Int. J. Peptide Protein Res. 38, 1991, 340-345

Solid phase synthesis of human growth hormone-releasing factor analogs containing a bicyclic /?-turn dipeptide

KAZUKI SATO,‘ MARI HO’ITA? MING-HUI DONG,’ HSIAO-YU HU,’ JOSEPH P. TAULENE,’ MURRAY GOODMAN: UKON NAGAI,’ and NICHOLAS LING’

‘Mitsubishi Kasei Institute of Life Sciences, Machiabshi, and *Tokyo Women’s Medical College, Shinjuku-ku, Tokyo, Japan; ’Department of Molecular Endocrinology, The Whittier Institute for Diabetes and Endocrinology, and

‘Department of Chemistry, University of California at San Diego, La Jolla, CA, USA

Received 25 November 1990, accepted for publication 10 May 1991

Three analogs derived from the N-terminal29-residue fragment of human growth hormone-releasing factor (hGRF) which contained a bicyclic fl-turn dipeptide (BTD) at 7-8,8-9, and 9-10 positions were synthesized by solid phase methodology to ascertain if the fl-turns are important for the biological activity of hGRF and also to show the applicability of the BTD unit to solid phase synthesis. All three analogs were obtained in good yield and purity indicating that the BTD unit can be used in the usual condition of solid phase synthesis. The capacity of these analogs to release growth hormone (GH) was tested in an in vitro bioassay using rat anterior pituitary cells. All three BTD-containing analogs showed the same maximal GH secretion with parallel dose-response curves to that of hGRF( 1-29)NH2, except their relative potencies were very low.

Key words: &turn; bicyclic 8-turn dipeptide; conformation; growth hormone-releasing factor; solid phase synthesis

To design an effective agonist or antagonist of a bio- logically active peptide, it is important to know the conformation of the peptide when it binds to its recep- tor. Most linear peptides possess a high degree of con- formational freedom. If such freedom can be restricted, it is possible to determine the conformational effects on biological activity. The high and selective activities of some cyclic analogs of somatostatin (l) , enkephalin (2), and a-melanocyte-stimulating hormone (3) are successful examples of such an approach. Another way of exploring conformational restriction is by the introduction of amino acid or dipeptide derivatives of low conformational flexibility (4).

Abbreviations according to IUPAC-IUB Commission (1984), Euro- pean J. Biochem. 138, 9-37, are used throughout. Additional abbreviations: Boc, ferf.-butyloxycarbonyl; BTD, bicyclic 8-turn dipeptide; CD, circular dichroism; DCC, dicyclohexylcarbodiimide; EtOAc, ethyl acetate; FAB-MS, fast atom bombardment mass spectrometry, GH, growth hormone; GRF, growth hormone- releasing factor; GS, gramicidin S; HOBt, N-hydroxybenzo- tyrazole; HPLC, high performance liquid chromatography; MBHA, p-methylbenzhydrylamine; NH,OAc, ammonium acetate; RIA, radioimmunoassay; TFA, trifluoroacetic acid; TFE, trifluoro- ethanol.

Recently, we reported the synthesis of a bicyclic &turn dipeptide (BTD) which was designed to sim- ulate the central part of type 11’ /?-turn (Fig. 1) (5). Since BTD is a type of dipeptide, it can be inserted into a peptide chain at any position and, as a result, restrict that position to assume a 8-turn conformation in the peptide chain owing to the semi-rigid nature of the bicyclic skeleton. This type of bicyclic dipeptide lactam was first reported by Wyvratt et al. (6) as an inhibitor of angiotensin converting enzyme. However, their elactam ring should be much more flexible than the d-lactam ring of BTD and no comment was given on the possible use of their compound in fixing 8-turns.

BTD has been successfully inserted into enkephalin (7), gramicidin S (GS) (8), luteinking hormone-releasing factor (9), and cyclic hexapeptide analog of somato: statin (10) by conventional solution phase procedure. However, incorporation of BTD into biologically active peptides by the solid phase procedure has not been attempted. As a result we prepared three human growth hormone-releasing factor (hGRF) analogs containing a BTD by the solid phase method.

Human GRF is a 44-residue linear peptide with an amidated C-terminus and its primary structure is H-

340

GRF analogs containing BTD

8 -

h - 6 - '- 0

.o E * 4- N

6 - F * - t 9 0 -

s -2-

U - X

E - -4 -

10 1

NH2 47?oo" 0

FIGURE 1 Structure of BTD.

Tyr- Ala- Asp- Ala-Ile-Phe-Thr- Asn-Ser-Tyr- Arg-Ly s- Val-Leu-Gly-Gln-Leu-Ser- Ala- Arg-Lys-Leu-Leu- Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser- Asn-Gln-Glu- Arg-Gly- Ala- Arg- Ala- Arg-Leu-NH, (1 1). Biological activity is located in the N-terminal part of the molecule and hGRF( 1-29)NH2 retains almost the full biological activity of the native mole- cule (1 2). Secondary structure prediction of hGRF by the Chou-Fasman procedure (1 3) suggests that hGRF may assume B-turns from positions 6 to 11 (Table 1). Biological data from a series of hGRF analogs which had the corresponding amino acid at each position of hGRF( 1-29)NH2 substituted by its D-isomer is consis- tent with the prediction that a /%turn might exist in this region (14). To ascertain whether the B-turns are, indeed, important for the biological activity of hGRF, we have synthesized [BTD'-*]-(l), [BTD*-9]- (2), and [BTD9-'']hGRF( 1 -29)NH2 (3) and determined their growth hormone (GH) releasing activity.

RESULTS AND DISCUSSION

The starting material for the synthesis of the three analogs was the protected hGRF( 1 1-29)-MBHA- resin prepared by stepwise solid phase methodology (15). Boc-BTD-OH was coupled to this resin at the appropriate positions with the DCC-HOBt (1 6) method and the reaction was monitored by the nin- hydrin test (17). The remaining peptide chain was assembled in a similar stepwise manner. After HF cleavage and subsequent purification by ion- exchange, gel filtration and partition chromatog-

TABLE 1 Secondary structures of hGRF(1-44)NH2 predicted by Chou and

Fasman algorithm"

1-6 3-8 6-9 8-11

10-14 14-31 22-26 33-36 35-38

a-helix 8-sheet 8-turn 8-turn

a-helix 8-sheet 8-turn p-turn

8-sheet

1.125 1.108 1.023 1.072 0.850 1.053 1.138 0.778 1.010 1.270 1.020 1.228 1.082 I .026 1.124 1.168 1.015 0.778 1.178 1.068 0.823 1.058

a ( PX ) = average propensity for x = a (helix), b (sheet), t (turn).

1 -6'1 , 1 . I . I . I . I . 1

190 2GO 210 220 230 240 250

Wavelength (nm) FIGURE 2 CD spectra of [BTD'-']- (- - -), [BTD*"] - (0 0 0 O), [BTD9-'0 I- hGRF(I-29)NH2 (- 0 -) and hGRF(I-29)NH2(-) in H,O (5 mM phosphate buffer, pH 6.8) and in TFE.

raphy, all three analogs were obtained in good overall yield (13%, 19%, and 11% for 1, 2, and 3 respectively) and purity (>98% pure by HPLC). Their structures other than the BTD residue were confirmed by amino acid analysis and sequencing up to the BTD residue. FAB-MS measurement showed that the BTD residue was not decomposed by repeated acid treatment with TFA and HF. Previously we had demonstrated that BTD was stable in single treatment with HCl/EtOAc (7), HCl/HCOOH (8), HBr/AcOH (8), and H F (9).

Though we didn't test the racemization of BTD residue in a coupling step, the possibility should be very low because of its semi-rigid bicyclic structure. If any racemization had occurred, the product contain- ing racemized BTD should take on a different confor- mation from that of the correct compound, and it can be easily detected by HPLC and removed by cFro- matographies. These results taken together indicate that the BTD residue is amenable to solid phase syn- thesis like the usual protected amino acids. In this connection, it is noteworthy that a monocyclic lactam was used successfully in solid phase synthesis of cyclic hexapeptide somatostatin analogs by Freidinger et al.

The conformatibns of the BTD-containing analogs in solution were studied by circular dichroism (CD) as

34 1

(18)-

K. Sato et al.

800

1 1 . I ' ' ' " I !0.14 10.13 10.12 lo.ll 1 0 . ~ loa 10.' l o6 1 0 . ~ l o 4

Peptide concentration (M)

FIGURE 3 Relative agonistic activity of [BTD'-*]- (O), [BTD8-9]- (0). and [BTDP-'']hGRF(1-29)NH2 (A) to hGRF(I-29)NH2 (0) in v i m . Each point represents the mean of triplicate cultures. SD of the actual release was less than 12% of the mean.

shown in Fig. 2. In H,O solution, all three analogs as well as hGRF( 1-29)NHZ showed weak CD intensities with minima at about 200nm, suggesting that they were essentially in a random conformation. In tri- fluoroethanol solution, hGRF( 1-29)NHz exhibited CD spectra with minima at 207nm and shoulder at around 220 nm typical of the a-helical conformation, whereas the BTD-containing analogs showed similar but weaker CD spectra to that of hGRF( 1-29)NH2. These results suggest that positions 6 to 11 in hGRF(I-29)NH2 are involved in a a-helix confor- mation, and the helix is broken by incorporation of the BTD unit. Although CD spectra suggested that the conformation of the BTD-containing analogs in solution were different from that of hGRF( 1-29)NH2, it did not necessarily mean that these analogs assumed a different conformation from that of hGRF( I - 29)NH2 when they bound to a hGRF receptor. CD spectra did not give us enough information to discuss in detail the conformation of a short part of relatively long linear peptides.

The capacity of these analogs to release GH was tested in an in vitro assay using rat anterior pituitary cells (19), and the results are shown in Fig. 3. All three BTD-containing analogs showed the same maximal GH secretion with parallel dose-reponse curves to that of hGRF( 1-29)NH,, except their relative potencies were very low. The calculated relative potencies are summarized in Table 2 together with the data from those analogs with single alanine substitution (20) in the predicted 8-turn positions. The fact that all three analogs retained full intrinsic activity suggests that the conformation of hGRF, when it binds to receptor, may involve 8-turns in the positions 6-1 I .

One of the reasons for the very low relative poten- cies of the analogs may be due to the lack of the

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side-chain functional groups of Thr', Asn*, Ser', and Tyr" residues in the BTD-containing compounds. The data from single alanine substitution indicate that the side chain functional group of Ser' can be removed without significant loss of activity, whereas replace- ment of other three residues, especially that of Tyr", resulted in large loss of activity. Assuming that these activity losses came from the decreased affinity to the GRF receptor, we calculated the relative binding energy of the analogs using general formula AG = - RT In K as shown in Table 2. Contribution of the effects of side chain deletion in the BTD- containing analogs were estimated by summation of the values of Ala-substituted analogs at each position. Binding energy of side chain of analog 2 was lower than 1 and 3, which agreed with the bioassay results that 2 has slightly higher potency than the other two analogs. However, the effects of side chain deletion can not fully explain such large loss of potencies as 4-5 orders of magnitude less than the parent GRF peptide. The difference between total binding energy and that of side chains, expressed as "others" in the Table 2, should mainly reflect the effects of conformational restriction. Among the three analogs, 3 showed lower binding energy of "others" than 1 and 2 do, suggesting that the position Ser9-TyrIo has a greater possibility to take on type 11' 8-turn than other positions do. However, apparently, none of these conformational restrictions were effective for binding.

While it may be that GRF has a 8-turn in the 6-1 1 region, alternate turn types such as type I1 can also fit the previous data. The BTD may be stabilizing the wrong turn type, and this incorrect backbone confor- mation plus the missing side chains would result in the extremely low potency. The fact that all three BTD analogs differ little in potency also suggests that none of them is in fact stabilizing the correct backbone conformation. It should also be considered that the BTD is introducing bulk in a region of the peptide that produces a steric interaction with the receptor, result- ing in lower potency.

In the case that BTD-containing analogs arC as potent as their parent compounds as has been shown

1-GS (8), we can conclude unam- biguously that type 11' /?-turn exists in their bioactive conformation. On the other hand, when BTD- containing analogs show quite low potencies as have been shown in the synthesis of an enkephalin analog (7) and a cyclic hexapeptide analog of somatostatin (lo), the rationalization is very difficult. In the case of enkephalin analog, H-Tyr-BTD-Phe-Leu-NH, , the major reason seemed to be that side chain bulk of BTD interfered in the interaction with the receptor because Gly3 in enkephalin cannot be substituted with other amino acids without significant loss of activity (21). In the case of somatostatin analog, cyclo(Phe-D- Trp-Lys-Thr-BTD), /?-turn type of original sequence

in [BTD~S,~-S

GRF analogs containing BTD TABLE 2

Relative potencies of GRF analogs in vitro"

GRF analogs Relative potency Relative binding energyb (kcal/mol)

Total Side chain Others

hGRF( 1-29)NH2 1 0 0 0 [BTD"*]hGRF(l-29)NH2 (1) 1.4 x 10-5 6.89 3.5T 3.37 [BTDG9]hGRF(1-29)NH, (2) 4.6 x 10-5 6.15 2.33' 3.82 [BTD""]hGRF( I-29)NH2 (3) 1.5 x 10-5 6.84 4.76' 2.08

hGRF( 1-27)NHZ [Ala']hGRF( 1 -27)NH2 [Ala8]hGRF( 1-27)NH2 [Ala9]hGRF( 1 -27)NH2 [Alal"]hGRF( I -27)NH2

1

0.051 (0.015-0.189)d 0.444 (0.172-1.842)d 0.001

0.064 (0.044-0.091)d 0 1.69 1.83 0.50 4.26

"hGRF(1-29)NH2 and hGRF(I-27)NH2 are the standards, respectively, for the analogs with 29 and 27 residues.

'Estimated by summation of the values of Ala-substituted analogs. d95% confidence limit.

Calculated by the general formula AG = - RT In K.

where BTD was incorporated seemed to be different from that of type 11' of BTD (22).

The present study points out the applicability of BTD to routine solid phase peptide synthesis. Additionally, it revealed the limitation of its use in fixing a fl-turn. To determine unambiguously whether hGRF assumes a fl-turn conformation in the region of positions 6 to 1 I , it is necessary to incorporate the rigid fl-turn unit with the proper functional groups to mimic the side chain moieties in native hGRF, and several types of fl-turn other than type 11' of BTD should also be examined.

EXPERIMENTAL PROCEDURES

Peptide synthesis Derivatized amino acids used in the synthesis reported here were of the L-configuration, unless stated other- wise, and were purchased from Bachem Inc., Torrance, CA. The iV-amino function was protected exclusively with the tert.-butyloxycarbonyl (Boc) group. Side chain functional groups were protected as follows: benzyl for threonine, serine, glutamic and aspartic acids; 2,6- dichlorobenzyl for tyrosine; p-toluenesulfonyl for arginine; 2-chlorobenzyloxycarbonyl for lysine. The side chain of the Asp3 residue was protected with the cyclohexyl group (23) (Peptide Institute, Inc., Osaka, Japan). p-Methylbenzhydrylamine (MBHA) resin was synthesized in our laboratory using a previously pub- lished procedure (24). The substitution was 0.55 mmol amine per gram by Gisin analysis (25). Amino acid compositions were determined on peptide hydro- lysates using a Kontron Liquimant I11 amino acid analyzer equipped with a post-column fluorescamine derivatization detection system (26). Hydrolyses were performed in 6 M HCI containing 2.5% thioglycollic

acid at 110" for 20h in tubes sealed under vacuum. Reversed-phase high performance liquid chromatog- raphy (HPLC) was performed on a Beckman model 322 liquid chromatography system using a 5 p particle size, 0.46 x 22 cm Spheri-5 RP-I 8 cartridge protected with a 0.46 x 3cm Spheri-I0 RP-18 guard column (Brownlee Labs, Inc., Santa Clara, CA) and a sol- vent system composed of buffer A: 0.25 M triethyl- ammonium phosphate at pH 3.00 and buffer B: 20% buffer A in acetonitrile by volume (27). The column was eluted at a flow rate of 1 .O mL/min and the column effluent detected by W absorption at 210nm with 0.5 absorbance unit at full scale and a recorder chart speed of 12 cm/h. Optical rotations were measured on a Jasco DIP-360 digital polarimeter. FAB mass spectra were measured on a Jeol HX-100 mass spec- trometer. Amino acid sequence was determined with an Applied Biosystem model 470A gas phase protein sequencer equipped with an on-line PTH analyzer. CD spectra were recorded with a computer-assisted Cary 61 dichrograph in optical cells of 0.01-cm ppth- length equipped with quartz windows. The results are reported as mean residue molar ellipticities [el.

[BTD"]hCRF(1-29)NHz (I). Protected hGRF(I 1- 29)-MBHA-resin (8.8 g ) was synthesized from MBHA-resin (3.6 g, 2 mmol) by solid phase method- ology (1 5 ) on a Beckman model 990 peptide synthesizer according to the procedure used in the synthesis of human hypothalamic GRF (1 Ic). Synthesis of 1 was continued by using 1.I.g (0.25 mmol equivalent) of this peptide-resin. After two more coupling cycles, Boc- BTD-OH (I58 mg, 0.5 mmol) was coupled to the resin in the presence of HOBt (135 mg, 1 mmol). The cou- pling reaction was monitored by ninhydrin test (17) and shown to be > 94% completion. After six more cou-

343

K. Sat0 et al.

plings, the N-terminal Boc-protecting group was removed and the protected peptide resin (1.24 g) was treated with a mixture consisting of 1.86 mL anisole, 0.31 mL methylethylsulfide and 12.4mL hydrogen fluoride at Oo for 1 h to cleave the peptide from the resin anchor as well as to deprotect the side chain functional groups. The resulting crude pep- tide (615 mg) was purified by CM-32 carboxymethyl- cellulose cation-exchange chromatography (Whatman) (1.8 x 20cm) developed with a gradient generated by adding 1 L of 0.3 M NH,OAc in 6 M urea at pH 6.5 to 400 mL of 0.01 M NH,OAc in 6 M urea at pH 4.5. The urea in the column fractions was removed by gel filtration on a Sephadex G-25 fine column (2.5 x 105 cm) developed in 0.5 M AcOH. The result- ing peptide (204 mg) was further purified by partition chromatography on a Sephadex G-25 fine column (2.5 x 105cm) using the solvent system 1-butanol/ acetic acid/water, 4: 1 : 5 (vol/vol) to give a highly purified peptide (107mg, 13% yield based on sub- stitution of the amino group on the MBHA resin). Analytical HPLC of the product showed a single peak eluting at 52% buffer B in a linear gradient from 0 to 80% buffer B in buffer A in 80 min. Sequence analysis showed that N-terminal six amino acids were correctly assembled and the sequencing terminated at the BTD residue. [a]g - 84.0' (c0.2 in 0.2 M AcOH); MH+ ,3342. C149H242041 N42S2 requires MH, 3341 or 3342. Amino acid ratios in acid hydrolysate: Asp 2.06 (2), Ser 3.07 (3), Glu 2.05 (2), Gly 1.05 (l), Ala 3.09 (3), Val 0.97 (l), Met 0.96 (l), Ile 1.91 (2), Leu 3.81 (4), Tyr 2.05 (2), Phe 0.99 (l), Lys 2.06 (2), Arg 2.94 (3).

[BTo"-']hGRF(1-29) NH, ( 2 ) . This peptide was syn- thesized as described in the synthesis of 1. The overall yield of the purified peptide (154mg) was 19%. Ana- lytical HPLC of the product showed a single peak eluting at 53% buffer B in the same condition as described for 1. Sequence analysis showed that the N-terminal 7 amino acids were correctly assembled and the sequencing terminated at the BTD residue. [a]g - 116O (c 0.2 in 0 . 2 ~ AcOH); MH' 3355. CIMH244041N42S2 requires MH, 3355 or 3356. Amino acid ratios in acid hydrolysate: Asp 1.95 (2), Thr 0.95 (l),Ser2.04(2),Glu 1.95(2),Gly0.98(1),Ala3.00(3), Val 1.05 (l), Met 0.98 (I), Ile 2.06 (2), Leu 4.03 (4), Tyr 2.03 (2), Phe 0.96 ( I ) , Lys 2.00 (2), Arg 3.02 (3).

[BTD9-'0]hGRF(1-29) NH, ( 3 ) . This peptide was synthesized as described in the synthesis of 1. The overall yield of the purified peptide (92 mg) was 1 1 %. Analytical HPLC of the product showed single peak eluting at 53% buffer B in the same condition as described for 1. Sequence analysis showed that the N-terminal 8 amino acids were correctly assembled and the sequencing terminated at the BTD residue. [a]: -90.4" (c 0.2 in 0 . 2 ~ AcOH); MH' 3306.

C145 H24, 041 N43 S2 requires MH, 3306 or 3307. Amino acid ratios in acid hydrolysate: Asp 2.85 (3), Thr 0.95 (l),Ser2.08(2),Glu2.00(2),Gly 1.04(1),Ala3.02(3), Val 1.02 (l), Met 0.97 (I), Ile 2.02 (2), Leu 3.94 (4), Tyr 1.02 (l), Phe 0.98 (l), Lys 2.03 (2), Arg 3.07 (3).

Bioassays The capacity of the GRF analogs of hGRF to release growth hormone was tested in 4-day-old rat anterior pituitary cells as described (19). Graded doses of the peptides were incubated in triplicates for 4h with the cells and the amount of growth hormone released into the culture medium at the end of the incubation period was determined by a RIA using a monkey anti-mouse growth hormone serum kindly provided by Dr. Y. Sinha (28) and a rat growth hormone preparation (RP-2) generously furnished by NIADDK. Potency estimates of the various analogs were calculated using the computer program BIOPROG (29).

ACKNOWLEDGMENTS

We thank M. Regno, R. Schroeder, D. Angeles, V. Bonora, and C. Wong for excellent technical assistance. Supply of the rat GH for RIA by the National Pituitary Program of the NIADDK is grate- fully acknowledged. The research was supported by National Institute of Health Grants (HD-09690 and AM-18811) and the Robert J. and Helen C. Kleberg Foundation.

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Address:

Dr. Kazuki Sat0 Mitsubishi Kasei Institute of Life Sciences 11 Minamiooya Machida-shi, Tokyo 194 Japan

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