Neuropeptide Technology: Synthesis, Assay, Purification, and Processing

volumes.

Methods in Neurosciences

Edited by P. Michael Conn

Volume 1. Gene Probes

Volume 2. Cell Culture

Volume 3. Quantitative and Qualitative Microscopy

Volume 4. Electrophysiology and Microinjection

Volume 5. Neuropeptide Technology: Gene Expression and Neuropeptide Receptors

Volume 6. Neuropeptide Technology: Synthesis, Assay, Purification, and Processing

Volume 7. Lesions and Transplantation (in preparation)

[1]

Large-Scale Synthesis of Gonadotropin-Releasing Hormone Antagonists for Clinical Investigations

Carl Hoeger, Paula Theobald, John Porter, Charleen Miller, Dean Kirby and Jean Rivier

Publisher Summary

This chapter discusses large-scale synthesis of gonadotropin-releasing hormone antagonists for clinical investigations. Once a new peptide has been discovered and its biological activities identified, a logical extension is to design analogs for the study and understanding of its structure–activity relationships and to identify molecules that are either more potent, longer acting, or competitive antagonists. Either an agonist or an antagonist of a given peptide may be examined as a potential candidate for the treatment of diseases arising from defects in the normal pathways that the native peptide governs. Such synthetic peptides and their analogs can be synthesized and purified in large-enough quantities for their successful and safe use in a clinical setting. The chapter discusses the development of two antagonists of gonadotropin-releasing hormone (GnRH): (1) Ac-D-2-Nal-D-Cpa-D-3-Pal-Ser-Arg-D-2-amino-5-oxo-5-(4-methoxyphenyl) pentanoic acid-Leu-Arg-Pro-D-Ala-NH2 (Nal-Glu antagonist), which has already been tested extensively in humans; and (2) Ac-D-2-Nal-D-Cpa-D-3-Pal-Ser-Lys(atz)-D-Lys(atz)-Leu-ILys-Pro-D-Ala-NH2 (Azaline), a member of the most recent generation of potent, water-soluble GnRH antagonists with little or no histamine-releasing activity.

Introduction

Advances in methods for the isolation and identification of new and novel neuropeptides by both classic chemical and the newer molecular biological techniques have aided in the initiation of our understanding of the molecular control of a number of pharmacological and physiological properties (1–6). As new peptides and proteins are isolated in minute quantities (or cloned) and characterized, a need for their duplication by total synthesis becomes imperative in order to study their biological properties. The technological advances that have arisen in the synthesis and chromatographic handling of peptides have led to a situation where it is now recognized that selected (10- to 40-residue) peptides can be readily synthesized on the solid phase pioneered by Merrifield (7) and purified by reversed-phase HPLC (8, 9) to yield quasihomogeneous preparations that are adequate for biological investigations. The need for scalar extrapolation from analytical (10–100 µg) to semipreparative (0.1–100 mg) and ultimately to the preparative scale (0.1 to 50 g) synthesis and separation of purified biologically active peptide and protein hormones was originally recognized and addressed at the analytical level by Burgus and Rivier (8) and later by many others (9–12). It is the ability to carry out this extrapolation, in order to satisfy our needs for large amounts of peptides for clinical investigations, that has been one of the trademarks of this laboratory (13).

Once a new peptide has been discovered and its biological activities identified, a logical extension is to design analogs for the study and understanding of its structure-activity relationships (14) and to identify molecules that are either more potent, longer acting, or competitive antagonists. During the course of these investigations, it is generally found that the desired properties described above may be obtained by substituting selected amino acids within the native sequence with unusual and/or unnatural amino acids. Either an agonist or an antagonist of a given peptide may be examined as a potential candidate for the treatment (in a clinical setting) of diseases arising from defects in the normal pathways that the native peptide governs. As one might imagine, the amounts of a given peptide analog needed to go from the initial investigations carried out in the laboratory to the quantities required for toxicological and human evaluation increase dramatically. Work in this laboratory has demonstrated that such synthetic peptides and their analogs [in our case, the releasing factors gonadotropin-releasing hormone (GnRH) (2), somatostatin (SS) (3), corticotropin-releasing factor (CRF) (4, 5), and growth hormone-releasing factor (GRF) (6)] can be synthesized and purified in large-enough quantities for their successful and safe use in a clinical setting. This chapter will deal with two antagonists of gonadotropin-releasing hormone (GnRH) developed in this laboratory∗: Ac-D-2-Nal-D-Cpa-D-3-Pal-Ser-Arg-D-2-amino-5-oxo-5-(4-methoxyphenyl)pentanoic acid-Leu-Arg-Pro-D-Ala-NH2 (Nal–Glu antagonist), which has already been tested extensively in humans (15, 16) and Ac-D-2-Nal-D-Cpa-D-3-Pal-Ser-Lys(atz)-D-Lys(atz)-Leu-ILys-Pro-D-Ala-NH2 (Azaline) (Fig. 1), a member of the most recent generation of potent, water-soluble GnRH antagonists with little or no histamine-releasing activity (17).

Fig. 1 Primary structure of (a) the Nal–Glu antagonist [Ac-D-2-Nal-D-Cpa-D-3-Pal-Ser-Arg-D-2-amino-5-oxo-5-(4-methoxyphenyl)pentanoic acid-Leu-Arg-Pro-D-Ala-NH2]; and (b) Azaline [Ac-D-2-Nal-D-Cpa-D-3-Pal-Ser-Lys(atz)-D-Lys(atz)-Leu-ILys-Pro-D-Ala-NH2]. The chirality of the individual residues is represented by an appropriate stereochemical label on the Ca carbons.

Nal-Glu Antagonist

A major thrust of this laboratory has been directed at the design and subsequent synthesis of analogs of GnRH (14). These peptides can be used for the treatment of a number of disease states, as well as for contraceptive purposes (18). Early work demonstrated that potent antagonists to GnRH could indeed be prepared; however, it was discovered that while these analogs were potent in an antiovulatory assay (AOA), they also elicited a strong anaphylactic response (19). Thus, the direction of GnRH antagonist design turned toward finding new analogs with high potency in the AOA but possessing low potency with regard to histamine release. A number of GnRH antagonists were developed in this laboratory out of research directed toward this goal; one analog in particular, the Nal–Glu antagonist (20) (Ac-D-2-Nal-D-Cpa-D-3-Pal-Ser-Arg-D-2-amino-5-oxo-5-(4-methoxyphenyl)pentanoic acid-Leu-Arg-Pro-D-Ala-NH2; Fig. la), is of current clinical interest (15, 16).

Synthesis

Nal–Glu contains six substitutions of the native GnRH structure (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), five of which are D-amino acids. The unusual amino acid present in the 6-position, D-2-amino-5-oxo-5-(4-methoxyphenyl)pentanoic acid, is prepared via an HF-mediated Friedel–Crafts acylation/modification of a D-glutamic acid with anisole on the fully assembled and deprotected precursor (Ac-D-2-Nal¹,D-Cpa²,D-3-Pal³, Arg⁵,D-Glu⁶,D-Ala¹⁰)GnRH (20-22). With the exception of this modified glutamic acid, the remaining amino acids are either commercially available or readily prepared in sufficient quantities for the synthesis of this peptide (20). The D-keto amino acid required in position 6 presents a unique challenge, since the free amino acid is known to undergo cyclization to form a dehydroproline derivative (22). This reaction is also responsible for the inability of the modified Glu to be detected upon amino acid analysis of the hydrolyzed peptide; therefore, full structural characterization would have to be highly dependant on other analytical and spectroscopic techniques. We chose to synthesize the Nal–Glu antagonist via the HF-mediated Friedel–Crafts acylation reaction, as this reaction is generally a reliable method for its preparation on a small scale (1–5 g). The only question that arises is one of reproducibility: the amount of this peptide required is considerable by academic standards (>50 g) and its end usage (toxicological and human clinical studies) requires that it be prepared to exacting standards. The viability of an approach incorporating the rather harsh conditions needed for the Friedel–Crafts reaction (vide infra) had yet to be demonstrated for the preparative scale synthesis of the Nal–Glu antagonist.

The first step in the extrapolation needed to make large quantities (>30 g) of the Nal–Glu antagonist required an assessment of the procedural and strategical aspects of its synthesis. Our general procedure for the synthesis of the Nal–Glu antagonist has been reported elsewhere (13, 20) as mentioned above. The critical aspect of this synthesis is based on a side reaction of glutamic acid first elucidated in 1975 (21, 22). Strategically (based on earlier yields), approximately 200 gof (Ac-D-2-Nal¹,D-Cpa²,D-3-Pal³, Arg⁵,D-Glu⁶,D-Ala¹⁰)GnRH (the precursor to the Nal–Glu antagonist) had to be obtained in order to gain 30 g of highly purified Nal–Glu antagonist. Consequently, at least 500 g of [Ac-D-2-Nal¹,D-Cpa²,D-3-Pal³, Ser⁴(Bzl), Arg⁵(Tos),D-Glu⁶-(OBzl), Arg⁸(Tos),D-Ala¹⁰]GnRH-resin would be needed. Therefore, because doubling of the initial resin weight in the synthesis of Nal–Glu antagonist was consistently obtained with a 0.92 (mEq NH2/g) substituted methyl benzhydrylamine resin (23), we started the synthesis of this large batch with 250 g of that resin. The amounts of amino acids needed for this synthesis were also derived from pilot experiments and are given in Table I.

Table I

Amino Acids Used in Synthesis of Nal–Glu Antagonist

aWeight of amino acid used is given in parentheses and corresponds to 275 mmol of protected amino acid.

bBoc-D-2-Nal, Boc-D-Cpa, and Boc-D-3-Pal were provided by Dr. P. Narasimha Rao (The Southwest Foundation for Biomedical Research, San Antonio, TX). All other amino acids were obtained from Bachem, Inc. (Torrance, CA).

cPal was recoupled; this amount of amino acid, corresponding to 113 mmol of Pal, is coupled using the BOP reagent of B. Castro, J. Dormoy, G. Evin, and C. Selve and N,N-diisopropylethylamine; see Tetrahedron Lett. 1219 (1975).

With the procedural and strategical approaches defined, the synthesis of [Ac-D-2-Nal¹,D-Cpa²,D-3-Pal³, Ser⁴(Bzl), Arg⁵(Tos),D-Glu⁶(OBzl), Arg⁸(Tos), D-Ala¹⁰]GnRH–resin could be carried out. This peptide–resin was synthesized utilizing standard solid-phase peptide synthesis (SPPS) techniques (23, 24) in a 3-liter reaction vessel using the usual excess of raw materials with only minor modifications to the standard techniques used, for example, in the large-scale synthesis of somatostatin analogs (25). Two notable adjustments were as follows: (1) the use of mechanical (overhead) stirring rather than the usual rocking method of mixing, and (2) the use of neat diisopropylcarbodiimide (DIIC) as a coupling reagent. The first adjustment was necessitated by the size of the vessel and the amount of resin involved while the second arose from a technical concern. Dicyclohexylcarbodiimide (DCC) is the most common coupling reagent used in SPPS; it suffers, however, from the fact that the by-product formed, dicyclohexylurea (DCU), is a highly dichlorome thane-insoluble material that tends, over the course of a synthesis, to plug the filtering frit used in synthesis vessels. The by-product of diisopropylcarbodiimide, diisopropylurea, on the other hand, does not have the same insolubility problem and thus filtering is much easier with fewer chances to block the frit. Employing these modifications, the synthesis of [AC-D-2-Nal¹,D-Cpa²,D-3-Pal³, Ser⁴(Bzl), Arg⁵(Tos),D-Glu⁶(OBzl), Arg⁸(Tos),D-Ala¹⁰]-GnRH–resin was accomplished in a rather straightforward manner using the tert-butyloxycarbonyl (Boc) group to protect the α -amino function of the individual amino acids and 60% trifluoroacetic acid (TFA) in CH2Cl2 in the presence of 1% ethanedithiol to effect its removal after each coupling cycle from the growing peptide–resin. Upon completion of the synthesis, the peptide–resin was washed with methanol and dried under vacuum; for the case at hand, 604 g of dry, peptide–resin was obtained.

Because of the large amount of peptide–resin obtained, the concurrent deprotection and cleavage of the resin-bound peptide by HF/anisole was carried out in a batchwise fashion. Approximately 200 ml of anhydrous HF is needed to cleave 50 g of peptide–resin, an optimum value of both resin and HF, as previously determined. The individual cleavage/deprotection steps are carried out at 0°C (bath temperature) for 90 to 120 min; after removal of the HF under vacuum, the peptide is treated with anhydrous ethyl ether (in which residual HF, anisole, and anisole derivatives are soluble), filtered, taken up in 1: 1 (v/v) acetonitrile–10% aqueous acetic acid, and lyophilized. The total weight of this crude Nal–Glu precursor obtained after all deprotection/cleavage steps were done is 227 g. The analytical HPLC–UV trace of this product run in 0.1% TFA/CH3CN is presented in Fig. 2; very few impurities are seen in this crude peptide preparation. The crude [AC-D-2-Nal¹,D-Cpa²,D-3-Pal³, Arg⁵,D-Glu⁶,D-Ala¹⁰]GnRH is pure enough, as it is, to carry on to the Friedel–Crafts acylation step, as the limited amount of peptidic and nonpeptidic impurities are not found to interfere to any significant extent with the subsequent conversion to and purification of the crude Nal–Glu antagonist. We have strong evidence that treatment of the peptide-resin at room temperature in an attempt to carry out the deprotection, cleavage, and acylation steps simultaneously gives significantly poorer yields than the two-step strategy presented here.

Fig. 2 Load: Crude, lyophilized (Ac-D-Nal,,D-Cpa²,D-Pal³, Arg⁵,D-Glu⁶,D-Ala¹⁰)-GnRH from HF cleavage (1.5 µl, ca. 5 µg). Column: Vydac 5 µm, C18, 25 × 0.46 cm i.d. Solvent: 0.1% TFA in water/acetonitrile. Gradient: 24–39% CH3CN over 25 min. Flow rate: 2.0 ml/min, 2000 psi back pressure. AUFS, Absorbance units full scale.

Therefore, the conversion of the crude lyophilized Nal–Glu precursor to the Nal–Glu antagonist is accomplished in a batchwise fashion as follows: approximately 20 g of [AC-D-2-Nall,D-Cpa²,D-3-Pal³, Arg⁵,D-Glu⁶,D-Ala¹⁰]-GnRH is placed into a graduated Kel-F HF cleavage vessel containing 20 ml of anisole and a magnetic stirring bar. After cooling the vessel for 5 min in a liquid nitrogen bath, it is evacuated and subsequently charged with between 160 and 180 ml of anhydrous HF. The reaction is then allowed to stir in a hood at ambient temperature (22°C) overnight (16–18 hr). This procedure, while potentially dangerous, is a convenient and facile method for the preparation of the Nal-Glu antagonist. After HF treatment of all of the crude [AC-D-2-Nal¹,D-Cpa²,D-3-Pal³, Arg⁵,D-Glu⁶,D-Ala,0]GnRH analog is accomplished, the weight of crude Nal–Glu antagonist obtained is 210 g. The absorbance profile obtained for this crude material is presented in Fig. 3a.

Fig. 3 (a) Load: Crude, lyophilized Nal–Glu antagonist (10 µl 10 µ g). Column: Vydac 5 µ m, C18, 25 × 0.46 cm i.d. Solvent: 0.1% TFA in water/acetonitrile. Gradient: 24–42% CH3CN over 30 min. Flow rate: 2.0 ml/min, 2000 psi back pressure. Starred peak is the desired compound, (b) Load: Crude, lyophilized Nal–Glu antagonist (10 µl, 10 µ g) after stirring at pH 6.5 for 30 min. Column: Vydac 5 µ m, C18, 25 × 0.46 cm i.d. Solvent: 0.1% TFA in water/acetonitrile. Gradient: 24–42% CH3CN over 30 min. Flow rate: 2.0 ml/min, 2000 psi back pressure. Starred peak is the desired compound.

Purification

The crude material obtained directly after the acylation reaction contains an approximate 1 : 1 mixture of the desired Nal–Glu antagonist (starred peak) and a closely associated hydrophilic impurity (Fig. 3a), in addition to a number of other hydrophobic/hydrophilic impurities. An examination of the mechanism and reaction conditions employed in the synthesis of the Nal–Glu antagonist brings to light the genesis of these impurities. The presumed intermediate in the conversion of the glutamic acid to the ketoamino acid in position 6 is the acylium ion; this reactive species can add not only to anisole but to other nucleophilic species in the reaction medium as well, the most prevalent being the amide nitrogens in the peptide backbone (21, 22). This can be minimized by using an optimal concentration of anisole in anhydrous HF [10% anisole in HF (v/v)] to peptide–substrate; in practice 20 g of peptide in 200 ml of anhydrous HF/10% anisole is employed. The most significant impurity, however, can be explained from the observation that peptides containing serine, when treated under acidic conditions at elevated temperatures for prolonged periods of time, can undergo an N- to O-acyl shift (26). This side reaction is not a serious problem, as adjustment of the pH of a solution of the crude peptide to 6.0–7.5 will result in a rapid reversal of this reaction (O- to N-acyl shift). In this case, the closely associated hydrophilic impurity arises from just such an N- to O-acyl shift between the D-3-Pal³ amide nitrogen and the Ser⁴ side-chain hydroxyl; stirring the crude Nal–Glu antagonist at pH > 6 for 30 min reverses this shift and simplifies the analytical HPLC–UV trace considerably, as shown in Fig. 3b (compare with Fig. 3a; the starred peak is the Nal–Glu antagonist). Thus an understanding of the chemistry aids in the simplification of the purification of the desired peptide from the crude material and in an increase in yield.

Purification of the Nal–Glu antagonist from the crude material is accomplished through HPLC procedures as described elsewhere (9, 27, 28). A typical HPLC profile of the preparative scale purification is shown in Fig. 4. Analytical isocratic conditions that will yield maximum information on the composition of the mixture eluting near the desired product are first determined; in general this is obtained when the desired product elutes with a solute capacity factor (k ’) between 4 and 8; in the case at hand, the ideal conditions employ a flow rate of 2 ml/min with an isocratic solvent composition of 36% CH3CN in H20 + 0.1% TFA (v/v). The analytical HPLC screening of various fractions obtained from the preparative HPLC run (depicted in Fig. 4) is presented in Fig. 5. A solvent system composed of 0.1% TFA/CH3CN is particularly convenient since the UV transparency of this solvent system at 210 nm allows for high sensitivity, in addition to providing extended column life span and reproducibly good separations, extremely important points when faced with purifications on the scale reported here. Determination of the isocratic analytical conditions allows successive and rapid assessment of the identity and purity of the fractions obtained from the individual preparative HPLC purification runs. Since we have found the triethylammonium phosphate (TEAP)/CH3CN solvent system to generally give higher resolution and different selectivity than the corresponding TFA systems, preparative scale purification is run first employing TEAP at pH 2.25 (TEAP 2.25) and acetonitrile, applying a gradient selected on the basis of the analytical chromatogram obtained for the crude material (see Fig. 3). Due to the strong elutropic characteristics of the TEAP buffer, preparative gradient conditions are generally started at about 10% lower acetonitrile concentration than the isocratic analytical conditions in TFA with a slope of 1% CH3CN increase/300 ml of solvent that is eluted. The fractions obtained from this purification step are screened and pooled based on the composition as determined from analytical HPLC; in this fashion three pools are obtained: Good (total impurities <1%), philic, and phobic (fractions containing, respectively, hydrophilic or hydrophobic impurities of 1–30%). From the preparative run depicted in Fig. 4, the relative fates of the fractions obtained are as follows: 1–5: waste; 6: philic; 9–11: phobic; and 7–8: good. Phobic and philic pools are then concentrated and separately repurified to obtain more purified material. This repurification, in the case of the Nal–Glu antagonist, is accomplished using either a TEAP 2.25/CH3CN solvent system and a different gradient or 0.5% HOAc/CH3CN. In general, a second purification in the same solvent system as the initial purification step is less desirable than switching to different counterions and pH values. We have accordingly developed and described elsewhere strategies employing other solvent systems (28). In the case of the Nal–Glu antagonist we have found that the use of either TEAP 2.25/CH3CN or 0.5% HOAc/CH3CN for this second step is quite satisfactory as no substantial gains are made in repurification of the philic and phobic pools in the other solvent systems that were tried (TEAP, pH 6.5 and 7.0, and 0.1% TFA). Although a high degree of purity can be achieved with the TEAP 2.25 system, a desalting step utilizing 0.5% HOAc/CH3CN is required to free the peptide from any TEAP salt. Purification in two systems (TEAP followed by HOAc) and analysis in yet another (TFA) was found in general to minimize the probability of missing any impurities and maximize the probability of obtaining a pure final product while offering good recovery.

Fig. 4 Load: Purify crude, lyophilized Nal–Glu antagonist after stirring at pH 6.5 for 30 min (3.0 g in 150 ml). Cartridge: 30 × 5 cm i.d., packed with Vydac 15–20 µ m, C18 material. Solvent: TEAP 2.25/acetonitrile. Gradient: 24–28% CH3CN over 20 min. Flow rate: 90 ml/min, 500 psi column back pressure. Fractions taken are indicated by slash marks on the trace, as well as boxes under the absorbing peak.

Fig. 5 Analytical screen of fractions 4–9 + 11 obtained from TEAP 2.25 purification shown in Fig. 4 (ca. 10 µl, 10 µ g). Column: Vydac 5 µ m, C18, 25 × 0.46 cm i.d. Solvent: 0.1% TFA in water/acetonitrile. Isocratic at 36% CH3CN. Flow rate: 2.0 ml/min, 2000 psi back pressure.

The conversion of the peptide from its TEAP salt to the corresponding acetate form is accomplished through HPLC procedures utilizing a 0.5% HOAc/CH3CN solvent system. This method of preparing acetate salts is based in part on an earlier procedure (28). Briefly, this material is desalted and converted to the acetate salt form in the following manner: A pool of acceptable fractions containing approximately 3 to 6 g of peptide (as determined by analytical HPLC) is diluted with an equal volume of 0.07 M NH4OAc (pH 4.5). This solution is then applied to the same Prep HPLC cartridge used in the purification above. After loading the peptide, the cartridge is washed with 1.5 liter of 6% CH3CN in H2O containing 0.5% HO Ac, after which the peptide is eluted from the cartridge by application of a gradient running from 6 to 54% CH3CN in H2O containing 0.5% HOAc over 15 min at a flow rate of 95 ml/min. This batchwise desalting procedure is repeated until all of the peptide has been converted to the corresponding acetate. We find that this desalting step for obtaining acetate counterions is both general and reliable for a wide range of peptides, and is an alternative to previously described peptide anion-exchange methodologies (29, 30). The fractions of the desired product that are deemed acceptable by analytical HPLC screening (data not shown) of the fractions obtained from these runs are collected and pooled for a final lyophilization carried out to ensure homogeneity of the final product. The purified peptide is dissolved in 2 liters of deionized, distilled water and stirred until a clear solution is obtained (15 min). The solution is then filtered into clean, acid-washed lyophilizer bottles so that the resulting solution prior to lyophilization contains no visible particulate matter. After lyophilization at ambient temperature (20–22°C), 36.2 g of the Nal–Glu antagonist is obtained as a fluffy white powder.

Azaline

In the continuing search for new and potent GnRH antagonists with an even more desirable histamine release profile than the Nal–Glu antagonist, investigations were geared at modifying the antagonist structural features in such a way as to accomplish this goal. One of the promising new GnRH antagonists to arise from these investigations is [AC-D-2-Nal¹,D-Cpa²,D-3-Pal³, Lys⁵(atz),D-Lys⁶(atz), ILys⁸,D-Ala¹⁰]GnRH (Azaline; Fig. lb) (31). In direct contrast to other GnRH antagonists [such as the Nal–Glu antagonist and the more recent Antide (32)], Azaline is freely soluble in water at pH 7.0, a highly desirable characteristic from a formulation and purification standpoint. Initial studies with this compound demonstrate that it has a desirable therapeutic index; therefore, in order to conduct more extensive tests a moderate amount (2–4 g) of this peptide is required.

Synthesis

Azaline, as was previously the case with the Nal–Glu antagonist (see above), contains a number of unusual and unnatural amino acids, not all of which are currently available from commercial sources or otherwise. The particular amino acids corresponding to the aminotriazole-functionalized lysines required in positions 5 and 6 are not available and must be prepared as a postsynthetic modification of a lysine. Therefore, the first step in the preparation| of Azaline is the synthesis of [AC-D-2-Nal¹,D-Cpa²,D-3-Pal³, Ser⁴(Bzl), Lys⁵(Fmoc), D-Lys⁶(Fmoc), ILys⁸(Z), D-Ala¹⁰] GnRH–resin accomplished through standard SPPS techniques.

Once the whole peptide is assembled, the Nε -Fmoc protecting group is removed by treatment of the peptide–resin with 20% piperidine in dimethylformamide (DMF) (5 and 20 min); this treatment selectively and quantitatively unmasks the Nε -amino functions of the lysines at positions 5 and 6, thus allowing their subsequent conversion to the aminotriazole moiety. The preparation of the aminotriazole moiety is based on the

Scheme 1

reaction between a primary amine, hydrazine, and the unique reagent diphenyl cyanocarbonimidate (PCI; see Scheme 1) (31, 33, 34). PCI can be reacted with a free amino function, such as the ε -amino of a lysine, to give the isourea derivative (I); this can then be reacted in a second step with a different amine to give the cyanoguanidine (II). The cyanoguanidino moiety is much less basic than the parent guanidino function; this has been put to use in the development of the potent H2 antagonist cimetidine (35, 36). The cyanoguanidine can be further functionalized by reaction of a third nucleophile at the — C ≡ N moiety; if this nucleophile is already present in the cyanoguanidine (II) one will obtain a heterocyclic structure (III) (33). In the case of Azaline, treatment of the partially protected peptide resin with PCI in DMF followed by anhydrous hydrazine gives the aminotriazole-functionalized lysines in positions 5 and 6. Cleavage from the resin support and concomitant removal of the remaining protecting groups (anhydrous HF/10% anisole/0°C for 1.5 hr) followed by extraction and lyophilization gives 8.4 g of crude Azaline. The analytical HPLC for the crude peptide obtained after the above-described series of steps is shown in Fig. 6. It should be noted that even though a number of nonclassical SPPS manipulations are carried out on the resin-bound peptide after its assembly and before its cleavage, an extremely clean crude material is obtained. This is a good example of a general observation noted in this laboratory: When extrapolating from experimental to preparative scale, the process (as well as the final results) should not be different from one scale to the other.

Fig. 6 Load: Crude, lyophilized Azaline from HF cleavage (5 µl, ca. 5 µ g). Column: Vydac 5 µ m, C18, 25 × 0.46 cm i.d. Solvent: 0.1% TFA in water/acetonitrile. Gradient: 18–42% CH3CN over 40 min. Flow rate: 2.0 ml/min, 2000 psi back