strategies for cyclizations of novel peptides on solid supports

ELSEVIER Reactive Polymers 22 (1994) 231-241

Reactive polymers

Strategies for cyclizations of novel peptides on solid supports *

Victor J. Hruby *, Susanne Wilke, Fahad AI-Obeidi , Ding Jiao, Ying Lin

Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA

(Received 27 November 1993; accepted 20 December 1993)

Abstract

Synthetic strategies and methods for the preparation of cyclic peptides on solid supports arc very important for peptide synthesis in general, and for the preparation of conformationally constrained biologically active peptides. In this paper we report synthetic methods for the preparation on solid supports of the cyclic and bicyclic biologically active peptides, Ac-[Nle 4, Asp 5, D-Phe, Lysl°]a-melanotropin(4-10)-NH2, [Lys 17, Lys ,~s~ sp21]glucagon-NH2, [/3- Mpa 1, G!u 4, Cys 6, Ly, sS]oxytocin and eyelo[Gly-Trp-N-MeNle-Asp-Phe] (cyclic CCK-5 analogue).

Key words: Solid-phase synthesis; Cyclization on solid supports; Cyclic lactams; Cyclic disulfides; Cyclic peptides; Cyclization of solid supports; Orthogonal synthesis

1. I n t r o d u c t i o n **

The importance of imposing conformational constraints on peptide structures has become a central theme in structure-biological activity studies of biologically active peptides, and in the development of rational approaches to the design of peptides and

* Corresponding author. * Dedicated to Professor Abraham Patchornik for his many contributions to peptide chemistry. **Abbreviations: Abbreviations used in this paper for amino acids, protecting groups and peptides follow the recommenda- tions of the IUPAC-IUB Commission on Biochemical Nomenclature and Symbols as described in J. Biol. Chem., 250 (1975) 3215-3225. All optically active amino acid are of the L variety unless otherwise stated. Other abbreviations include: aMSH, a-melanotropin; Nle, norleucine; Boc, tert.-butyloxy- carbonyl; Born, benzyloxymethyl; N-MeNIe, N-methylnorleu- cine; Dpr, L-2,3-diaminopropionic acid; OFm, O-fluoroen- ylmethyl; Fmoc, fluorenylmethyloxycarbonyl; OT, oxytocin; CCK, cholecystokinin.

peptidomimetics [1-4]. Cyclic peptides are of critical importance in assessing the important structural, conformational and dynamic properties that are critical to the biological potency, efficacy and receptor (acceptor) se- lectivity of bioactive peptides and proteins. In addition, cyclization generally promotes increase of metabolic stability and bioavail- ability of peptides. These and other advantages of cyclization are critical for ultimately obtaining insight into the pharmacophore of a bioactive peptide or protein ligand and its "bioactive" conformation. The use of monocyclic and bicyclic disulfide and /o r amide- bridged peptide structures especially of linear (but also of cyclic) structures that are conformationally constrained has led to a number of outstanding successes [5-10]. Cyclic peptides also have been important for the design of peptides with specific biochemical/biophysical properties such as chelation

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232 V.J. Hruby et aL / Reactive Polymers 22 (1994) 231-241

of a particular metal ion, stabilization of an a-helix or/3-turn, or stabilization of a protein from denaturation.

In many cases there is a need to introduce multiple disulfide a n d / o r lactam or other bridges in the same molecule. This can often lead to difficult complex syntheses with cur- rent conventional solution synthesis methods. Consequently, this has led to the development of novel strategies for cyclization such as the peptide cyclization on oxime resins [11,12] and to direct regioselective side-chain-side-chain cyclization on solid supports [13]. However, these and more recently developed methods are still inade- quate and often do not proceed in high yields making isolation and structure elucidation difficult. This is particularly a critical problem as one attempts to introduce conformational constraints into large peptide libraries. Thus, there is a need to establish mild, effi- cient synthetic strategies that can lead to the generation of clean cyclic peptides on solid supports and that are compatible with auto- mated peptide synthesis. The development of strategies to accomplish these goals and an illustration of some of the progress to date with examples from our laboratory is the subject of this communication.

The work reported here emphasizes side- chain to side-chain and backbone to backbone cyclizations, but it also is applicable to backbone to side-chain cyclizations. We will present results of cyclization on the resin without concomitant cleavage from the resin, and cyclization on solid supports with concomitant cleavage from the resin.

2. Experimental

2.1. General synthetic and analytical procedures

Synthesis of the peptide was performed using a Vega 1000 semiautomatic synthe-

sizer. The p-methylbenzhydrylamine (p- MBHA) resin (1% divinylbenzene cross-linked polystyrene) was purchased from Pep- tides International (Louisville, KY, USA) or was prepared by previously reported [14,15] methods with the amine substitution indicated. N'~-Boc amino acids were purchased from Bachem. All amino acids not purchased were prepared by the literature procedure of Tarbell et al. [16]. Purity of each amino acid was established by the ninhydrin test [17], melting point, and thin-layer chromato- graphy (TLC) on Baker-flex silica gel 1 B-F plates in three solvent systems: chloroform- methanol (95 : 5), ch loroform-methanol (1:1), and acetone-acetic acid (98:2). De- tection was with ultraviolet (UV) (254 nm) using iodine and ninhydrin. All N~-Boc amino acids were coupled with diisopro- pylcarbodiimimide (DIC)-N-hydroxybenzo- triazole (HOBT)-diisopropylethylamine (DIEA), using standard solid-phase peptide synthesis methodology [18]. Progress in the condensation reaction was monitored by the ninhydrin test [17].

Purity of the peptides was assessed by analytical reversed-phase high-performance liquid chromolography (RP-HPLC) performed on a Spectra Physics Model 8400 instrument with variable-wavelength detector. A Vydac reversed-phase C 4 or C18 column (17 nm, 25 cm x 4.55 mm I.D.) was used with a mobile phase consisting of acetonitrile in aqueous 0.1% trifluoroacetic acid (TFA). Amino acid analyses were carried out by the method of Spackman et al. [19] on a Beck- man 120 C amino acid analyzer following hydrolysis of the peptide material for 22-48 h at 100°C with either 6 M HCI or 4 M 2-methanesulfonic acid (MSA) and subse- quent neutralization with an equivalent of 3.5 M NaOH [20]. No corrections were made for the destruction of amino acids during hydrolysis. Optical rotation values were measured at the mercury green line (547 nm) by using a Rudolph Research Auto-pol II po-

VJ. Hruby et aL / Reactive Polymers 22 (1994) 231-241 233

larimeter. Molecular masses of the peptides were determined by fast atom bombardment mass spectrometry (FAB-MS) using a Cratos MS-50 triple analyzer equipped with Kratos DS-55 data system. Mass spectral determina- tions were performed either at the University of Arizona Biotechnology Center or by the Midwest Center for Mass Spectrometry at the University of Nebraska, a National Sci- ence Foundat ion Regional Instrumentation Facility. Capillary melting points were determined on a Hoover melting point apparatus and are uncorrected. TLC was performed on silica gel G plates with use of the following solvent systems: (A) 1-butanol-acetic acid- water (4 :1 :5 , upper phase only); (B) 1- butanol-acet ic acid-pyr idine-water (15:3 : 10 : 12); (C) 1 - p e n t a n o l - p y r i d i n e - w a t e r (7 : 1 : 6); (D) 1-bu tanol -pyr id ine-ace t ic acid-water (5 : 1 :4 : 5); (E) ethanol-chloroform (1:3); (F) ch loroform-methanol -ace t ic acid (9:1:0.5); (G) ethyl ace ta te-pyr id ine- acetic acid-water ( 2 : 4 : 4 . 2 : 2 2 ) ; (H) 1- butanol-acet ic acid-pyr idine-water (4:1 : 1:3). Detection was made by UV, iodine, ninhydrin, or C12-o-toluidine. Single symmetrical spots were observed. Preparative RP-HPLC was performed on a Perkin-Elmer Series 3B liquid chromatograph equipped with an LC-75 spectrometric detector and an LCI-100 laboratory computer integrator. A Vydac C 4 or ClS semipreparative column (10 nm, 25 cm x 1.0 cm I.D.) was used with a mobile phase of acetonitrile in aqueous 0.1% TFA. Detection of the peptide material was made by monitoring the UV absorbance of the peptide backbone at 214 nm and the tyrosine side-chain at 280 nm.

2.2. Synthesis of the cyclic lactam analogue of 4 a-MSH, Ac-[Nle -Asp-His-D-Phe-Arg- Trp-

~pr-NH 2 (1, Ac-[Nle 4, Asp s, o-Phe 7 Dpr '°] a-MSH(4-10)-NH 2)

The peptide was synthesized by solid-phase methods similar to those previously reported

[7,21]. The N"-Boc amino acids N"-Boc-Nle, N'~-Boc-Asp(OFm), N~-Boc-His(N=-Bom), N"-Boc-D-Phe, N"-Boc-Arg(N~-Tos), N "- Boc-Trp(Nin-For) and N"-Boc-Dpr(NS- Fmoc) [21] were used for the synthesis and the solid support used was p-MBHA resin. The substitution of the first amino acid was controlled to be between 0.30 and 0.40 m M / g resin and all remaining amino groups were acylated by acetic anhydride in N,N-di- methylformamide (DMF). N~-Boc-Dpr(N tL Fmoc) was coupled to the resin at the level of 0.35 m M / g resin using DIC and HOBT as the coupling reagent. Following removal of the N"-Boc protecting group and neutralization with 10% DIEA in dichloromethane (DCM), and using standard methods of solid-phase synthesis, the amino acids listed above were added one at a time to the growing peptide chain on the resin (0.66 g, 0.35 m M/g resin) to give the following intermedi- ate protected peptide-resin: N"-Boc-Asp - (OFm)-His(N ~-Bom)-D-Phe-Arg(N G-Tos)- Trp(N in-For)-Dpr(NtLFmoc)-p-MBHA resin. The OFm and Fmoc side-chain protecting groups were removed by use of 50% piperidine in DMF for 1 h. Following extensive washings with DMF (3 x 10 ml), DCM (3 x 10 ml), 10% DIEA in DCM (3 × 10 ml) and DCM (3 x 10 ml), the peptide was sus- pended in 5 ml of DMF and mixed with a six-fold excess of (benzotriazolyloxy)tris(di- methylamino)phosphonium hexafluorophos- phate (BOP) reagent in the presence of an eight-fold excess of DIEA for 6 h. The cyclization was repeated to give the cyclic peptide resin N"-Boc-Asp-His(N=-Bom)-D-Phe - Arg(N G-Tos)-Trp(N in-Fmoc)-Dpr-p-MBHA- resin, which gave a negative ninhydrin test [17]. The N~oBoc protecting group was removed and, following neutralization, N "- Boc-Nle was coupled to the growing peptide chain as usual. The N~-Boc protecting group was removed and the terminal amino group acylated with 10 ml of 25% acetic anhydride in DCM for 30 min. The peptide resin was

234 V.J. Hruby et al . / Reactive Polymers 22 (1994) 231-241

treated with 12 ml of anhydrous HF in the presence of anisole (10%) and 0.8% 1,2-di- thioethane (to remove the N in-For group [22]) at 0°C for 45 min. Following removal of the HF and anisole in vacuo at 10°C, the peptide resin mixture was washed with anhydrous diethyl ether and the peptide extracted into 30% aqueous acetic acid. The peptide was lyophilized to give an off-white powder. The final product was purified by preparative RP-HPLC on a C18 bonded silica column (Vydac 218TP1010, 25 cm x 1.0 cm I.D.) eluted with a linear gradient of acetonitrile (20-40%) and aqueous 0.1% TFA (v/v). The effluent was monitored at 260 nm and the major peak was collected and lyophilized to give a white powder (54% yield); [a]229 = - 3 3 ° (c = 0.045, 10% acetic acid); TLC, R F = 0.77 (A), 0.78 (B), 0.87 (D); RP-HPLC, k' = 3.45 (20-30% acetonitrile in 0.1% TFA, 15 min); FAB-MS (M + H+), 983.0 (984); amino acid analysis, Nle 1.10 (1.0), Asp 1.00 (1.0), His 1.10 (1.0), Phe 0.90 (1.0), Arg 0.94 (1.0), Trp 0.95 (1.0), Dpr 0.91 (1.0).

2.3. Synthesis o f [Lys z7 Lys~, Glu21]gluca -

gon-NH 2 (2)

The cyclic glucagon analogue was synthesized by using solid-phase peptide synthesis procedures developed in our laboratory for glucagon analogues and cyclic lactam-containing peptides with modifications. The solid support used was p-MBHA-polystyrene cross-linked with 1% divinylbenzene (0.35 mmol /g or 0.64 mmol/g) (Bachem, Tor- rence, CA, USA). The protected amino acids were coupled to the growing peptide chain using DIC and HOBT (Aldrich, Milwaukee, WI, USA), and was monitored by the ninhydrin test. Cyclizations were accomplished on the resin using BOP reagent (Peptides Inter- national, Louisville, KY, USA). After being cleaved from the resin using the low-high HF procedure [29], the peptides were purified by dialysis, gel filtration on Sephadex

G-15 using 10% acetic acid, and RP-HPLC (Spectra Physics Model 8700 instrument equipped with a Model 8400 variable-wavelength detector). Vydac 214TP1010 C a semipreparative (10 mm, 25 cm x 10 mm I.D.) columns were used in the purification. De- tection was done by monitoring the UV absorbance at 214 nm for the peptide backbone and at 280 nm for tyrosineside-chains. The preparative RP-HPLC conditions were as follows: a linear gradient elution starting with 30% acetonitrile and going to 55% acetonitrile in 0.1% TFA ( l % / m i n for 25 min) at a flow-rate of 3 ml /min, followed by the elution with 90% acetonitrile in 0.1% TFA for 5 min. The column was equilibrated for 10 min with 30% acetonitrile in 0.1% TFA.

In the low-high HF procedure, 1.5 g of the peptide resin were placed into an HF reaction vessel along with 20 ml of dimethylsulfide, 2.25 ml of p-cresol, and 0.8 ml of thiocresol. The mixture was chilled to liquid nitrogen temperature, 7.5 ml of HF were then distilled into the reaction vessel, and the mixture was stirred at 0°C for 1 h. After HF and dimethylsulfide were evaporated off in vacuo, the resin was washed with anhydrous ether, 3 x 30 ml (yield 1.2 g). This resin was then treated with high HF (15 ml of HF containing 1.5 ml of p-cresol) for 40 min at 0°C. The HF and p-cresol were distilled off at high vacuum.

The crude peptide mixture was dissolved in 10% acetic acid solution and transferred to a dialysis bag. The dialysis membrane had a 1000 or 2000 molecular weight cut-off (Spectrum, Los Angeles, CA, USA; 5 m x 50 mm sulfur-free EDTA-treated cellulose dialysis tubing in 0.05% sodium azide, record 132118). This permeable membrane allows only compounds below 1000 or 2000 molecular weight to pass into an aqueous solvent bath. By frequently changing the solvent, the mixture loses it lower-molecular-weight components removing most of the small organic and inorganic components. The dialysis was

V.J. Hruby et al. / Reactit~e Polymers 22 (1994) 231-241 235

done in a cold room at 5°C in a 10% aqueous acetic acid solution with constant stirring. The bath was changed every 6 h for 18 h. Then the solution inside the dialysis bag was transferred to a round-bottom flask and lyophilized overnight. A fluffy white solid was obtained. The lyophilized product was again dissolved in 10% acetic acid and purified with RP-HPLC. One major peak was obtained with a k' value of 1.24. This major peak, which is 18.8% of total peak area of the injection, was collected and lyophilized to yield the title peptide as its TFA salt [3-5].

The determination of the structure of the cyclic glucagon analogues was accomplished by FAB peptide mapping using endoproteinase Asp-N digestion by the procedures given below. Glucagon or glucagon analogue (0.2 mg) was dissolved in 250 ml of 1 M NHaHCO 3 buffer (pH 8.1), and 2 mg of endoproteinase Asp-N (sequencing grade) were added to this solution giving an en- zyme/subs t ra te ratio of 1:100 (w/w). The mixture was incubated at 37°C for 30 h and lyophilized. The lyophilized mixture was ana- lyzed by FAB-MS on a double-focusing (BE) mass spectrometer designed and built by AMD Intectra (Harpstedt, Germany). The data acquisition and instrument control were accomplished using two AM 68K computers (KWS Computer Systems, Ettlingen, Ger- many), and software was developed by AMD Intectra. The ionization involved the use of a 2.0-mA primary beam of Cs + ions acceler- ated to 7.0 kV. The samples were placed on the target of the inlet probe, and the target temperature was maintained at 12°C using chilled methanol -water (1: 1, v/v). The mass range 100-2500 was scanned at 60 s /decade with a resolution of 2000 (10% valley defini- tion). The digest mixtures were dissolved in 20 ml of 10% CF3COOH. Then 1 ml of this solution was mixed with 1 ml of glycerol on the target. The mass spectra obtained are the result of five accumulated scans. The molec-

ular weights of the glucagon analogues were measured by scanning the magnet from m / z 3000 to m / z 4000 at 60 s /decade at a resolution of 2000. The mass values are mono- isotopic mass except where noted. Spectra are not subtracted.

The particular synthesis of 2 was accomplished using 5.7 g of p-MHBA resin with 0.35 m m o l / g substitution. The N"-Boc strategies used was for 21 and 18, respectively. Following addition of residue 18, the side-chain groups were moved by treatment with piperidine after N'~-Boc-Glu(OFm) and N"-Boc-Lys(N'-Fmoc) had been placed in position. The cyclization reaction for Lys ~s and Glu 2~ residues took only two BOP-mediated treatments and less than 8 h to complete. After synthesis, the resin weighed 9.1 g. The low-high HF procedure was used to cleave the peptide from the resin, and the above method was used for purification. The yield of the total synthesis was 4%. Amino acid analysis for 2: Ala 0.99 (1.00), A s p / A s n 3.12 (3.00), G l u / C l n 3.92 (4.00), Gly 1.(12 (1.00), His 0.98 (1.00), Leu 2.02 (2.00), Lys 2.91 (3.00), Met 1.06 (1.00), Phe 2.00 (2.00), Ser 3.72 (4.00), Thr 3.12 (3.00), Tyr 2.08 (2.00), Val 0.83 (1.00); TLC, R F = 0.67 (G), 0.76 (D), 0.63 (H); RP-HPLC, k ' = 0.85 (C42147r1010), a ce ton i t r i l e -0 .1% T F A (25:75), k' = 1.02 (C~82187rP1010), acetoni- tri le-0.1% TFA (25:75). MS following Asp- N digestion gave the following results: 1-8 864.2 (864.4), 788.9 (788.4), 15-29 1807.0 (1807.0) B; FAB-MS (M + 1) A 3422.4 (3422.8).

2.4. Synthesis of the bicyclic oxy. tocin (OT) analogue cyclo 4,8-[[3-Mpa 1, Glu 4, Cys 6, LysS]OT (3)

S-4-Methy lbenzy l - f l -mercap toprop ion ic acid was prepared by a modification of the procedure of Hope et al. [23] and Berman et al. [24] from S-benzyl-/3-mercaptopropionic acid. From /3-mercaptopropionic acid (5 ml,

236 V.J. Hruby et al . / Reactive Polymers 22 (1994) 231-241

57.4 mmol) there was obtained 11.9 g (99%) of the crystalline title compound: m.p. 74- 76°C, literature [24] 74-76°C; nuclear mag- netic resonance (NMR) (60 MHz, CDC13) , 2.3 (3H, s), 2.6 (4H, t) 3.7 (2H, s), 7.1 (4H, s).

The synthesis of 3 was accomplished starting with 2.85 g of p-MBHA resin (0.35 m M amine per g resin). The N"-Boc amino acids were coupled to the resin with D I C - H O B T in D C M - D M F . The side-chain protecting groups for the Lys 8 and Glu 4 residues involved in the cyclization are N~-Fmoc and OFm, respectively. After each amino acid was coupled, the reaction was monitored for completion of coupling by the ninhydrin test [17] and completion of the coupling was indicated at each coupling. Following these procedures, assembly of the linear protected peptide resin was completed up to N~-Boc - Glu(OFm)-Asn-Cys(S-4-MeBzl)-Pro-Lys(e- Fmoc)-GIy-MBHA resin. At this step, the peptide resin was treated with 20% piperidine in 50% D C M - D M F for 20 min in order to remove both the Fmoc and OFm protecting groups on the two side-chains involved in the lactam formation, Lys 8 and Glu 4, respectively. The piperidyl salt was exchanged to the DIEA salt by a standard wash with 10% DIEA in DCM (3 × ) to ensure faster ring closure in the BOP-mediated cyclization step. The washings were repeated three times. Ring closure was brought about by BOP reagent (5 equiv.), HOBT (5 equiv.) and DIEA (10 equiv.) in DCM. After 2 h the reaction was 80% complete as judged by the ninhydrin test. The coupling was repeated for another 4 h to completion. The synthesis was completed by further coupling of Boc-Ile, Boc-Tyr(O-2,6-ClzZ) and /3-Mpa(S-MeBzl); for N'~-Boc deprotection of the cyclized peptide 48% TFA, 2% anisole in DCM was used. The peptide was extensively washed, and dried in vacuo (4 g). The resin was treated with anhydrous HF-p- th iocresol -m- thiocresol (10 : 1 : 1) (40 ml) for 1 h at 0°C in order to remove the peptide from the resin

and, simultaneously, to remove all protecting groups. After evaporation of the HF and scavenger in uacuo, the residue was washed with anhydrous diethyl ether (3 × 30 ml). The resin was the extracted with deaerated glacial acetic acid (50 ml), and three 30-ml portions each of 30% acetic acid, 0.2 N acetic acid and water. The aqueous extracts were combined and lyophilized to yield the crude de- protected peptide. The white powder was dissolved in 0.1% acetic acid (50 ml) which was deaerated and bubbled with nitrogen for 90 min. The pH of the solution was adjusted to 8.5 with 3 N NHaOH. Another solution (500 ml) of 0.1% acetic acid was prepared and brought to pH 8.5 with 3 N NHaOH; 2.5 equiv, of oxidizing agent K3Fe(CN) 6 were added. The 50 ml of dissolved monocyclic peptide were taken up into a 50-ml syringe and added at a rate of 2 m l / h to the 500 ml of buffered solution containing the oxidant. After the complete addition of the peptide, the solution was continuously stirred for a further 1-2 h. The pH was then adjusted to 4.8 with 30% acetic acid, and anion-exchange resin (Amberlite IRA-45; Chemical Dynam- ics Corporation) was added (30 ml settled volume) to remove excess ferro- and ferri- cyanide. The suspension was stirred for 1-2 h, and the resin was removed by filtration and washed (3 × 25 ml of 20% acetic acid). The filtrate was reduced in volume by rotary evaporation and then lyophilized to yield the crude bicyclic peptide. The crude peptide was initially purified by gel filtration on a Sephadex G-25 column (100 cm × 2.65 cm I.D.). The peptide was dissolved in 3 N acetic acid (4 ml) and then applied onto the column. The column was eluted with 3 N acetic acid at a flow-rate of 10 m l / h . The fractions were monitored for peptide material at 280 nm and the fractions containing the major peak were pooled and lyophilized to yield a fluffy white powder. The peptide was further purified by preparative RP-HPLC by using a linear gradient of 10-40% acetonitrile in

V.J. Hruby et al. /Reactiue Polymers 22 (1994) 231-241 237

aqueous 0.1% TFA (0.66%/min). The title peptide was obtained as its TFA salt: [a]~ 2 = - 6 9 ° (c = 0.2, 1 M acetic acid); the purified peptide gave single uniform spots (UV, iodine, Cl2-toluidine) on TLC, R F = 0.20 (A) , 0.57 (B), 0.58 (C); RP-HPLC, k ' = 1.44 [iso- cratic, 0.1% aqueous TFA-ace toni t r i le (78:22)]. Amino acid analysis (hydrolysis for 48 h at 110°C in 4 M MSA), Tyr 1.01 (1.00), Ile 0.93 (1.00), Glu 0.95 (1.00), Asp 1.00 (1.00), Mpa-Cys 1.08 (1.00), Pro 1.01 (1.00), Lys 0.99 (1.00), Gly 1.02 (1.00). FAB-MS (MH+), calculated 990, found 990.

2.5. Synthesis of the cholecystokinin (CCK) analogue cyclo[Gly- Trp-N-MeNle-Asp-Phe] (s)

3-Nitrooxime resin was prepared as follows. A 500-ml three-necked round-bottom flask containing 100 ml of dry 1,2-dichloro- ethane and 7.0 g of dry, thoroughly washed Biobeads SX-1 was equipped with a mechan- ical stirrer and a refluxing condenser at- tached to a drying tube. While stirring and refluxing of the mixture, 1.2 g of 3-nitroben- zyl chloride was added into the flask. Then 9.0 ml of 1 M aluminum chloride in ni- trobenzene were added over 10 min, The reaction mixture was refluxed for 10 h. The resin was filtered and thoroughly washed with 4 x 100 ml of dioxane-4 M HC1 (3 : 1, v/v), 3 x 100 ml of dioxane-water (3 : 1, v/v), 3 x 100 ml of DMF and 6 x 50 ml of methanol. Yield, 7.9 g. The weight gain of 0.9 g indicated that there is 0.76 mmol of substitution per g of resin. The formation of ketone was shown by the strong absorptions of carbonyl and nitro groups in the IR spectra: 1670 cm -1 (C=O), 1540, 1320 cm 1 ( N O 2 ) . The same set up was used in the next step. Hy- droxylamine hydrochloride (7.0 g), pyridine (10 ml) and absolute ethanol (50 ml) were added into the flask. The 7.9 g of resin obtained in the last step was added into the refluxing solution bit by bit over 30 rain to

avoid formation of a big clump of resin. The reaction mixture was stirred at reflux for 8 h. After filtration, the resin was washed with 4 x 100 ml of methanol-water (3:1, v/v), 3 x 1 0 0 ml of DMF and 4 x 5 0 ml of methanol Yield, 7.91 g. The formation of oxime was followed by the disappearance of the strong C=O absorption at 1670 cm-~ and the appearance of a strong oxime OH absorption at - 3500 cm-~ in its IR spectra. Elemental analysis of the nitrogen content of the resin (2.09%) indicated that there was 0.75 mmol of oxime in each gram of resin, which was consistent with that in the previ- ous step where 0.76 mmol /g was observed and the conversion from ketone to oxime was almost quantitative.

The above oxime resin (3.0 g) was used to couple the first amino acid, N~-Boc-Gly. N ~- Boc-Gly (0.6 g, 3 mmol) and DIC (3 mmol) were shaken with the resin in DCM for 12 h. The N~-Boc-Gly-resin (3.39 g) gave a degree of substitution of 0.73 mmol/g. N'~-Boc - Gly-resin (1.65 g) was used to continue the synthesis using a symmetric anhydride procedure. The N~-Boc protecting groups were removed using 25% TFA in DCM followed by neutralization with purified DIEA. After the peptide on the resin Trp-N-MeNle- Asp(O-Bn)-Phe-Gly-oxime resin was obtained, cyclization was performed under the catalysis of acetic acid (10 equiv.) in DCM over two days. The peptide resin was filtered off and the resin was thoroughly washed with DCM, methanol, acetic acid, methanol. All filtrates were combined and the solvents were evaporated in uacuo. The crude product was dissolved in a mixture of DMF (4.0 ml) and acetic acid (2.0 ml), and 210 mg of Pd-C (10%) and 210 mg of ammonium formate were added to the solution. The mixture was stirred and warmed to 40-50°C to initiate the reaction. The reaction stopped after 5 h as indicated by analytical HPLC. The solvents were evaporated in uacuo. The peptide was purified by RP-HPLC. The gradient used

238 F.J. Hruby et aL / Reactive Polymers 22 (1994) 231-241

for the C18 semipreparative column was 35- 55% acetonitrile in 0.1% aqueous solution over 20 min. The peak with a retention time of 15.8 min was collected. After lyophiliza- tion, the peptide was obtained as a white powder; MS (M + H), 633 (632.7); HPLC, k' = 1.81 (25-55% acetonitrile in 0.1% aqueous TFA; 25-55% over 30 min); TLC, R e --- 0.72 (A), 0.53 (E), 0.12 (F); amino acid analysis, Gly 1.1 (1.0), Asp 1.0 (1.0), Phe 1.0 (1.0), N-MeNle 0.7 (1.0). 1H NMR (250 MHz, [2H6]DMSO) /~, Gly, NH 8.89 ( J = 7.3 Hz), a ' H 4.13 ( J = 5 . 1 Hz), a " H 2.94 ( J = 13.6 Hz), Trp NH 7.64 ( J = 9 . 3 Hz), a H 4.96 (J = 6.8 Hz), /3'H 3.04 (J = 6.8 Hz), fl" 2.95 ( J = 14.5 Hz), NH in 10.91 ( J = 2.1 Hz), 2 'H 7.19 (J = 7.9 Hz), 4'H 7.69 (J = 1.0 Hz), 5'H 6.95, 6'H 7.02, 7 'H 7.28; N-MeNIe, NCH 3 2.98, a H 3.37 (J = 9.1 Hz), fl 'H 1.86 (J = 6.1 Hz), f f 'H 1.76 (m), ~'H 1.15 (m), t~"H 1.05 (m), ~ CH 2 1.21 (m), to CH 3 0.83 (m); Asp NH 8.01 ( J - 8.1 Hz), a H 4.45 (J = 9.8 Hz), f l 'H 2.78 ( J = 3.4 Hz), /3"H 2.66 ( J = 16.7 Hz); Phe NH 7.84 ( J = 6 . 8 Hz), a H 4.30 ( J = 9 . 8 Hz), /3'H 3.08 ( J = 3 . 8 Hz), f l " H 2.82 (J = 12.4 Hz).

3. Results and discussion

Syntheses of cyclic lactam analogues of a-melanotropin resulted in compounds with very high potency and in many cases pro- longed biological activity [21,25]. In these cases, a N~-Boc strategy has been used in which protecting groups on side-chain moieties (except for the e-carboxyl and amino side-chain groups to be cyclized) are protected with benzyl-based protecting groups. The side-chain moieties to be cyclized are protected with groups that can be removed orthogonally, the e-carboxyl group by an OFm group and the e-amino group by an Fmoc group. An example of the methodology used is illustrated in Fig. 1 which outlines the

Boc-Dpr(NP-Fmoc)-p-MB HA-resin

1. Boc-Trp(Nin-For)

2. [ Boc-Arg(NG-Tos) 3. / Boc-O-Phe

4. I Boc'His(Nin'Bom) 5. ~Boc-Asp(O-Fm)

Boc.Asp(O-Frn).His(Nim-Born)-~-p he-Arg(NG-Tos)-Trp(Nin-For)-Dpr(NILFmoc)-p-M B HA-resin

6. / 50% Pipeddine in DMF

7. I BOP, DIEA, OMF, 6 hr

Boc-Asl p-His(Nim-Bom)-D- Phe-Arg(NG-Tos )-Trp(Nin-For)-D/r~-MB HA-resin

. [ Boc-Nle

~, Ac20, DCM 10. HF

11. Purification

Ac.Nle-ALsp-His-D-p he-A rg-Trp-D/r-NH2 (1) (65%)

Fi~. 1. Scheme for the synthesis of Ac-[NIe 4, As"P"5-~ - Phe7DI~rl°]-a-MSH(4-10)-NH2 via cyclization on a solid support.

synthesis of N~-Ac-[Nle 4, Asp 5, o-Phe 7, D-~prI°]-a-MSH(4-10)-NH2 (1, see Experi- mental for details). The cyclization on the solid support proceeded to completion based on the ninhydrin test, and RP-HPLC purification led to the desired peptide in high yield and excellent purity. The methodology utilized works very well, even though the most problematic amino acids (His, Arg, Trp) are part of the sequence.

This success has led us to the idea that we might be able to use a similar strategy for the synthesis of cyclic lactam analogues of glucagon. The reason for optimism was further enhanced by the studies of Felix et al. [26] on growth hormone releasing factor. Previously we had shown that substitution in glucagon of A r g 17 and A r g is with Lys 17 and Lys 18 and A s p 21 with G I u 21 increased the helicity to a superpotent glucagon agonist [27]. Molecular modeling based on these findings [28] has led us to prepare cyclic glucagon analogues in an effort to further stabilize the a-helical structures further. Pre-

V.J. Hruby et al . / Reactive Polymers 22 (1994) 231-241 239

viously we had adapted an N~-Boc strategy for the synthesis of glucagon [29], and the synthetic approach discussed above was adapted for the synthesis of the cyclic glucagon analogue [Lys 17, L ~ 2 1 ] g l u c a - gon-NH 2 (2) [18]. Interestingly, though this analogue was found to have increased a- helical content relative to the glucagon it was also found to bind with decreased potency to glucagon receptors when compared to glucagon [30].

Previously, Hill et al. [31] have reported that the conversion of the weak cyclic disulfide agonist [fl-Mpa 1, Gly 4, LysS]-oxytocin (3), /3-Mpa-Tyr-Ile-Glu-Asn-Cys-Pro-Lys- GIy-NH 2) to the highly potent bicyclic antagonist analogue [/3-Mpa 1, G ! u 4, Cys 6, Lys8] - oxytocin (4) gave a highly potent antagonist. The synthetic methodology used to prepare this compound involved first the formation of the disulfide-containing compound in solution at low concentration and second the cyclization reaction to the bicyclic lactam at high dilution in solution. The overall yield was very low (less than 10%). We have found a much more effective method for synthesiz- ing this compound utilizing the procedure outlined in Fig. 2. In this methodology the cyclic lactam portion of the structure is assembled first on the solid support using a mixed N'~-Boc-N~-Fmoc strategy as outlined in Fig. 2. Then the rest of the peptide chain is assembled on the solid support by the N'~-Boc strategy. The peptide is cleaved from the solid support with concomitant removal of the side-chain protecting group using HF cleavage conditions (Fig. 2), and the disulfide ring is formed in aqueous solution under oxidative conditions. The final product is obtained in a good overall yield (about 45%), over 400% better than that obtained in the initial approach.

In many cases it is desirable to prepare N-terminal to C-terminal cyclic peptides, N- terminal to side-chain cyclic, or C-terminal to side-chain cyclic peptides. For this pur-

Boc-Gly-pMBHA-resin (0.35 mM/g resin)

1 ~ Boc_Lys(Nf_Fmoc), DIC. HOBT

2 ] Boc-Pro

3. Boc-Cys (S-4'-MeSn)

4. Boc-Asn

5. Boc-Glu(O-Fm)

BOC-Glu(O-Fm)-Asn-C ys(S-4'-MeBn)-Pro-Lys(N~ Fmoc)-Gly-p-MB HA <e sm 6 20% Piperidme/OMF

7 t DIEA/CH2CI2 8. ~ BOP, DIEA, HOBT/DMF

I I Boc-Glu-Asn-Cys(S-4'-MeBn)-Pro-Lys-Gly-p-MB HA-resin

9, Boc-Ile

10. I Boc-Tyr(O-2', 6'-CI2Bn ) I 11. / 13-Mpa /

12 { HF, 5% p-cresol, 5% thiocresol, O ° 1 h r i

I]-Mpa(SH)-Tyr-Ile-Glu-Asn-Cys (S H)-Pro-Lys-Gly-N H 2 13./K 3 Fe(CN)6, pH 8.5, " rng/mL

14.1 Purification

~-M~)a-Tyr-Ue-Gll-As~Cys-Pro-Llys-Gly-NH2 (45°/°) (4)

Fig. 2. Synthesis of a bicyclic oxytocin analogue containing a disulfide and a lactam bridge.

pose the procedure first suggested by De- Grado and Kaiser [11] and Taylor and Osa- pay [12] can be most useful. Recently as part of efforts to understand the conformational requirements for interaction of cholecystokinin (CCK) with its receptors, we have prepared a variety of cyclic CCK fragment analogues [32] including cyclo[Gly-Trp-N- MeNle-Asp-Phe] (5). For this purpose we have adapted the procedure of DeGrado and Kaiser [11] as outlined in Fig. 3 (see Experi- mental for details). An N~-Boc strategy using preformed symmetrical anhydrides was cho- sen. The N%Boc protecting group was removed after each coupling step with 25% TFA in DCM. Neutralization of the TFA salt was accomplished with the non- nucleophilic base DIEA (carefully purified by distillation from ninhydrin to remove all secondary and primary amines). Following assembly of the peptide on the resin, the N%Boc was removed, the amine terminal

240 V.J. Hruby et aL / Reactive Polymers 22 (1994) 231-241

HO \ N O 2 ~ Resin

" | AC20, DIEA

B ° c ' G l y ' O ' ~ R e s i n

I NO2 3.] Boc-Phe Anhydride 4.1 Boc-Asp(O-Bn) Anhydride 5.1 Boc-N-MeNle Anhydride 6.~ Boc-Trp Anhydride

Boc-Trp-N-MeNle-Asp(O-Bn)-Phe-Gly-O-~ Resin

7. 25% TFA/CH2CI 2 ~ ~'NO 2 8. I 1.5 eq. DIEA 9. ~ 10 eq. AcOH/CH2CI 2, 2 Days

FGly-Trp-N-MeNle-Asp(O-Bn)-ehe~

lo IN.,o.~-.. lOOjo P~,c

Fig. 3. Synthesis of cyclo[Gly-Trp-N-MeNle-Asp-Phe] on a solid support.

salt neutralized, and the cyclization initiated by acetic acid in methylene chloride. The cyclic peptide formed slowly over a two-day period to provide the cyclic product. To ob- tain the final product 5, the O-benzyl ester on the side-chain of the aspartic acid residue was removed by catalytic reduction. A major problem with this procedure was that some peptide was lost during each step of the synthesis due to the lability of the oxime peptide resin. Nonetheless, this is a poten- tially powerful method to make cyclic peptides because, even if the yield is reduced, the final product is generally obtained in a quite pure form.

The synthetic methodologies discussed above and a number of other methods developed in our and other laboratories have pro-

vided synthetic cyclic peptides and peptidomimetics in good yields. In view of the importance of cyclic peptides and peptidomimetics, continued development of methodologies for cyclization on solid supports that utilize the inherent advantages of such supports for macrocyclization should provide increasingly useful methods, not only for the synthesis of the cyclic lactams and N-terminal to C-terminal peptides reported here, but also of cyclic lactams, sulfides, disulfides, ethers, and other macrocyclic structures. We look forward to participating in the development of these methodologies.

Acknowledgements

This research was supported by grants from the U.S. Public Health Service DK 17420, DK 21085 and DK 36289.

References

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Poleveda, F.W. Holly, R.G. Strackam, R.F. Nutt, B.H. Arison, C. Homnick, W.C. Randall, M.S. Glitzer, R. Saperstein and R. Hirschmann, Nature, 292 (1981) 55-57.

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[8] H.I. Mosberg, R. Hurst, V.J. Hruby, K. Gee, H.I. Yama- mura, J.J. Galligan and T.F. Burks, Proc. Natl. Acad. Sci. U.S.A., 80 (1982) 5871-5874.

[9] J.T. Pelton, K. Gulya, V.J. Hruby, S.P. Duckies and H.I. Yamamura, Proc. Natl. Acad. Sci. U.S,4., 82 (1985) 236- 239. J. Rivier, G. Kupryszewski, J. Varga, J. Porter, C. Rivier, M. Perrin, A. Hagler, S. Struthers, A. Corrigan and W. Vale, J. Med. Chem., 31 (1988) 677-682. W.F. DeGrado and E.T. Kaiser, J. Org. Chem., 45 (1980) 1295-1300. J.W. Taylor and G. Osapay, Ace. Chem. Res., 23 (1990) 338-344.

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[14] V.J. Hruby, D. Upson and N.S. Agarwal, J. Org. Chem., 42 (1977) 3552-3557.

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[16] D.S. Tarbell, Y. Yamamoto and B.M. Pope, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 730-732.

[17] E. Kaiser, R.L. Colescott, C.D. Bossinger and P.I. Cook, Anal, Biochem., 34 (1976) 595-602.

[18] D.A. Upson and V.J. Hruby, J. Org. Chem., 41 (1976) 1353-1358.

[19] D.H. Spackman, W.H. Stein and S. Moore, Anal. Chem., 30 (1958) 1190-1206.

[20] R.J. Simpson, N.R. Neuberger and T.Y. Liu, J. Biol. Chem., 251 (1976) 1936-1940.

[21] F.A. AI-Obeidi, A.M. de L. Castrucci, M.E. Hadley and V.J. Hruby, J. Med. Chem., 32 (1989) 2555-2561.

[22] G.R. Matsunda, Int. J. Peptide Protein Res., 20 (1982) 26-34.

[23] D.B. Hope, V.V.S. Murti and V. du Vigneaud, Z Biol, Chem., 237 (1962) 1563-1566.

[24] J.M. Berman, J.T. Pelton, C.F. Hassman, S.H. Buck, P.J. Shea, P. Ertl, E.F. Heminger and R.J. Broersma, Int. J. Peptide Protein Res., 32 (1988) 47-55.

[25] M.E. Hadley, M.M. Marwan, F. AI-Obeidi, V.J. Hruby and A.M. de L. Castrucci, Pigment Cell Res.~ 2 (1989) 478-484.

[26] A.M. Felix, C.T. Wang, E.P. Heimer and A. Fournier, Int. J. Peptide Protein Res., 31 (1988) 231-238,

[27] J.E. Krstenansky, D. Trivedi, D. Johnson and V.J. Hruby, J. Am. Chem. Soc., 108 (1986) 1696-1698.

[28] Y. Lin, Thesis University of Arizona, Tucson, AZ, 1992. [29] J.L. Krstenansky, D. Trivedi and V.J. Hruby, Biochem-

is'tO', 25 (1986) 3833-3839. [30] Y. Lin, V.J. Hruby and D. Trivedi, unpublished results. [31] P.S. Hill, D.D. Smith, J. Slaninova and V.J. Hruby, .l.

Am. Chem. Soc., 112 (1990)3110-3113. [32] D. Jiao, Thesis, University of Arizona, Tucson, AZ, 1993.

strategies for cyclizations of novel peptides on solid supports

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