Synthesis and assignment of stereochemistry of the antibacterial cyclic peptide xenematide

Abstract

The synthesis of the antimicrobial cyclic peptide xenematide was accomplished by Fmoc solid phase peptide synthesis and the key esterification reaction was achieved using a modified Yamaguchi esterification. Comparison of the optical rotation and NMR data of the synthesized diastereomers to that of the natural product confirmed the structure of xenematide to be PA-L-[Thr-L-Trp-D-Trp-β-Ala]. (PA = phenylacetyl).

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1. Introduction

Xenematide is a cyclodepsipeptide isolated from the bacteria Xenorhabdus nematophilus in 2008.¹Xenematide exhibits potent antibacterial activity against several bacterial strains including Erwinia amylovora, the pathogen which causes fire blight, a contagious disease that causes the death of apple and pear trees.^1,2 Fire blight is an ongoing horticultural problem both internationally and locally. The presence of fire blight in New Zealand restricts fruit exportation to foreign markets; thus effective control over the disease is required. Fire blight is currently controlled with copper solutions, the aminoglycoside antibiotic streptomycin or the less effective antibiotic oxytetracycline.³Streptomycin is used at concentrations of 50–200 μM and is by far the most efficient treatment although streptomycin-resistant strains of E. amylovora have been observed.^4,5 Development of novel and effective antibacterial agents such as xenematide is therefore crucial in order to prevent outbreaks of this disease in local horticultural industries. Xenematide has a molecular weight of 662.3 g mol⁻¹ and was shown to consist of one β-alanine (β-Ala) residue, two tryptophan (Trp) residues, one threonine (Thr) residue and one phenylacetyl (PA) group. The threonine residue was determined to be of the L-configuration, whereas both L-and D-tryptophan residues were present in the structure. The order of the L-and D-tryptophan residues within the cyclic peptide could not be assigned at the time of isolation thus two diastereomers are possible. Chemical synthesis of the two diastereomers, namely PA-L-[Thr-D-Trp-L-Trp-β-Ala] and PA-L-[Thr-L-Trp-D-Trp-β-Ala] would allow comparison of their optical rotation and NMR spectra to that of the isolated natural product such that the correct relative and absolute stereochemistry can be unequivocally assigned.

Xenematide (1) can be synthesized by late stage macrolactam formation after cleavage from the resin, via intramolecular cyclization, of the N-terminal β-alanine onto the C-terminal tryptophan residue of resin-bound PA-L-Thr-(L,D)-Trp-(D,L)-Trp-β-Ala 2. Peptide 2 is obtained via esterification of the secondary alcohol on threonine of tripeptide 3 with the carboxylic acid of Boc-β-Ala-OH. Tripeptide 3 in turn is prepared using Fmoc solid phase peptide synthesis (SPPS) starting from either L-or D-Trp-2-ClTrt-resin 4 (Scheme 1).^6–8 2-Chlorotrityl-resin (2-ClTrt-resin) was chosen in the current synthesis to minimize C-terminal racemization and the formation of alkylated by-products resulting from cations that can be generated from benzyl-based linkers during TFA-mediated cleavage.⁹

Scheme 1 Proposed retrosynthesis of xenematide (1).

2. Results and discussion

The synthesis of diastereomer PA-L-[Thr-D-Trp-L-Trp-β-Ala] 11 began with amide coupling of Fmoc-D-Trp(Boc)-OH to commercially available H₂N-L-Trp-2-ClTrt-resin 5 using HBTU and DIPEA in DMF. After Fmoc deprotection with 20% piperidine solution, dipeptide 6 was coupled to PA-L-Thr-OH prepared according to literature procedure¹⁰ affording tripeptide 7. Esterification between the carboxylic acid group on Boc-β-Ala-OH and the hydroxyl group on threonine of tripeptide 7 was then attempted using DIC/DMAP¹¹ but the reaction did not proceed even after extended reaction times under microwave irradiation (Scheme 2).

Scheme 2 Synthesis of tripeptide 7 and attempted esterification of Boc-β-Ala-OH with L-threonine. Reagents, conditions and yields: i) Fmoc-D-Trp(Boc)-OH (3 equiv.), HBTU, DIPEA, DMF, rt, 45 min; ii) 20% piperidine/DMF, rt; iii) PA-L-Thr-OH (3 equiv.), HBTU, DIPEA, DMF, rt, 45 min; iv) Boc-β-Ala-OH (3 equiv.), DIC, DMAP, DMF, Δ, microwave.

After extensive investigation of the formation of the ester bond between threonine and Boc-β-Ala-OH, it was found that the use of modified Yamaguchi esterification conditions¹² (BzCl, Et₃N and catalytic DMAP in THF) afforded the desired dipeptide 9 in satisfactory yield in the solution phase reaction (Scheme 3). Unfortunately, subsequent removal of the benzyl group by hydrogenolysis followed by coupling to dipeptide 6 using HBTU and DIPEA in DMF resulted in the formation of a product with m/z value of 462.2. This undesired product 10, identified as β-Ala-D-Trp-L-Trp, was postulated to form viaβ-Ala transfer from threonine to tryptophan during the peptide coupling process (Scheme 3).

Scheme 3 Esterification using modified Yamaguchi conditions and formation of undesired product 10. Reagents, conditions and yields: i) Et₃N, BnBr, DMF, rt, 16 h, 73%; ii) Boc-β-Ala-OH, BzCl, Et₃N, DMAP, THF, rt, 88 h, 71%; iii) H₂, 10% Pd/C, MeOH, rt, overnight, 93%; iv) compound 6, HBTU, DIPEA, DMF, rt, 45 min; v) TFA : H₂O : TIPS (95 : 2.5 : 2.5), 1 h.

Esterification using modified Yamaguchi conditions was next performed on resin-bound peptide 7, forming the desired peptide 8 in excellent yield with none of the undesired product 10 being detected (Scheme 4). CH₂Cl₂ was found to be a superior solvent to DMF and use of excess reagents resulted in a shortened reaction time with quantitative conversion. Following cleavage from the resin using a mixture of TFA/H₂O/TIPS (95 : 2.5 : 2.5), intramolecular cyclization was carried out using BOPCl and DMAP¹³ to give the diastereomer PA-L-[Thr-D-Trp-L-Trp-β-Ala] 11 (Scheme 4).

Scheme 4 Synthesis of PA-L-[Thr-D-Trp-L-Trp-β-Ala] 11. Reagents, conditions and yields: i) Boc-β-Ala-OH (20 equiv.), BzCl (20 equiv.), Et₃N (40 equiv.), CH₂Cl₂, rt, 18 h; ii) TFA : H₂O :TIPS (95 : 2.5 : 2.5), 1 h; iii) BOPCl, DMAP, CH₂Cl₂–MeOH, 0 °C to rt, 19 h, 8% from compound 5.

Alternatively, the other possible diastereomer PA-L-[Thr-L-Trp-D-Trp-β-Ala] 12 was synthesized in a similar manner starting from D-Trp(Boc)-2-ClTrt-resin 13 (Scheme 5). Upon comparison of the optical rotation and NMR data (¹H and ¹³C) of the synthesized peptides to that of the natural product (Tables 1–3), it was established that xenematide contains the peptide sequence PA-L-[Thr-L-Trp-D-Trp-β-Ala] 12.

Scheme 5 Synthesis of PA-L-[Thr-L-Trp-D-Trp-β-Ala] 12. Reagents, conditions and yields: i) Fmoc-L-Trp(Boc)-OH (3 equiv.), HBTU, DIPEA, DMF, rt, 45 min; ii) 20% piperidine/DMF, rt; iii) PA-L-Thr-OH (3 equiv.), HBTU, DIPEA, DMF, rt, 45 min; iv) Boc-β-Ala-OH (20 equiv.), BzCl (20 equiv.), Et₃N (40 equiv.), CH₂Cl₂, rt, 18 h; v) TFA : H₂O : TIPS (95 : 2.5 : 2.5), 1 h; vi) BOPCl, DMAP, CH₂Cl₂–MeOH, 0 °C to rt, 19 h, 4% from compound 13.

Table 1 δ _H and δ_C(600 MHz; DMSO-d₆) of isolated xenematide (1)¹

Position	δ C, multiplicity	δ C (J in Hz)	Position	δ C, multiplicity	δ C (J in Hz)
a PA = phenylacetyl. [α]^20D +45.0 (c 0.20 in MeOH).
β-Ala	169.2, qC		Trp–4	109.3, qC
	33.9, CH2	2.52, m	–5	127.0, qC
		2.40, m	–6	118.2, CH	7.55, d (7.8)
	34.6, CH2	3.39, m	–7	118.2, CH	7.00, td (7.8, 1.0)
		3.32, m	–8	121.0, CH	7.08, m
β-Ala-NH		7.39, t (6.1)	–9	111.3, CH	7.36, bd (8.0)
Trp–1	171.0, qC		–10	136.0, qC
–2	54.5, CH	4.19, ddd (10.0, 7.9, 3.7)	–11		10.7, s
–3	25.7, CH2	3.18, m	–12	123.4, CH	7.09, m
		2.87, m	Trp-NH		8.81, d (6.7)
–4	110.6, qC		Thr	170.2, qC
–5	126.9, qC			54.0, CH	4.65, dd (9.2, 2.2)
–6	117.9, CH	7.49, d (7.8)		72.0, CH	5.11, qd (6.3, 2.3)
–7	118.2, CH	6.96, m		16.2, CH3	1.05, d (6.2)
–8	120.8, CH	7.05, td (7.8, 1.0)	Thr-NH		8.09, d (9.5)
–9	111.3, CH	7.33, bd (8.0)	PA^a	170.6, qC
–10	135.9, qC			41.7, CH2	3.65, d (14.1)
–11		10.6, s			3.55, d (14.1)
–12	123.1, CH	6.96, m		136.3, qC
Trp-NH		8.72, d (6.7)		129.0, CH	7.26, m
Trp–1	172.0, qC			128.1, CH	7.29, m
–2	54.0, CH	4.53, q (7.3)		126.1, CH	7.19, m
–3	25.7, CH2	2.82–2.92, m

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Table 2 δ _H(300 MHz; DMSO-d₆) and δ_C(75 MHz; DMSO-d₆) of PA-L-[Thr-D-Trp-L-Trp-β-Ala] 11

Position	δ C, multiplicity	δ C (J in Hz)	Position	δ C, multiplicity	δ C (J in Hz)
a PA = phenylacetyl. b δ H Peaks for PhCH2CON are not included as they are obscured by DMSO-d6. [α]^20D −36.9 (c 0.52 in MeOH).
β-Ala	169.70, qC		Trp–4	109.98, qC
	34.77, CH2	2.28–2.44, m	–5	127.59, qC
	35.21, CH2	3.55–3.59, m	–6	118.77, CH	7.46, d (7.8)
β-Ala-NH		7.22–7.35, m	–7	118.77, CH	6.91–6.95, m
Trp–1	171.52, qC		–8	121.39, CH	6.91–6.95, m
–2	55.32, CH	4.30–4.34, m	–9	111.85, CH	7.22–7.35, m
–3	26.45, CH2	2.74–2.95, m	–10	136.47, qC
–4	110.93, qC		–11		10.69, s
–5	127.59, qC		–12	123.66, CH	6.91–6.95, m
–6	118.54, CH	7.41, d (7.8)	Trp-NH		8.44–8.59, m
–7	118.54, CH	6.91–6.95, m	Thr	171.30, qC
–8	121.33, CH	6.91–6.95, m		56.59, CH	4.44–4.48, m
–9	111.85, CH	7.22–7.35, m		70.43, CH	5.41–5.43, m
–10	136.47, qC			16.95, CH3	1.03, d (6.0)
–11		10.69, s	Thr-NH		7.82, d (7.8)
–12	123.66, CH	6.91–6.95, m	PA^a	171.45, qC
Trp-NH		8.44–8.59, m		42.56, CH2	—^b
Trp–1	171.76, qC			136.85, qC
–2	53.70, CH	4.53–4.57, m		129.48, CH	7.22–7.35, m
–3	27.04, CH2	3.12–3.17, m		128.75, CH	7.22–7.35, m
				126.90, CH	7.22–7.35, m

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Table 3 δ _H(300 MHz; DMSO-d₆) and δ_C(75 MHz; DMSO-d₆) of PA-L-[Thr-L-Trp-D-Trp-β-Ala] 12

Position	δ C, multiplicity	δ C (J in Hz)	Position	δ C, multiplicity	δ C (J in Hz)
a PA = phenylacetyl. [α]^20D +61.0 (c 0.58 in MeOH).
β-Ala	169.78, qC		Trp–4	109.82, qC
	34.48, CH2	2.37–2.49, m	–5	127.49, qC
			–6	118.81, CH	7.54, d (7.8)
	35.13, CH2	3.39–3.41, m	–7	118.81, CH	6.93–7.09, m
			–8	121.53, CH	6.93–7.09, m
β-Ala-NH		7.17–7.42, m	–9	111.90, CH	7.17–7.42, m
Trp–1	171.61, qC		–10	136.54, qC
–2	55.07, CH	4.17, ddd (10.2, 6.6, 3.6)	–11		10.72, s
–3	26.25, CH2	3.16–3.21, m	–12	123.96, CH	6.93–7.09, m
–4	111.17, qC		Trp-NH		8.82, d (6.9)
–5	127.40, qC		Thr	170.83, qC
–6	118.49, CH	7.48, d (7.8)		54.53, CH	4.62–4.65, m
–7	118.81, CH	6.93–7.09, m		72.54, CH	5.09–5.11, m
–8	121.38, CH	6.93–7.09, m		16.70, CH3	1.04, d (6.0)
–9	111.84, CH	7.17–7.42, m	Thr-NH		8.13, d (6.9)
–10	136.54, qC		PA^a	171.20, qC
–11		10.63, s		42.17, CH2	3.65, d (14.1)
–12	123.70, CH	6.93–7.09, m			3.54, d (14.1)
Trp-NH		8.76, d (6.9)		136.83, qC
Trp–1	172.58, qC			129.55, CH	7.17–7.42, m
–2	54.44, CH	4.52, q (7.2)		128.64, CH	7.17–7.42, m
–3	26.25, CH2	2.82–2.95, m		126.78, CH	7.17–7.42, m

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In addition to the two possible diastereomers of the natural cyclic peptide xenematide, two more unnatural diastereomers with the peptide sequences, PA-L-[Thr-L-Trp-L-Trp-β-Ala] and PA-L-[Thr-D-Trp-D-Trp-β-Ala], were also synthesized using the same synthetic strategy. It was envisaged that these additional cyclic peptides could be used to probe the bioactivity of xenematide.

3. Conclusions

The synthesis of the antimicrobial cyclic peptide xenematide was accomplished by Fmoc solid phase peptide synthesis with the key esterification reaction being achieved using a modified Yamaguchi esterification. Comparison of the optical rotation and NMR data of the synthesized diastereomers to that of the natural product confirmed the structure of xenematide to be PA-L-[Thr-L-Trp-D-Trp-β-Ala]. (PA = phenylacetyl). The antibacterial activity of the synthetic peptides and the design of further peptidomimetic analogues of xenematide are currently being investigated.

4. Experimental

4.1 Synthesis of (2S,3R)-benzyl 3-[3–(tert-butoxycarbonyl amino)propanoyloxy]-2-(2-phenylacetamido)butanoate 9^10,12

4.1.1 Synthesis of (2S,3R)-benzyl 2-hydroxy-2-(2-phenyl acetamido)butanoate (PA-L-Thr-OBn). To a solution of L-threonine (5.42 g, 45.49 mmol) in 1 M aqueous NaOH solution (150 mL) at 0 °C was added phenylacetyl chloride (7.85 mL, 59.12 mmol) dropwise, and the reaction was stirred at 0 °C for 1 h. Phenylacetyl chloride (7.85 mL, 59.12 mmol) was added at 0 °C and the reaction mixture was warmed to room temperature and stirred for 18 h. After extraction with ethyl acetate (90 mL), the aqueous layer was acidified with 3 M HCl solution to pH 2 and was extracted with ethyl acetate (3 × 120 mL). The combined organic extracts were dried over anhydrous Na₂SO₄, filtered, concentrated in vacuo to give a colourless solid which was washed with cold diethyl ether and used without further purification. To a solution of the above solid (6.03 g, 25.44 mmol) in DMF (28 mL) at room temperature was added triethylamine (4.25 mL, 30.49 mmol), benzyl bromide (6.63 mL, 30.52 mmol), and the reaction mixture was stirred for 16 h. Water (24 mL) and dichloromethane (60 mL) were added and the suspension was stirred for 15 min. The organic layer was separated and the aqueous layer was extracted with dichloromethane (3 × 60 mL). The combined organic extracts were dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Washing with cold diethyl ether afforded the desired product (6.07 g, 73%) as a colourless solid; R_f 0.41 (1 : 1 EtOAc–hexane); mp 150.5–152.8 °C; v_max(film)/cm⁻¹ 3469, 3349, 1705, 1648, 1529, 1279, 1001, 725 and 693; [α]²⁰_D −3.6 (c 1.11 in CDCl₃); δ_H(300 MHz; CDCl₃) 1.04 (3H, d, J 6.0, Thrβ–CH₃), 3.55 (2H, s, PhCH₂CON), 4.25 (1H, dq, J 3.0 and 6.0, Thrβ–CH), 4.48–4.52 (1H, m, Thrα–CH), 5.07 (2H, s, PhCH₂CO₂), 6.88 (1H, d, CONHCH), 7.17–7.27 (10H, m, Ph); δ_C(75 MHz; CDCl₃) 19.6 (CH₃, Thrβ–CH₃), 42.95 (CH₂, PhCH₂CON), 43.0 (CH₂, PhCH₂CON), 57.6 (CH, Thrα–CH), 57.65 (CH, Thrα–CH), 67.1 (CH₂, PhCH₂CO₂), 67.2 (CH, Thrβ–CH), 126.9 (CH, Ph), 127.9 (CH, Ph), 128.1 (CH, Ph), 128.2 (CH, Ph), 128.4 (CH, Ph), 128.6 (CH, Ph), 129.0 (CH, Ph), 129.6 (CH, Ph), 132.75 (quat., Ph), 134.4 (quat., Ph), 135.1 (quat., Ph), 170.6 (quat., CHCO₂CH₂), 172.1 (quat., CH₂CON), 172.2 (quat., CH₂CON); m/z (EI) 328.1531 (MH⁺, C₁₉H₂₂NO₄ requires 328.1543), 328 (MH⁺, 6%), 350 (MNa⁺, 100%), 351 (20) and 352 (2).

4.1.2 Synthesis of (2S,3R)-benzyl 3-[3-(tert-butoxycarbonyl amino)propanoyloxy]-2-(2-phenylacetamido)butanoate 9. To a solution of PA-L-Thr-OBn (1.70 g, 5.19 mmol), Boc-β-Ala-OH (0.98 g, 5.19 mmol), and benzoyl chloride (0.61 mL, 5.25 mmol) in dry THF (47 mL) at room temperature under N₂ was added dropwise triethylamine (1.45 mL, 10.38 mmol) and DMAP (0.16 g, 1.31 mmol), and the reaction mixture was stirred for 88 h. The solvent was removed in vacuo and the residue was purified by flash column chromatography (EtOAc–hexane 1 : 2) to give compound 9 (1.85 g, 71%) as a yellow oil; R_f 0.39 (1 : 2 EtOAc–hexane); v_max(film)/cm⁻¹ 3302, 1739, 1663, 1519, 1164, 731 and 697; [α]²⁰_D +17.3 (c 2.61 in CDCl₃); δ_H(300 MHz; CDCl₃) 1.17 (3H, d, J 6.0, Thrβ–CH₃), 1.44 [9H, s, C(CH₃)₃], 2.21–2.30 (2H, m, CO₂CH₂CH₂NH), 3.16–3.27 (2H, m, CO₂CH₂CH₂NH), 3.67 (2H, s, PhCH₂CON), 4.79 (1H, d, J 9.0, Thrα–CH), 5.09 (2H, d, PhCH₂CO₂), 5.38 (1H, d, J 3.0, Thrβ–CH), 7.26–7.37 (10H, m, Ph); δ_C(75 MHz; CDCl₃) 16.9 (CH₃, Thrβ–CH₃), 28.3 [CH₃, C(CH₃)₃], 34.6 (CH₂, β–Ala–CH₂), 35.9 (CH₂, β–Ala–CH₂), 43.3 (CH₂, PhCH₂CON), 55.5 (CH, Thrα–C), 67.5 (CH₂, PhCH₂CO₂), 70.6 (CH, Thrβ–C), 79.4 [quat., C(CH₃)₃], 127.3 (CH, Ph), 128.3 (CH, Ph), 128.5 (CH, Ph), 128.6 (CH, Ph), 128.8 (CH, Ph), 129.3 (CH, Ph), 129.9 (CH, Ph), 133.1 (CH, Ph), 134.5 (quat., Ph), 134.9 (quat., Ph), 155.8 (quat., NCO₂C), 169.5 (quat., CHCO₂CH₂, CHCO₂CH₂), 170.5 (quat., CH₂CON); m/z (EI) 521.2256 (MNa⁺, C₂₇H₃₄N₂NaO₇ requires 521.2258), 521 (MNa⁺, 100%), 522 (48), 523 (10), 537 (20) and 538 (4).

4.2 Synthesis of H₂N-D-Trp(Boc)-2-ClTrt-aminomethyl polystyrene resin 13^6–8

To aminomethyl polystyrene resin (0.1 g) was added a mixture of 2-chloro-4′-carboxytriphenylmethanol (68.8 mg, 0.2 mmol) and N,N-diisopropylcarbodiimide (21.37 μL, 0.2 mmol) in DMF (1 mL), and the reaction was stirred for 1 h. The resin was washed with DMF (2 × 1 mL), dichloromethane (2 × 1 mL), methanol (3 × 1 mL), diethyl ether (2 × 1 mL) and then dried under N₂. To the above resin was added dry thionyl chloride/dichloromethane (1 : 1 v/v, 4 mL) dropwise, and the reaction was gently stirred at room temperature for 3 h. The resin was washed with DMF (2 × 1 mL), dichloromethane (3 × 1 mL) and then dried for 10 min. To this resin was added a solution of Fmoc-D-Trp(Boc)-OH (0.12 g, 0.2 mmol) and DIPEA (92.1 μL, 0.5 mmol) in dry dichloromethane (2 mL), and the reaction was gently stirred at room temperature for 30 min. After washing with DMF (2 × 1 mL), a solution of CH₂Cl₂/MeOH/DIPEA (80 : 15 : 5 v/v, 10 mL) was added, the reaction was stirred for 10 min and repeated. After washing with DMF (3 × 1 mL), a 20% piperidine/DMF solution (v/v, 10 mL) was added and the reaction was stirred for 3 min and then repeated for 20 min. The resin was washed with DMF (6 × 1 mL), iPrOH (3 × 1 mL), hexane (4 × 1 mL), air dried for 15 min and then dried under N₂. The loading of Fmoc-D-Trp(Boc)-OH was found to be 0.39 mmol g⁻¹ (theoretical loading = 0.58 mmol g⁻¹, 67%) using the Fmoc assay.¹⁴

4.3 Synthesis of PA-L-[Thr-D-Trp-L-Trp-β-Ala] 11

4.3.1 Synthesis of PA-L-Thr-D-Trp-L-Trp-2-ClTrt-resin 7. To H₂N-L-Trp-2-ClTrt-resin (0.56 mmol g⁻¹) (0.18 g, 0.1 mmol) was added a mixture of Fmoc-D-Trp(Boc)-OH (0.16 g, 0.31 mmol), HBTU (0.11 g, 0.29 mmol) and DIPEA (0.11 mL, 0.61 mmol) in DMF (1 mL), and the reaction was agitated for 45 min. The resin was washed with DMF (6 × 1 mL), isopropanol (3 × 1 mL), hexane (4 × 1 mL) and then dried under N₂. A solution of 20% piperidine/DMF (v/v, 10 mL) was added and the reaction was gently agitated for 5 min. The resin was washed with DMF (6 × 1 mL), and the same procedure was repeated for 10 min. After washing with DMF (6 × 1 mL), a mixture of PA-L-Thr-OH (0.08 g, 0.33 mmol), HBTU (0.11 g, 0.29 mmol) and DIPEA (0.11 mL, 0.61 mmol) in DMF (1 mL) was added, and the reaction was agitated for 45 min. The resin was washed with DMF (6 × 1 mL), isopropanol (3 × 1 mL), hexane (4 × 1 mL) and then dried under N₂.

4.3.2 Synthesis of PA-L-Thr(Boc-β-Ala)-D-Trp-L-Trp-2-ClTrt-resin 8. To peptide 7 was added a mixture of Boc-β-Ala-OH (0.39 g, 2.05 mmol), benzoyl chloride (0.24 mL, 2.04 mmol), triethylamine (0.57 mL, 4.08 mmol) and DMAP (4.7 mg, 0.04 mmol) in dichloromethane (10 mL) and the reaction was agitated for 18 h. The resin was washed with DMF (6 × 1 mL), isopropanol (3 × 1 mL), hexane (4 × 1 mL) and dried under N₂.

4.3.3 Synthesis of PA-L-[Thr-D-Trp-L-Trp-β-Ala] 11¹³. To peptide 8 was added a mixture of TFA/H₂O/TIPS (95 : 2.5 : 2.5 v/v, 10 mL) and the reaction was agitated for 1 h. The solution was filtered and concentrated in vacuo. To the resultant yellow residue (69.5 mg, 0.1 mmol) was dissolved in dichloromethane–methanol (4 : 1 v/v, 139 mL) at 0 °C, BOPCl (0.13 g, 0.51 mmol) and DMAP (0.11 g, 0.92 mmol) were added, and the reaction was stirred for 19 h. 1 M HCl solution (30 mL) was added and the aqueous layer was extracted with dichloromethane (3 × 80 mL). The combined organic extracts were dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Purification by semi-preparative RP–HPLC (using a linear gradient of 40% B to 70% B) yielded the title compound (8% from H₂N-L-Trp-2-ClTrt-resin) as an off-white amorphous solid in >99% purity according to analytical RP-HPLC; R_t 10.77 min (XTerra C18, 4.6 × 150 mm, 30% to 75% B over 15 min, 1 mL min⁻¹); mp 150.2–156.4 °C; v_max(film)/cm⁻¹ 3315, 1736, 1649, 1514, 1165, 1060, 743 and 697; [α]²⁰_D −36.9 (c 0.52 in MeOH); δ_H(300 MHz; DMSO-d₆)^* 1.03 (3H, d, Thrβ–CH₃), 2.28–2.44 (2H, m, β–Ala–CH₂), 2.74–2.95 (3H, m, Trpβ–CH₂), 3.12–3.17 (1H, m, Trpβ–CH₂), 3.55–3.59 (2H, m, β–Ala–CH₂), 4.30–4.34 (1H, m, Trpα–CH), 4.44–4.48 (1H, m, Thrα–CH), 4.53–4.57 (1H, m, Trpα–CH), 5.41–5.43 (1H, m, Thrβ–CH), 6.91–6.95 (6H, m, Trp–H7, Trp–H8, Trp–H12), 7.22–7.35 (8H, m, β–Ala–NH, Trp–H9, PA–Ph), 7.41 (1H, d, J 7.8, Trp–H6), 7.46 (1H, d, J 7.8, Trp–H6), 7.82 (1H, d, J 7.8, Thr–CONH), 8.44–8.59 (2H, m, Trp–CONH), 10.69 (2H, s, Trp–H11); δ_C(75 MHz, DMSO-d₆) 16.95 (CH₃, Thrβ–CH₃), 26.45 (CH₂, Trpβ–CH₂), 27.04 (CH₂, Trpβ–CH₂), 34.77 (CH₂, β-Ala–CH₂), 35.21 (CH₂, β-Ala–CH₂), 42.56 (CH₂, PhCH₂CON), 53.70 (CH, Trpα–CH), 55.32 (CH, Trpα–CH), 56.59 (CH, Thrα–CH), 70.43 (CH, Thrβ–CH), 109.98 (quat., Trp–C4), 110.93 (quat., Trp–C4), 111.85 (CH, Trp–C9), 118.54 (CH, Trp–C6 and C7), 118.77 (CH, Trp–C6 and C7), 121.33 (CH, Trp–C8), 121.39 (CH, Trp–C8), 123.66 (CH, Trp–C12), 126.90 (CH, Ph), 127.51 (quat., Trp–C5), 127.59 (quat., Trp–C5), 128.75 (CH, Ph), 129.48 (CH, Ph), 136.47 (quat., Trp–C10), 136.85 (quat., PA–Ph), 169.70 (quat., β–Ala–CON), 171.30 (quat., Thr–CON), 171.45 (quat., PhCH₂CON), 171.52 (quat., Trp–CON), 171.76 (quat., Trp–CON); m/z (EI) 663.2918 (MH⁺, C₃₇H₃₉N₆O₆ requires 663.2926), 663 (MH⁺, 21%), 682 (30), 685 (MNa⁺, 100%), 686 (40), 687 (9) and 701 (42). (^* 1H peaks for PhCH₂CON are not included as they are obscured by DMSO-d₆.)

4.4 Synthesis of PA-L-[Thr-L-Trp-D-Trp-β-Ala] 12

The title compound [4% from resin 13 (0.39 mmol g⁻¹) (0.25 g, 0.1 mmol)] was obtained as a colourless amorphous solid in >99% purity according to analytical RP-HPLC; R_t 10.70 min (XTerra C18, 4.6 × 150 mm, 30% to 75% B over 15 min, 1 mL min⁻¹); mp 181.9–184.6 °C; v_max(film)/cm⁻¹ 3276, 1739, 1634, 1547, 1261, 1233, 1187 and 742; [α]²⁰_D +61.0 (c 0.58 in MeOH); δ_H(300 MHz; DMSO-d₆) 1.04 (3H, d, J 6.0, Thrβ–CH₃), 2.37–2.49 (2H, m, β–Ala–CH₂), 2.82–2.95 (3H, m, Trpβ–CH₂), 3.16–3.21 (1H, m, Trpβ–CH₂), 3.39 (2H, m, β–Ala–CH₂), 3.54 (1H, d, J 14.1, PhCH₂CON), 3.65 (1H, d, J 14.1, PhCH₂CON), 4.17 (1H, ddd, J 10.2, 6.6 and 3.6, Trpα–CH), 4.52 (1H, q, J 7.2, Trpα–CH), 4.62–4.65 (1H, m, Thrα–CH), 5.09–5.11 (1H, m, Thrβ–CH), 6.93–7.09 (6H, m, Trp–H7, Trp–H8, Trp–H12), 7.17–7.42 (8H, m, β–Ala–NH, Trp–H9, PA–Ph), 7.48 (1H, d, J 7.8, Trp–H6), 7.54 (1H, d, J 7.5, Trp–H6), 8.13 (1H, d, J 6.9, Thr–CONH), 8.76 (1H, d, J 6.9, Trp–CONH), 8.82 (1H, d, J 6.6, Trp–CONH), 10.63 (1H, s, Trp–H11), 10.72 (1H, s, Trp–H11); δ_C(75 MHz; DMSO-d₆) 16.70 (CH₃, Thrβ–CH₃), 26.25 (CH₂, Trpβ–CH₂), 34.48 (CH₂, β–Ala–CH₂), 35.13 (CH₂, β–Ala–CH₂), 42.17 (CH₂, PhCH₂CON), 54.44 (CH, Trpα–CH), 54.53 (CH, Thrα–CH), 55.07 (CH, Trpα–CH), 72.54 (CH, Thrβ–CH), 109.82 (quat., Trp–C4), 111.17 (quat., Trp–C4), 111.84 (CH, Trp–C9), 111.90 (CH, Trp–C9), 118.49 (CH, Trp–C6), 118.81 (CH, Trp–C6 and C7), 121.38 (CH, Trp–C8), 121.53 (CH, Trp–C8), 123.70 (CH, Trp–C12), 123.96 (CH, Trp–C12), 126.78 (CH, Ph), 127.40 (quat., Trp–C5), 127.49 (quat., Trp–C5), 128.64 (CH, Ph), 129.55 (CH, Ph), 136.45 (quat., Trp–C10), 136.54 (quat., Trp–C10), 136.83 (quat., PA–Ph), 169.78 (quat., β–Ala–CON), 170.83 (quat., Thr–CON), 171.20 (quat., PhCH₂CON), 171.61 (quat., Trp–CON), 172.58 (quat., Trp–CON); m/z (EI) 663.2913 (MH⁺, C₃₇H₃₉N₆O₆ requires 663.2926), 663 (MH⁺, 20%), 685 (MNa⁺, 100%), 686 (32), 687 (8) and 701 (35).

4.5 Synthesis of PA-L-[Thr-L-Trp-L-Trp-β-Ala]

The title compound (6% from H₂N-L-Trp-2-ClTrt-resin) was obtained as an off-white amorphous solid in >99% purity according to analytical RP-HPLC; R_t 10.46 min (XTerra C18, 4.6 × 150 mm, 30% to 75% B over 15 min, 1 mL min⁻¹); mp 162.6–167.4 °C; v_max(film)/cm⁻¹ 3324, 1727, 1649, 1522, 1457, 1177, 1069, 742 and 703; [α]²⁰_D −30.7 (c 0.81 in MeOH); δ_H(300 MHz; DMSO-d₆) 1.13 (3H, d, J 6.3, Thrβ–CH₃), 2.29–2.37 (1H, m, β-Ala–CH₂), 2.60–2.70 (1H, m, β–Ala–CH₂), 2.93–3.03 (2H, m, Trpβ–CH₂), 3.05–3.10 (1H, m, Trpβ–CH₂), 3.24–3.35 (1H, m, Trpβ–CH₂), 3.36–3.41 (2H, m, β–Ala–CH₂), 3.61 (2H, m, PhCH₂CON), 4.15–4.28 (2H, m, Trpα–CH), 4.61–4.65 (1H, m, Thrα–CH), 5.03–5.06 (1H, qd, J 6.3 and 1.5, Thrβ–CH), 6.53 (2H, s, Trp–H8), 6.83 (2H, s, Trp–H12), 7.02–7.08 (1H, m, Trp–H9), 7.12–7.17 (1H, m, Trp–H9), 7.19–7.38 (7H, m, Trp–H7 and PA–Ph), 7.43–7.47 (2H, m, Trp–H6); δ_C(75 MHz; DMSO-d₆) 16.16 (CH₃, Thrβ–CH₃), 24.48 (CH₂, Trpβ–CH₂), 25.80 (CH₂, Trpβ–CH₂), 34.28 (CH₂, β–Ala–CH₂), 34.99 (CH₂, β–Ala–CH₂), 42.85 (CH₂, PhCH₂CON), 54.61 (CH, Thrα–CH), 55.22 (CH, Trpα–CH), 55.94 (CH, Trpα–CH), 72.25 (CH, Thrβ–CH), 108.70 (quat., Trp–C4), 110.68 (quat., Trp–C4), 111.00 (CH, Trp–C9), 111.15 (CH, Trp–C9), 117.94 (CH, Trp–C6), 118.09 (CH, Trp–C7), 118.72 (CH, Trp–C6), 118.95 (CH, Trp–C7), 121.32 (CH, Trp–C8), 121.62 (CH, Trp–C8), 122.90 (CH, Trp–C12), 123.20 (CH, Trp–C12), 126.71 (quat., Trp–C5), 127.12 (quat., Trp–C5), 127.21 (CH, Ph), 128.61 (CH, Ph), 129.03 (CH, Ph), 134.24 (quat., PA–Ph), 136.00 (quat., Trp–C10), 136.12 (quat., Trp–C10), 169.96 (quat., β–Ala–CON), 171.06 (quat., Thr–CON), 171.42 (quat., PhCH₂CON), 171.99 (quat., Trp–CON), 172.08 (quat., Trp–CON); m/z (EI) 663.2913 (MH⁺, C₃₇H₃₉N₆O₆ requires 663.2926), 663 (MH⁺, 20%), 685 (MNa⁺, 100%), 686 (32), 687 (8) and 701 (35).

4.6 Synthesis of PA-L-[Thr-D-Trp-D-Trp-β-Ala]

The title compound [8% from resin 13 (0.39 mmol g⁻¹) (0.25 g, 0.1 mmol)] was obtained as an off-white amorphous solid in >90% purity according to analytical RP-HPLC; R_t 10.53 min (XTerra C18, 4.6 × 150 mm, 30% to 75% B over 15 min, 1 mL min⁻¹); mp 155.2–159.2 °C; v_max(film)/cm⁻¹ 3315, 1727, 1657, 1527, 1457, 1166, 1061, 743 and 698; [α]²⁰_D +51.1 (c 0.99 in MeOH); δ_H(300 MHz; DMSO-d₆) 0.92 (3H, d, J 6.6, Thrβ–CH₃), 2.33–2.36 (2H, m, β-Ala–CH₂), 2.88–3.03 (2H, m, Trpβ–CH₂), 3.05–3.19 (2H, m, Trpβ–CH₂), 3.42–3.54 (2H, m, β–Ala–CH₂), 3.78–3.83 (2H, m, PhCH₂CON), 4.35–4.44 (3H, m, Trpα–CH and Thrα–CH), 5.48–5.54 (1H, qd, J 6.6 and 4.5, Thrβ–CH), 6.40 (2H, s, Trp–H8), 6.80 (2H, s, Trp–H12), 7.04–7.10 (2H, m, Trp–H9), 7.12–7.21 (2H, m, Trp–H6), 7.27–7.42 (7H, m, Trp–H7 and PA–Ph); δ_C(75 MHz; DMSO-d₆) 16.09 (CH₃, Thrβ–CH₃), 25.26 (CH₂, Trpβ–CH₂), 26.19 (CH₂, Trpβ–CH₂), 34.57 (CH₂, β–Ala–CH₂), 35.36 (CH₂, β–Ala–CH₂), 42.84 (CH₂, PhCH₂CON), 54.13 (CH, Trpα–CH), 54.94 (CH, Trpα–CH), 56.11 (CH, Thrα–CH), 69.50 (CH, Thrβ–CH), 108.22 (quat., Trp–C4), 111.17 (CH, Trp–C9), 111.32 (CH, Trp–C9), 117.74 (CH, Trp–C6), 118.43 (CH, Trp–C6), 118.93 (CH, Trp–C7), 119.47 (CH, Trp–C7), 121.61 (CH, Trp–C8), 121.88 (CH, Trp–C8), 122.82 (CH, Trp–C12), 123.44 (CH, Trp–C12), 126.94 (quat., Trp–C5), 127.21 (CH, Ph), 127.38 (quat., Trp–C5), 128.72 (CH, Ph), 128.81 (CH, Ph), 134.66 (quat., PA–Ph), 135.98 (quat., Trp–C10), 136.05 (quat., Trp–C10), 169.99 (quat., β–Ala–CON), 171.39 (quat., Thr–CON and PhCH₂CON), 171.89 (quat., Trp–CON), 172.63 (quat., Trp–CON); m/z (EI) 663.2920 (MH⁺, C₃₇H₃₉N₆O₆ requires 663.2926), 663 (MH⁺, 100%), 664 (40), 665 (10), 666 (2), 685 (MNa⁺, 60%), 686 (32) and 687 (8).

Acknowledgements

We thank the Maurice Wilkins Centre for Molecular Discovery for financial support.

Notes and references

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7. M. Harre, K. Nickisch and U. Tilstam, React. Funct. Polym., 1999, 41, 111–114.
8. K. Barlos and D. Gatos, in Fmoc Solid Phase Peptide Synthesis, ed. W. C. Chan and P. D. White, Information Press, Oxford, U. K., 2000, ch. 9, pp. 216.
9. K. Barlos, O. Chatzi and D. Gatos, Int. J. Peptide Protein Res., 1991, 37, 513–520.
10. G. P. Nora, M. J. Miller and U. Möllmann, Bioorg. Med. Chem. Lett., 2006, 16, 3966–3970.
11. M. Stawikowski and P. Cudic, Tetrahedron Lett., 2006, 47, 8587–8590.
12. I. Dhimitruka and Jr. J. SantaLucia, Org. Lett., 2006, 8, 47–50.
13. A. K. Ghosh and C. Liu, Org. Lett., 2001, 3, 635–638.
14. W. C. Chan and P. D. White, in Fmoc Solid Phase Peptide Synthesis, ed. W. C. Chan and P. D. White, Information Press, Oxford, U. K., 2000, ch. 9, pp. 62.

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Footnote

† Electronic supplementary information (ESI) available: The NMR (¹H and ¹³C) spectra of PA-L-Thr-OBn, compound 9, isolated xenematide and the four diastereomers of xenematide, and the HPLC chromatograms of peptides 8, 10 (after cleavage from resin) and the four diastereomers of xenematide are provided. See DOI: 10.1039/c0ob00315h

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