Glu(OtBu)-Glu(OtBu)Met-OH (95 g, yield 84.7%, enantiomeric excess 92.4%). m.p. 127.2-130.0°C. TLC: Rf=0.3, EtOAc:HEX:MeOH: AcOH=10:10:1:1. HPLC: A (0.1% TFA/ACN), B (0.1% TFA/H2O), 30 min 1090%, 19.83 min, purity 95.03%. ESI-MS for [M] calcd: 561.3, found: 584.48 ([M+Na]+, 100%); 528.52 ([M-34], 40%). [α]D20.5-19.35 (c=1.0, CH3OH). 1H NMR (400 MHz, MeOD) δ = 4.55 (dd, J=9.2, 4.4, 1H), 4.37 (ddd, J=18.3, 8.5, 5.7, 2H), 2.63 – 2.51 (m, 2H), 2.37 (ddd, J=14.9, 9.2, 5.1, 4H), 2.22 – 2.13


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(m, 2H), 2.10 (s, 3H), 2.08 – 2.04 (m, 1H), 2.02 (d, J=8.9, 3H), 1.99 – 1.95 (m, 1H), 1.91 (dd, J=15.3, 6.8, 2H), 1.47 (d, J=1.9, 18H). Fmoc-Gln(Trt)-Arg-OH (B-1) Fmoc-Gln(Trt)-OH (122 g, 0.2 mol) and HOSu (27.6 g, 0.24 mol) were dissolved in dry THF (975 mL) under stirring. The resulting solution were placed in an ice bath. DCC (53.6 g, 0.26 mol) in THF (245 mL) was gradually added. The reaction mixture was stirred until complete reaction as indicated by TLC. The filtrate was used without further purification.[13] H-Arg-OH (38.4 g, 0.22 mol) and NaHCO3 (25.5 g, 0.3 mol) were dissolved in H2O (490 mL). The solution was stirred and Fmoc-Gln(Trt)-OSu (obtained from the previous step) was added slowly. The reaction was continually stirred until complete reaction as indicated by TLC (≈12 h). The resultant solution was then concentrated in vacuum. The residue was acidified to pH 2-3 with 20% citric acid aqueous solution and extracted with EtOAc (200 mL, 4×). The combined organic layers were then extracted with water (200 mL, 2×), saline (200 mL, 2×) and dried with sodium sulfate. The organic layer was concentrated to obtain an off-white solid (approximately 138 g). The crude product was purified by mashing with EtOAc (200 mL) to get Fmoc-Gln(Trt)-Arg-OH (125 g, yield 81.5%). m.p. 161.0-164.3°C. TLC: Rf=0.3, EtOAc:HEX:MeOH: AcOH=10:10:1:1. HPLC: A (0.1% TFA/ACN), B (0.1% TFA/H2O), 30 min 10-90%, 23.31 min, purity 94.78%. ESI-MS for [M] calcd: 766.34, found: 767.94 ([M+H], 100%); 769.14 ([M+3H], 95%); 766.93 ([M], 60%). 1H NMR (400 MHz, MeOD) δ = 7.81 (d, J=7.5, 2H), 7.68 (dd, J=7.1, 3.5, 2H), 7.39 (d, J=7.5, 2H), 7.35 – 7.10 (m, 17H), 4.40 (dd,

J=10.4, 7.3, 1H), 4.33 (t, J=8.5, 1H), 4.25 (dt, J=14.3, 5.9, 2H), 4.15 – 4.03 (m, 1H), 3.14 (d, J=5.6, 2H), 2.45 (dd, J=15.2, 7.4, 2H), 2.13 – 1.99 (m, 1H), 1.92 – 1.82 (m, 2H), 1.71 (dd, J=13.4, 7.3, 1H), 1.61 (s, 2H). H2N-Gln(Trt)-Arg-OH (B-2) Fmoc-Gln(Trt)-Arg-OH (110 g, 0.143 mol) was added to a solution of DEA in DCM (10% v/v, 400 mL) and stirred for approximately 20h.[17] After TLC measurements indicated the complete reaction of the


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dipeptide, EtOAc (250 mL) was slowly added and a white solid precipitated. The solution was filtered and washed with EtOAc to yield H2N-Gln(Trt)-Arg-OH (73 g, yield 93.7%, enantiomeric excess 96.3%) as a white pulverous solid. m.p. 118.4-112.6°C. TLC: Rf=0.2, n-BuOH:AcOH:H2O: Ninhydrine (0.05 g/mL in ethanol)=8:3:1:1. HPLC: A (0.1% TFA/ACN), B (0.1% TFA/H2O), 30 min 10-90%, 16.36 min, purity 96.29%. ESI-MS for [M] calcd: 544.21, found: 544.62 ([M], 80%); 545.49 ([M+H], 40%); 164.82 (100%). [α]D20.5+21.51 (c=1.0, CH3OH). 1H NMR (400 MHz, MeOD) δ = 7.28 (dd, J=13.8, 6.7, 15H), 4.45 (s, 1H), 3.90 (d, J=6.4, 1H), 3.21 (t, J=6.8, 2H), 2.72 (t, J=7.2, 2H), 2.10 (d, J=6.8, 2H), 1.96 (s, 1H), 1.76 (s, 1H), 1.69 (d, J=6.7, 2H). Ac-Glu(OtBu)-Glu(OtBu)-Met-ONp (A-4)[13] HONp (24.5 g, 0.176 mol) was added to the solution of Ac-Glu(OtBu)-Glu(OtBu)-Met-OH (90 g, 0.16 mol) in DCM (800 mL). The obtained solution was cooled under ice bath and DCC (43 g, 0.21 mol) in DCM (200 mL) was added with stirring. The reaction mixture was stirred at room temperature overnight. The separated DCU was filtered off and washed with DCM (30 mL, 3×). The filtrate was concentrated to a faint yellow solid, Ac-Glu(OtBu)-Glu(OtBu)-Met-ONp (109 g, yield 99.0%). TLC: Rf=0.4, EtOAc:HEX: MeOH:AcOH=10:10:1:1. Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg-OH (AB) The solution of H2N-Gln(Trt)-Arg-OH (B-2, 70.6 g, 0.13 mol) and NaHCO3 (15.8 g, 0.187 mol) in H2O (700 mL) was slowly added to a solution of Ac-Glu(OtBu)-Glu(OtBu)-Met-ONp (102.4 g, 0.15 mol) in DMF (1000 mL). The mixture was stirred until a complete reaction was indicated by TLC (≈ 24 h). After extracted with a mixture of EtOAc and PE (2 : 1, 200 mL, 2×). The aqueous phase was acidified with 30% citric acid and further extracted with a mixture of EtOAc and MeOH (3 : 1, 300 mL, 1× and 200 mL, 3×). The organic layer was washed with water (400 mL, 3×) and saline (200 mL, 2×), dried with magnesium sulfate and then evaporated to yield a pale yellow solid. The residue was soaked in ice ether, the precipitate was filtered and then washed with ice ether to yield a yellowish powder (92.5 g, yield 65.6%). m.p.125.7-129.2°C. TLC: Rf=0.2, EtOAc:HEX:MeOH:AcOH=8:3:1:1. HPLC: A


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(0.1% TFA/ACN), B (0.1% TFA/H2O), 30 min 10-90%, 24.15 min, purity 74.23%. ESI-MS for [M] calcd: 1087.54, found: 1088.60 ([M+H], 50%); 1089.27 ([M+2H], 30%). [α]D20.5-31.17 (c=1.0, CH3OH). 1H NMR (400 MHz, MeOD) δ = 7.40 – 7.13 (m, 15H), 4.48 – 4.36 (m, 2H), 4.25 (ddd,

J=21.9, 13.0, 6.7, 3H), 3.86 – 3.77 (m, 2H), 3.71 (s, 1H), 3.64 (dd, J=10.9, 5.9, 1H), 3.25 – 3.13 (m, 2H), 2.54 (dd, J=14.5, 7.1, 4H), 2.34 (dd, J=15.7, 7.4, 4H), 2.12 (s, 2H), 2.10 (s, 3H), 2.05 – 1.99 (m, 2H), 1.97 (s, 3H), 1.91 (s, 2H), 1.76 (s, 1H), 1.67 (d, J=7.2, 2H), 1.46 (d, J=1.1, 18H). NH2-Arg-NH2 (C)[18] Arginine (26.13 g, 0.15 mol) was suspended in absolute methanol (200 mL) and cooled under ice bath. SOCl2 (3 mL, 0.18 mol) was added slowly. The reaction mixture was stirred at room temperature and monitored by TLC. The solvent was removed under reduced pressure to obtain H-Arg-OMe as yellowish paste. It was used without further purification. TLC: Rf=0.4, n-BuOH:AcOH:H2O: Ninhydrine (0.05 g/mL in ethanol) = 8:3:1:1. ESI-MS for [M] calcd: 188.12, found: 188.74 ([M], 100%); 189.41 ([M+H], 40%). 1H NMR (400 MHz, MeOD) δ = 4.13 (t, J=6.5, 1H), 3.88 (s, 3H), 3.28 (t, J=7.0, 2H), 2.09 – 1.89 (m, 2H), 1.88 – 1.64 (m, 2H). After evaporation, saturated ammonium hydroxide aqueous solution (29%, 27.7 mL, 180 mmol) was added to the paste. The resulting solution was continuously stirred until reaction completion, then concentrated by rotary evaporation. The residue was purified by recrystallization from ice ether to get H-Arg-NH2 (22.2 g, yield 84.7%, enantiomeric excess 99.0%). m.p.187.5-192.2°C. TLC: Rf=0.2, EtOAc:HEX:MeOH:AcOH=4:4:1:1. HPLC: A (0.1% TFA/ACN), B (0.1% TFA/H2O), 30 min 10-90% (275nm), 19.60 min, purity 93.91%. ESI-MS for [M] calcd: 173.12, found: 173.65 ([M], 70%); 197.25 ([M+Na], 100%); 236.94 (100%). [α]D20.5+23.14 (c =1.0, MeOH). 1H NMR (400 MHz, MeOD) δ = 4.01 (t, J=6.4, 1H), 3.30 – 3.24 (m, 1H), 2.04 – 1.88 (m, 2H), 1.76 (dd, J=14.7, 7.4, 2H). Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg-Arg-NH2 (ABC)[19] HOBt (8.1 g, 0.06 mol), EDC·HCl (11.5 g, 0.06 mol) and NMM (22 mL, 0.2 mol) were added to the solution of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg-OH (54.4 g, 0.05 mol) and H-Arg-CONH2


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(13.0 g, 0.075 mol) in mixed solutions of DMF and DMSO (1:1, 500 mL) under ice bath. After standing for 30 h, the reaction media was added to 500 mL ice water and acidified to a pH of 3-4 with 3mol/L hydrochloric acid solution, then extracted with the mixtures of EtOAc and MeOH (3 : 1, 200 mL, 3×). The organic phase was washed with water (300 mL, 3×) and saline solution (200 mL, 2×) and dried. The solvent was evaporated and treated with ice ether, filtered and dried in vacuum to provide the protected hexapeptide (42g, yield 67.7%). m.p. 182.3-185.5°C. TLC: Rf=0.2, EtOAc:HEX:MeOH: AcOH = 8:2:3:1. HPLC: A (0.1% TFA/ACN), B (0.1% TFA/H2O), 30 min 10-90%, 15.82 min, purity 84.85%. ESI-MS for [M] calcd: 1242.65, found: 1243.97 ([M+H], 30%); 1245.00 ([M+2H], 100%). Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 (Acetyl hexapeptide-3) The protected hexapeptide (42 g, 0.034 mol) was treated with 50% TFA in DCM (200 mL).[3c] After reaction media clarification, Tis (40 mL) was added and the solution was stirred constantly for 5-6 h. The reaction mixture was concentrated to a small volume and treated with ice ether (150 mL). The precipitate was collected, washed with ice ether and dried under vacuum. The crude product (approximately 28.4 g, yield 94.3%) was dissolved in the minimum volume of 0.02 mol/L acetic acidsodium acetate aqueous buffer (pH 4, K = 0.36) and applied to a column (50 × 400 mm) of SP sepharose fast flow which had been equilibrated previously with the same buffer. The column was eluted with buffer (pH 4) of 0.02mol/L acetic acid-sodium acetate containing 1mol/L sodium chloride aqueous from 50% to 70% for 60min at a flow rate of 30 mL/min and the effluent monitored for UV absorption at 215 nm. Fractions were pooled and evaporated. Desalting was done on a SBC MCI GEI column (50 x 400 mm) with buffer A (0.25% TFA/ACN) and B (0.25% TFA/H2O). The column was eluted with buffer A from 17%-27% for 30 min at a flow rate of 30 mL/min and monitored as in the case of cation exchange chromatography. The fractions containing the major component were pooled and evaporated, which was further lyophylized at -50°C for 3 days to obtain chromatographically homogeneous Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 (25g, yield 88.0%, enantiomeric excess >96.8%, 36.5% overall yield) as a white crystalline solid. M.p. 202.7-205.7°C. HPLC: A(0.1% TFA/ACN),


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B(0.1% TFA/H2O), 25min 5-65%, 7.85min, purity 98.31%. ESI-MS for [M] calcd: 888.42, found: 889.51([M+H], 100%); 890.66([M+2H], 30%); 446([M+H]/2, 70%); 444.60(40%). [α]D20.5-43.74 (c=0.88, CH3OH). 1H NMR (400 MHz, D2O) δ = 4.25 (dd, J=8.4, 5.9, 1H), 4.14 (ddd, J=14.1, 8.9, 5.5, 3H), 4.06 (dd, J=14.4, 8.4, 2H), 3.07 (t, J=6.4, 4H), 2.49 (dd, J=13.4, 7.0, 1H), 2.45 – 2.35 (m, 1H), 2.31 – 2.21 (m, 2H), 2.20 – 2.07 (m, 4H), 2.00 (dd, J=14.6, 7.9, 1H), 1.96 (s, 3H), 1.94 (s, 1H), 1.92 (s, 3H), 1.90 (s, 1H), 1.88 (s, 1H), 1.86 (s, 1H), 1.78 (s, 1H), 1.73 (d, J=5.0, 2H), 1.65 (dd, J=9.7, 3.8, 2H), 1.59 – 1.42 (m, 4H). 4. CONCLUSIONS In conclusion, the synthesis of acetyl hexapeptide-3 required overcoming the difficulties encountered in fragment condensation was successively achieved via an active ester mediated full solution phase synthesis. Arg-Arg-NH2 was split to avoid the formation of an inner salt. The process optimization on batch production lead to a satisfied yield approximately 36.5% with 98.3% purity. Chiral HPLC analysis confirmed that appearance of epimerization during this entirely solution process with less than 2.0%. The good level of yield with scaling up potential and operational simplicity highlighted this practical procedure represents the most efficient, atom economical and sustainable synthesis of this stereochemically complex molecule reported to date, and is well suited to facilitate the synthesis of peptide analogues and medicinal chemistry development efforts in a time- and resource-efficient manner. This convergent approach are currently under investigation to the syntheses of acetyl hexapeptide-3 on a large-scale, and the application of the product in cosmetic enhancements are also underway in our group. ACKNOWLEDGEMENTS We gratefully acknowledge the National Natural Science Foundation of China (No. 20872118, 30070905), Innovative Research Team in University (No. IRT1174), the Foundation of the Education Department of Shaanxi Province (12JK1010) and Technology innovation fund of Xi'an City


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(No.CX13120) for financial support. We also thank R & D Center of Shaanxi East-Star Biochemical Technology Co., Ltd. for technical assistance. SUPPORTING INFORMATION AVAILABLE Experimental details and spectroscopic data for all compounds described here. This information is available free of charge via the Internet at http://pubs.acs.org/ AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; Tel.: +86 (0)29 88302013; fax: +86 (0)29 88303572 Present Address *

College of Life Science, Northwest University, TaiBai road No. 229, Xi’an, 710069, P.R. China.

Notes The authors declare no competing financial interest NOTES AND REFERENCES

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peptides in preventing or treating aged skin. Int. J. Cosmetic Sci. 2009, 31, 327; c) Fields, K.; Falla, T. J.; Rodan, K.; Bush, L. Bioactive peptides: signaling the future. J. Cosmet. Dermatol. 2009, 8, 8; d) Cheung, R.; Ng, T.; Wong, J. Marine peptides: bioactivities and applications. Mar. Drugs 2015, 13, 4006; e) Xiao, Y.; Jie, M.; Li, B.; Hu, C.; Xie, R.; Tang, B.; Yang, S. Peptide-based treatment: a promising cancer therapy. J. Immunol. Res. 2015, 2015, 1. (2) For reviews on SPPS, see: a) Bruce, M. Solid-phase peptide synthesis. Peptides 1995, 1995, 93; b) Marshall, G. R. Solid-phase synthesis: a paradigm shift. J. Peptide Sci. 2003, 9, 534. (3) For reviews on the development of SPPS and reagents, see: a) Gravert, D. J.; Janda, K. D. Organic synthesis on soluble polymer supports: liquid-phase methodologies. Chem. Rev. 1997, ACS Paragon Plus Environment

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97, 489; b) Montalbetti, C.; Falque, V. Amide bond formation and peptide coupling, Tetrahedron 2005, 61, 10827; c) Isidro-Llobet, A.; Alvarez, M.; Albericio, F.; Amino acidprotecting groups. Chem. Rev. 2009, 109, 2455; d) White, C.; Yudin, A. Contemporary strategies for peptide macrocyclization. Nat. Chem. 2011, 3, 509; e) A. El-Faham, F. Albericio, Peptide coupling reagents, more than a letter soup. Chem. Rev. 2011, 111, 6557; f) Behrendt, R.; White, P.; Offer, J. Advances in Fmoc solid-phase peptide synthesis. J. Pept. Sci. 2016, 22, 4. (4) a) Bray, B. L. Large-scale manufacture of peptide therapeutics by chemical synthesis. Nat. Rev. Drug Discov. 2003, 2, 587; b) Bruckdorfer, T.; Marder, O.; Albericio, F. From production of peptides in milligram amounts for research to multi-tons quantities for drugs of the future. Curr. Pharm. Bio. 2004, 5, 29. (5) Blanes-Mira, C.; Clemente, J.; Jodas, G.; Gil, A.; Fernández-Ballester, G.; Ponsati, B.; Gutierrez, L.; Pérez-Payá, E.; Ferrer-Montiel, A. A synthetic hexapeptide (Argireline) with antiwrinkle activity. Int. J. Cosmet. Sci. 2002, 24, 303. (6) a) Ruiz, M. A.; Clares, B.; Morales, M. E.; Cazalla, S.; Gallardo, V. Preparation and stability of cosmetic formulations with an anti-aging peptide. J. Cosmet. Sci. 2007, 58, 157; b) Wang, Y.; Wang, M.; Xiao, X.; Huo, J.; Zhang, W. The anti-wrinkle efficacy of Argireline. J. Cosmet. Laser Ther. 2013, 15, 237; c) Wang, Y.; Wang, M.; Xiao, S.; Pan, P.; Li, P.; Huo, J. The antiwrinkle efficacy of argireline, a synthetic hexapeptide, in chinese subjects. Am. J. Clin. Dermatol. 2013, 14, 147. (7) Lungu, C.; Considine, E.; Zahir, S.; Ponsati, B.; Arrastia, S.; Hallett, M. Pilot study of topical acetyl hexapeptide-8 in the treatment for blepharospasm in patients receiving botulinum toxin therapy. Eur. J. Neurol. 2013, 20, 515. (8) a) Ruiz, M. A.; Clares, B.; Morales, M. E.; Cazalla S.; Gallardo, V. Preparation and stability of cosmetic formulations with an anti-aging peptide. J. Cosmet. Sci. 2007, 58, 157; b) Kiris, E.;


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TOC Graphic

A brief abstract

Title Authors’ Names

Full solution-phase synthesis of acetyl Hexapeptide-3: a practical syntheses that combines high efficiency of atom economy with high levels of environmental-friendly and scalability.

Full solution-phase synthesis of acetyl hexapeptide-3 by fragments coupling strategy Teng Zhang,a Wei Song,b Jinli Zhaob and Jianli Liu*,c

Graphical Information

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Full Solution-Phase Synthesis of Acetyl Hexapeptide-3 by Fragments

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