Semisynthetic Derivatives of Glucagon THE CONTRIBUTION OF HISTIDINE-1 TO HORMONE CONFORMATION AND ACTIVITY*

Semisynthetic iV-acetimidoglucagon was prepared from the [des-His'lanalogue by coupling the N-hydrox-ysuccinimide ester of N"-tBoc-~""'*-DNP-L-histi- dine to the peptide in dimethylformamide in the presence of 1-hydroxybenzotriazole. The deprotected, pu- rified product was chemically identical to W-acetimi-doglucagon and equipotent to and native glucagon in its ability to activate adenylate cyclase and displace ['asI]iodoglucagon from rat liver plasma membranes. Semisynthetic [Phe'l-, [Ala']-, and [des-His']glucagons prepared similarly achieved 85, 55, and 35% of the maximal activity and 22,2, and 6% of the binding potency of iV-acetimidoglucagon. The biological assays indicate that the amino group is involved to a greater extent in transduction than in binding, but the aromatic nature and hydrogen bond-ing capability of the imidazole ring of histidine-1 are important for both binding and transduction.

The amino-terminal region of glucagon mediates the action of the hormone by transducing the receptor-binding signal into the activation of adenylate cyclase (Pohl et Rodbell et al., 1971) as evidenced by the observation that [des-His']glucagon is a partial agonist (Lin et al., 1975;Bregman and Hruby, 1979). The mechanism by which this transduction is accomplished remains enigmatic. The role of histidine-1 has been evaluated by derivatization of the amino terminus (Desbuquois, 1975;Bregman et al., 1980;Epand et ai., 1981;Liepniecks and Epand, 1982;Flanders et al., 1982), but the modifications introduce artificial moieties into the molecule making it difficult to ascertain accurately the functions of separate features of the amino terminus for binding and transduction. For this purpose, semisynthesis is an appropriate approach since only a single coupling is required OH 44106. reducing the chance of racemization and deletion of amino acids that might otherwise occur during total synthesis.
The strategy adopted for this semisynthesis bears resemblance to studies of insulin (Weinert, 1969;Saunders and Offord, 1977;Cosmatos et al., 1978), myoglobin (Garner and Gurd, 1975;DiMarchi et al., 1979DiMarchi et al., , 1980, ferredoxin (Lode et al., 1974(Lode et al., ,1976, and phospholipase Az (Slotboom and de Haas, 1975;Slotboom et aC, 1977Slotboom et aC, , 1978 which also involve replacement of readily accessible amino-terminal residues. In this study, the amino-terminal histidine was removed from glucagon and reincorporated through the coupling of the active ester of Nu-tBoc-N'"-DNP-histidine' to [des-His']glucagon in which the e-amino group of the single lysine is blocked with an acetimidyl group. The chemical and biological characterization reported here and elsewhere (Flanders et al., 1982) show W-acetimidoglucagon regenerated by semisynthesis to be identical to that prepared by acetimidation of native glucagon as well as to native glucagon itself. Histidine-1 was replaced with alanine lacking the imidazole ring system and with phenylalanine which possesses a hydrophobic aromatic ring. Secondary structure differences between the semisynthetic hormones are revealed by circular dichroic spectra. Functional studies show that the amino group and the imidazole ring system have separate roles, but each is involved in both binding and transduction.

Materials
Crystalline porcine glucagon  was provided through the courtesy of Eli Lilly and Co. Trifluoroacetic acid (Sequanal grade), N,N-dicyclohexylcarbodiimide, and l-hydroxybenzotriazole monohydrate were purchased from Pierce Chemical Co. Dithiothreitol, N-ethylmorpholine, Trizma base, leucine aminopeptidase (microsomal), L-amino acid oxidase (Type V), 2-hydroxy-5nitrobenzyl bromide, and the Nu-tBoc, N-hydroxysuccinimide esters of alanine and phenylalanine were obtained from Sigma Chemical Co. Nu-tBoc-N"-DNP-L-histidine was from Fluka Chemical Corp. and N-hydroxysuccinimide obtained from Aldrich was recrystallized from ethyl acetate. The purities of the amino acid derivatives were confirmed by TLC analysis in ch1oroform:methanol (9:1), chloroform:acetic acid (19:1), and ch1oroform:methanol:acetic acid (172:l) using plates with an indicator fluorescent at 254 nm from Sybron and Brinkmann. The sources of other materials were as published (Flanders et al., 1982).

7031
of 50 mM ammonium acetate in 6 M urea, pH 5.2. A flocculent white precipitate was observed in the fractions corresponding to the native glucagon peak and direct application of the suspension to a gel peptide as also has been reported by Carrey and Epand (1982). The filtration column to remove the urea resulted in poor recovery of fractions corresponding to the native glucagon peak were pooled and ammonium hydroxide was added dropwise until the precipitate was completely solubilized. It was then desalted on a Sephadex G-25F column (2.6 X 100 cm) eluted with 5% acetic acid. The peptide fraction was lyophilized and stored at -20 "C.
Preparation and Purification of N-Acetimidoglucagon-The acetimidation of native glucagon and the anion-exchange purification step were performed as previously described (Flanders et al., 1982). Since amino acid analysis of the material recovered from the anionexchange column revealed low levels of histidine, the sample was further subjected to cation-exchange chromatography following the procedure described here for the purification of native glucagon to remove an impurity in which the a-amino group is blocked. The peptide-containing fractions from this column contained no precipitate making the addition of ammonium hydroxide before desalting unnecessary.

Preparation of /des-Hisl]N"Acetimidoglucagon-[des-Hisl]N"
Acetimidoglucagon was prepared via an Edman degradation of N" acetimidoglucagon performed according to Flanders et al. (1982). Cation-exchange purification was omitted unless amino acid analysis or isoelectric focusing indicated contamination with W-acetimidoglucagon. However, the [des-Hisl]N"acetimidoglucagon used in the biological assays was purified by cation-exchange chromatography to remove any small amount of deamidated material that may have been generated.

Preparation of N"-tBoc-N'"-DNP-L-histidine-ONSu-W-tBoc-
N'"-DNP-histidine (50 mg; 0.119 mmol) and N-hydroxysuccinimide (15 mg; 0.130 mmol) were dissolved in 0.7 ml of cold dimethoxyethane:methylene chloride (1:l) and N,N-dicyclohexylcarbodiimide (27 mg; 0.131 mmol) in 0.35 ml of methylene chloride was added (Nakajima and Okawa, 1973). The reaction was stirred in the dark at 4 "C for 4 h and then was terminated by filtration. The yellow filtrate was evaporated under a nitrogen stream for approximately 1 h until an oily residue formed since attempts to crystallize the active ester were unsuccessful. Upon TLC analysis of this material, the N"-tBoc-N'"-DNP-His-ONSu was found to migrate with an RF of 0.70 (chloroform:methanol, 9:l). The residue was dissolved in 2 ml of dimethylformamide for immediate use in a coupling reaction. Based on 90% formation of active ester as judged by visual inspection of the TLC plate under ultraviolet light, the concentration of N"-tBoc-N'"-DNP-His-ONSu in the sample was 53 pmol/ml. Preparation of Semisynthetic N'-Acetimidoglucagon-The coupling of Nu-tBoc-N'"-DNP-L-histidine-ONSu to [des-His']N"acetimidoglucagon was carried out under conditions similar to those employed by Saunders and Offord (1977) in coupling various amino acids to insulin. [des-Hisl]N"acetimidoglucagon (4.6 mg; 1.36 pmol) was dissolved in approximately 1 ml of trifluoroacetic acid in a nitrogen atmosphere. The sample was vortexed immediately and the trifluoroacetic acid was removed rapidly under a stream of nitrogen. To the oily residue was added 840 pl of dimethylformamide containing a 10fold molar excess of 1-hydroxybenzotriazole (2.09 mg; 13.6 pmol) over peptide. Enough N-ethylmorpholine (approximately 10 pl) was added to adjust the apparent pH determined as described by Rees and Offord (1976) to 8-8.5. A 10-fold molar excess of Nu-tBoc-N'"-DNP-His-ONSu (7.3 mg; 13.6 pmol) was added to the peptide solution in the appropriate volume (250 pl) of active ester solution prepared as described above. The reaction was stirred in the dark for 12 h while the apparent pH was maintained at 8-8.5 by the addition of 1-2 pl of N-ethylmorpholine. After the reaction had proceeded for 12 h, the sample was treated at 37 "C for 30 min with a 1000-fold molar excess of 2-mercaptoethanol (0.96 ml; 13.6 mmol) over the amount of active ester to remove the DNP group (Shaltiel and Fridkin, 1970;Goren and Fridkin, 1978). These conditions led to essentially complete deprotection of Nu-tBoc-N'"-DNP-histidine as monitored by the appearance of S-DNP-2mercaptoethanol, the thiolytic by-product (Amx = 341 nm; t341 = 12,500 M" cm") (Shaltiel and Fridkin, 1970). Lower temperatures as also suggested by Shaltiel and Fridkin (1970) were less efficient. Since N'"-DNP-His elutes at the position of Trp from the PA-35 column, procedures were employed to destroy Trp in order to quantitate N'"-DNP-His. The peptide was desalted on a Sephadex G-25F column (1.6 x 100 cm) eluted with 5% acetic acid and lyophilized. After the peptide (60 nmol) was hydrolyzed in 6 N HCl and the residue dissolved in citrate buffer, 50 pl of a 100 mM solution of 2-hydroxy-5-nitrobenzyl bromide in acetone was added to destroy Trp (Henkart, 1971). Ten minutes at room temperature destroyed all Trp remaining in a native glucagon hydrolysate without affecting the amount of N'"-DNP-His added to control samples.
The tBoc group then was removed from the peptide by the addition of trifluoroacetic acid (1.5 g) in the presence of dithiothreitol (12 mg) and anisole (8 p1) (Bodansky et al., 1976;DiMarchi et al., 1980), which reduce oxidation as well as the alkylation of susceptible amino acids by the tertiary butyl cation formed during deprotection (Lundt et al., 1978). The sample was incubated in a nitrogen atmosphere at room temperature in the dark for 30 min, evaporated under a nitrogen stream and further dried for 30 min in vacuo. The resulting residue was dissolved in 2 ml of first buffer and purified twice by cation exchange using the conditions given above for the purification of native glucagon.
Preparation and Purification of /Ala']and [Phe']N"Acetimidoglucagons-The conditions used to couple tBoc-Ala-ONSu and tBoc-Phe-ONSu to [des-His']N"acetimidoglucagon were the same as those used in the histidine coupling above, except that, because of the lack of steric hindrance, the reaction time required for the coupling was only 3 h. The reaction was terminated by gel filtration and following lyophilization, purification of the crude Nu-tBoc semisynthetic derivative was carried out by cation-exchange chromatography on a CMcellulose column under conditions similar to those used for the purification of native glucagon except that the buffer pH values were 4.5. The major peptide fraction was desalted, the tBoc group was removed, and the crude mixture was purified by cation-exchange chromatography under the conditions given above. An additional cation-exchange purification of [Phel]N"acetimidoglucagon was required to obtain a homogeneous derivative.
Determination of Extent of Racemization-L-Amino acid oxidase digests were performed using modifications of procedures of Syrier and Beyerman (1974) and Jones and Ramage (1979). Peptide samples (175 nmol) were hydrolyzed in acid, the sample w,as evaporated to dryness, the residue was dissolved in water (1 ml) and enough sodium bicarbonate solution was added to adjust the pH to approximately 7 on range 1-11 pH paper. After evaporation, the residue was dissolved in 400 p1 of 0.2 M Tris-HC1 (pH 7.5). Toluene (5 pl) and 0.4 unit of L-amino acid oxidase in 10 pl of 0.2 M Tris-HC1 (pH 7.5) were added to an aliquot which was incubated with stirring at 37 "C for 12 h. At this time, a second aliquot of enzyme was added and the incubation continued for another 12 h. Amino acid analyses were performed both before and after oxidase treatment.
Zsoelectric Focusing-Isoelectric focusing was carried out on 7.5% polyacrylamide gels (5 X 105 mm) containing 6 M urea and 2% (w/v) pH 3-10 ampholytes as described previously (England et at, 1982). The sensitivity of the Coomassie blue G-250 staining determined by focusing known amounts of native glucagon yielded a detection limit of 1-2 pg.
Amino Acid Analysis-Routine acid hydrolysates and enzymatic digests with leucine aminopeptidase were performed and analyzed as described by Flanders et al. (1982).
Circular Dichroism-Circular dichroism measurements were performed as previously described (Rothgeb et al., 1978). The protein concentrations of stock solutions were determined by their absorbance at 278 nm using a molar absorptivity of 8310 M" cm" and diluted with 0.01 N HC1 and 2-chloroethanol to 0.3 mg/ml or less. The spectra were analyzed by the method of Greenfield and Fasman (1969) using the reference spectra of Chen et al. (1974) taken at 1nm intervals over the range of 250-205 nm.
formed as described by England et al. (1982), except that the incu-Biological Assays-Binding and adenylate cyclase assays were perbation buffer used in the binding assay was 30 mM Tris-HC1 (pH 7.5), 1 mM dithiothreitol, and 0.1% bovine serum albumin. Results of the adenylate cyclase assay are expressed as percentage of activation over basal level which averaged about 0.4 nmol of CAMP formed/mg of membrane protein in 10 min. Maximum activity was 3-4 times the basal level activity. For each set of assays, maximum stimulation of adenylate cyclase by native glucagon was determined for use as a control. In the binding assays, nonspecific binding measured in the presence of excess unlabeled peptide was 5-10% of the total binding and was subtracted from the total to give specific binding. Results are expressed as the percentage of maximum specific binding which was about 1 X lo' cpm/mg of membrane protein. In both assays, the standard deviation for individual points was approximately 5%.

Coupling of Ne-tBoc-N"-DNP-L-Ht-ONSu to [des-Hisl]
N-Acetimidoglwagon-Due to the insolubility of glucagon in organic solvents, initial couplings of histidine were performed in dimethylformamide:H20 (3:l). Despite a 24-h coupling and a 50-fold molar excess of active ester at pH 8.8, a disappointing 15% incorporation of histidine occurred even though similar conditions for coupling various amino acids to ferredoxin (Hong and Rabinowitz, 1970;Lode et al., 1976) led to essentially quantitative coupling. In an attempt to increase coupling yields by performing the reactions in a totally organic medium, the [des-His'IW-acetimidoglucagon was solubilized by forming the trifluoroacetate salts of the amines which are liberated by subsequent treatment with N-ethylmorpholine (Bodansky et ai., 1967).
1-Hydroxybenzotriazole was included in the coupling reaction Geiger, 1972, 1973) to accelerate the rate of active ester coupling of the sterically hindered, doubly protected histidine in dimethylformamide. Even with as much as a 25-fold molar excess of active ester, in the absence of 1hydroxybenzotriazole the coupling resulted in the incorporation of only 0.77 residue of histidine.
The conditions chosen were optimized to obtain maximum acylation at the NH2 terminus with minimum 0-acylation which has been reported in some N-hydroxysuccinimide active ester couplings (Slotboom and de Haas, 1975;Bodansky et al., 1977;DiMarchi et al., 1979DiMarchi et al., , 1980. Isoelectric focusing after deprotection showed the major band to focus with N'acetimidoglucagon. Minor bands corresponded to [des-His'] N-acetimidoglucagon and deamidated material. A band focusing at a position more positive than native glucagon, presumably 0-acylated material, was also noted. The alanine and phenylalanine couplings were carried out under conditions essentially identical to those used in the histidine coupling, but were complete with a much shorter reaction time, indicating that steric hindrance was involved in the coupling of the doubly protected histidine. Purification and Chemical Characterization of Semisynthetic N-Acetimidoglucagon-As shown in Fig. 1, a major peak (ZV) corresponding to N-acetimidoglucagon by amino acid analysis (Table 1)   These values were obtained from analysis on the lithium citrat,e column following digestion of the derivatives with leucine aminopeptidase.
These values were obtained from analysis on the PA-35 column following digestion of the derivatives with leucine aminopeptidase.
conditions did not affect its elution position from the cationexchange column.
Isoelectric focusing of the once purified semisynthetic material showed that a major product focused with W-acetimidoglucagon prepared from native glucagon (PI 6.5). Two minor bands (-5% of total material) were seen; one at the position of (des-His'JW-acetimidoglucagon and the other at a position more positive than native glucagon such as would be expected with 0-acylation. A second identical cation-exchange purification removed these contaminants. The overall yield of semisynthetic hormone after two cation-exchange purifications starting from purified native glucagon is 5-10%. The results of amino acid analysis of material from peak ZV following acid hydrolysis are shown in Table I. The expected number of residues of Thr, Ser, and Tyr were recovered upon leucine aminopeptidase digestion of the peptide, indicating the absence of significant 0-acylation. The basic residues were quantitated on the PA-35 column following digestion of the peptide with leucine aminopeptidase to analyze for an absence of lysine since N-acetimidoglucagon is partially destroyed during an acid hydrolysis (Flanders et al., 1982). Less than 0.01 residue of lysine was detected and within the error of amino acid analyses a full residue of histidine was incorporated. In further control experiments with Ne-acetimidoglucagon, the acetimidyl group was shown to be stable to the coupling and deprotection conditions. Preparation and Chemical Characterization of [Ala' ] and [Phe'] Analogues-Following the coupling reactions and desalting, the crude reaction mixtures were purified by cationexchange chromatography as shown in Fig. 2 (top). This purification separates the semisynthetic glucagon derivative in which the a-amino group is blocked by the tBoc group (peak I ) from unreacted [des-His']N'-acetimidoglucagon (peak ZZ). Since the tBoc group of tBoc-alanine was shown to be stable to the buffer conditions used for the separation, the tBoc group on the peptide would be expected to remain intact during the purification procedure.
Removal of the tBoc group from an aliquot of the fraction containing the protected [Ala'lderivative and subsequent isoelectric focusing revealed impurities, presumed to be 0acylated and deamidated material. To remove these contam- inants, a second ion-exchange purification was performed following removal of the tBoc group; cation-exchange chromatography at pH 4.5 (Fig. 2) (bottom) yielded one major species (peak IZ). Peak Z is presumed to be deamidated material. Both the [Ala'] and [Phe'lanalogues showed similar elution profiles, as expected.
The results of amino acid analysis of these derivatives following hydrolysis with 6 N HCl are also presented in Table  I. Both analogues showed the incorporation of a full residue of either alanine or phenylalanine and other amino acids were quantified as expected. With isoelectric focusing of [Ala'IN" acetimidoglucagon (1OO-Fg sample), a single band with a PI of 5.5 was present indicating a purity >98%. Similar analysis of [Phe'l W-acetimidoglucagon showed minor contamination with deamidated and 0-acylated products. A third identical cation-exchange purification yielded a single component on isoelectric focusing of 100 kg of sample.
Determination of Extent of Racemization-In control studies, L-amino acid oxidase treatment destroyed 99.8% of L-His and L-Phe samples, 49.7% of a DL-His sample, and 52% of a DL-Phe sample. L-Alanine was found to be a poor substrate for L-amino acid oxidase as reported by Greenstein et al. (1953). Native glucagon as well as semisynthetic material was subjected to HCI hydrolysis since some racemization occurs during this treatment (Neuberger, 1948). Amino acid analysis after the L-amino acid oxidase treatment showed the native glucagon sample to contain 2.0% D-His and 1.87% D-Phe/ Phe residue. The semisynthetic W-acetimidoglucagon sample contained 2.1% D-His while [Phe']N"acetimidoglucagon showed 1.70% D-Phe/Phe residue. These results indicate that essentially no racemization of the coupled amino acids had occurred during the extensive procedures necessary for its preparation. No racemization would be expected with [Ala'] N'acetimidoglucagon as it was prepared identically to the [Phe'lderivative and alanine shows no enhanced propensity to racemize. Circular Dichroism Analysis of Derivatives-The CD spectra of dilute solutions of the modified derivatives obtained in 0.01 N HCl and various concentrations of the helix-forming solvent, 2-chloroethanol, are shown in Fig. 3. As expected, all derivatives demonstrated an increase in a-helicity in this solvent (Gratzer and Beaven, 1969;Srere and Brooks, 1969;Epand, 1972a). In 0.01 N HCl in the absence of 2-chloroethanol, the percentage of p-sheet content was approximately equivalent to that of a-helical content for all peptides while in 2-chloroethanol no @-structure was found and the secondary structure was composed entirely of a-helix and random coil. Also in the absence of 2-chloroethanol in 0.01 N HCl native glucagon, W-acetimidoglucagon and [Phe'IW-acetimidoglucagon exhibited 13-15% a-helix, while surprisingly [Alal]N"acetimidoglucagon and [des-His']N"acetimidoglucagon showed almost 30% helicity. The concentrations used (approximately 0.3 mg/ml) were more dilute than that at which an increase in helicity is observed due to the aggregation of native glucagon into trimers which stabilize the a-helical configuration (Epand, 1972b;Panijpan and Gratzer, 1974). was only 0.18 mg/ml due to its limited solubility even with warming.

The concentration of [Phe'IW-acetimidoglucagon studied
To determine if the higher helical content of [Ala'IN" acetimidoglucagon, as compared with native glucagon, was due to aggregation to trimers, the CD spectra of diluted samples of native glucagon, [ Phe'IW-acetimidoglucagon and [Ala']N"acetimidoglucagon in 0.01 N HCl were obtained. The a-helical content of the [Ala'lderivative was found to decrease with dilution while the native glucagon and [Phe'lanalogue did not display this phenomenon. This suggests that the higher helical content of [Ala']N"acetimidoglucagon at 0.3 mg/ml may be due to the self-association of the derivative into trimers with a concomitant increase in helicity.
Biological Characterization of Glucagon Derivatives-Both W-acetimidoglucagon prepared from native hormone and twice-purified semisynthetic W-acetimidoglucagon were essentially identical with native glucagon in their abilities to activate adenylate cyclase and to bind to rat liver plasma membranes as shown in Fig. 4, A and B, respectively. The peptide concentration giving half-maximal activation was 2.1 X M for native glucagon, 1.9 X lo-' M for W-acetimidoglucagon, and 2.4 X lo-' M for semisynthetic W-acetimidoglucagon, while the concentration displacing 50% of mono['251]iodoglucagon from membranes was 1.4 X lo-' M for native glucagon, 1.4 X lo-' M for Ne-acetimidoglucagon, and 2.3 X lo-' for semisynthetic W-acetimidoglucagon. All values have an error of f l x lo-' M. The half-maximal binding and activation values previously reported from this laboratory for W-acetimidoglucagon were approximately 50% of those for native glucagon (Flanders et al., 1982). The closer agreement of the values reported here probably result from the cationexchange purification added in the preparation of N-acetimidoglucagon which removes an impurity that may have been present in the first preparation. Fig. 4, A and B, also shows the activation and binding exhibited by [des-His']N"acetimidoglucagon. The concentration of derivative displacing 50% of mono['251]iodoglucag~n from membranes was 3.5 X lo-' M, giving a relative binding affinity of 4.0% while the concentration required for halfmaximal activation was 3.3 X lo-' M, giving a relative biological potency of 5.7% when compared with W-acetimidoglucagon. As can be seen in Fig. 4A, this derivative is a partial agonist, attaining only 35% of the maximum activation elicited by native glucagon and the other derivatives. This is in accord with other reports (Lin et al., 1975;Bregman and Hruby, 1979) in which preparations of [des-His']glucagon were found to be partial agonists, but the degree of maximum activation is more than in our more highly purified preparation, perhaps due to slight contamination with fully active native glucagon.
A separate series of adenylate cyclase activation and receptor-binding assays were performed to characterize [Ala']-and [Phe']N"acetimidoglucagons and the results are shown in Fig. 5, A and B, respectively. Table I1 reports the derivative concentrations required for half-maximal activation and displacement of mono['251]iodoglucag~n bound to membranes along with those values for W-acetimidoglucagon and native glucagon assayed for control. Since the derivatives were partial agonists, Table I1 also gives the per cent maximal activation as compared to Ne-acetimidoglucagon. All derivatives exhibited high dose inhibition (England et al., 1983), ensuring that maximal stimulation was attained. These points were not included in computer fits of the data which were used to calculate the half-maximal activation concentration and per cent maximal activity.

DISCUSSION
The regeneration of fully active W-acetimidoglucagon and the preparation of [Ala']-and [Phe']N"acetimidoglucagon have been accomplished. The coupling of histidine posed special problems due to the limited solubility of glucagon, the slow rate of coupling of the sterically hindered histidine active ester, and the susceptibility of histidine to racemization which necessitates the use of both N" and N" protection. The solubilization of [des-His']N"acetimidoglucagon in dimethylformamide and the use of 1-hydroxybenzotriazole as a catalyst Geiger, 1972, 1973;Saunders and Offord, 1977;Geiger et al., 1978;Kisfaludy, 1979) are required for the quantitative coupling of the sterically hindered histidine in a reasonable time.
Protection of the imidazole nitrogen of histidine with DNP (Henkart, 1971) virtually eliminates its racemization (Erickson and Merrifield, 1976;Bodansky et al., 1976). Furthermore, the thiolytic deprotection conditions are sufficiently mild to avoid additional damage to the molecule (Shaltiel and Fridkin, 1970). The use of the tBoc group prevents undesired racemization, presumably by eliminating oxazolinone formation (Erickson and Merrifield, 1976;Bodansky et al., 1976;Barany and Merrifield, 1979) as well as acylation. These protecting groups proved effective as only 0.1% D-histidine was detected in the semisynthetic peptide.
Structural Studies-Although the substitutions at the amino-terminal residue would not, a priori, be expected to alter the secondary structure of the peptide (Chou and Fas-  * Relative biological potency equals (N"acetimidog1ucagon concentration required for half-maximal activation)/(derivative concentration required for half-maximal activation) X 100. Relative binding affinity equals (W-acetimidoglucagon concentration required for half-maximal displacement of the 'Z61-glucagon)/ (derivative concentration required for half-maximal displacement of the '*'I-glucagon) X 100. man, 1974), conformational differences between the derivatives are marked, as evidenced by CD studies in 0.01 N HC1.
The concentrations of native glucagon used in the CD studies are below those at which trimerization and helix induction are expected to occur (Epand, 1972b;Panijpan and Gratzer, 1974). However, the increased helicity of the [Ala'] derivative appears to be the result of trimerization as evidenced by the decrease in a-helix content upon dilution (Bregman et al., 1980). The [Phellderivative with its even lower solubility appears to aggregate without an increase in secondary structure comparable to that seen with the [Ala'lderivative.
Since the [Ala'] and [des-His'lderivatives exhibit an increased helical content over native glucagon in acid while the [Phe'lanalogue and other glucagon derivatives with aminoterminal modifications do not (Epand and Wheeler, 1975;Bregman et at., 1980;Liepnieks and Epand, 1982), it appears that the bulky ring system at residue 1 may limit helix formation under these conditions even though the amino terminus does not itself participate in the helix. Chou and Fasman (1975) have predicted that residues 5-10 of glucagon exist as a @-sheet, while residues 2-5 are in a @-turn. Since such regions bring together segments of proteins to allow long range interactions to stabilize secondary structure (Lewis et al., 1971), it is possible that the ability of residues 2-5 of glucagon to form a P-bend may affect the amount of secondary structure in the remainder of the molecule and hence its propensity to self-associate. A ring system at the side chain of position 1 may provide enough steric hindrance to make flturn formation difficult, thereby prohibiting the formation of a greater degree of secondary structure.
In the presence of 8-chloroethanol, all of the peptides increased in a-helix content (Beaven et al., 1969) with a concomitant decrease in B-sheet content, indicating that the structure at the amino terminus does not affect this phenomenon. Thus, the increase in helicity in organic solvents which is not associated with self-association differs markedly from the increased extent of helicity of the [Ala'lderivative which appears to be due to trimerization.
Functional Studies-Biological characterization of the [des-His'], [Ala'] and [Phe'lderivatives showed them to be partial agonists with each analogue exhibiting comparable decreases in binding and activation potencies. [des-His']N"acetimidoglucagon transduces poorly either because of the loss of the side chain or of the displacement of the a-amino group as a result of shortening the peptide. However, the [Ala'lderivative with an a-amino group in the original location has greater transducing ability than the [des-His']analogue, suggesting that the position of the a-amino group is important in transduction. Moreover, the side chain of the residue at position 1 must also be involved in transduction since, as the side chain is replaced by the methyl group of Ala', the nonhydrogenbonding, planar, aromatic ring system of Phe' and ultimately, the aromatic, hydrogen-bonding imidazole ring, the maximum activation of adenylate cyclase increases.
A large aromatic ring system at the side chain of the aminoterminal residue also seems to be very important for proper receptor binding inasmuch as the [Phe'lderivative shows only a slight reduction in binding ability when compared to N eacetimidoglucagon and native glucagon. Paradoxically, [Ala'] W-acetimidoglucagon shows less potency than the [des-His'] analogue. Perhaps, the hydrogen-binding capability of the side chain of either His-1 or Ser-2 also contributes to the binding potential. Clearly, some compensatory mechanism of receptor interaction must be present in the [des-His'lderivative which, even in the absence of the first residue, allows it to bind with a higher affinity than the [Ala'lderivative.
Although formal analyses were not attempted, the slopes of the binding curves of the derivatives appear to be similar to those of native glucagon and other derivatives modified throughout the molecule (England et al., 19831, suggesting that these derivatives, like those studied previously, interact with the receptor in a noncooperative manner. Furthermore, at high concentrations, inhibition of adenylate cyclase activity is characteristic of these derivatives. As pointed out previously (England et al., 1983), these features differentiate glucagonreceptor interactions from those of the adrenergic system and must be taken into account in the development of models of the glucagon-receptor system.
The results presented here indicate that the a-amino group of glucagon is involved to a greater extent in the transduction of adenylate cyclase than in the binding process. The imidazole moiety of histidine-1 participates in both processes with its planar ring system and hydrogen-bonding ability contributing to its function. Alteration of the amino terminus affects the way in which that region interacts with the receptor. However, the modification can influence the remainder of the molecule but these effects cannot be predicted from our data. Small perturbations in the structure of the hormone caused by the alternative residues at the amino terminus may cause intramolecular interactions which alter the conformation of another area of the hormone whose structural integrity is important in receptor binding (Yeung et al., 1979). Although the results of the circular dichroism studies do not correlate directly with the results of the biological assays, they do indicate that the amino-terminal substitutions may cause unexpected structure changes. Clearly, the semisynthetic preparation of a variety of amino-terminal derivatives is required in attempts to delineate the binding and transduction messages of this region of the hormone. This information may lead to the development of a potent glucagon antagonist, as well as contribute to our understanding of the mechanism of the transduction process in this and other peptide hormone systems.