A detailed study of the L2beta long-loop region of human chorionic gonadotrophin suggests it to be spatially close to, but not part of, the receptor-binding site.

An in-depth study of the L2beta long-loop region of human chorionic gonadotrophin (hCG), earlier identified to be a conformational bioneutralization epitope and receptor-binding site of the hormone, was carried out. The linear 38-57 hCGbeta peptide and the corresponding cyclic disulphide peptide were synthesized and antipeptide antibodies developed. Binding studies with antibodies to the linear peptide, and with hCGbeta, hCG and human LH suggest that part of the region is buried at the alpha/beta interface and part exposed in hCG. Observation of the surface exposure of residues 47-53 from the crystal structure of hCG was confirmed by epitope mapping studies of the region. The region is not unique to hCG as a majority of the antibodies to both the linear and cyclic peptides did not exhibit the required specificity. Competitive inhibition studies with the linear and cyclic peptides failed to show inhibition of radiolabelled hCG binding to its receptors. However, both the antipeptide antibodies were able to bioneutralize the hormone in an in vivo assay. Taken together, these results seem to indicate that the L2beta long-loop region is not a receptor-binding site of hCG but spatially close to it.

Beta-subunit of baboon chorionic gonadotropin. Continuous flow Fmoc-polyamide synthesis of the C-terminal 37-peptide.

The synthesis of a 37-peptide fragment derived from the carboxyl terminal of the beta-subunit of baboon chorionic gonadotropin has been accomplished by the continuous flow Fmoc-polyamide solid phase method. The use of N alpha-fluorenylmethoxy-carbonyl-glutamine with its sidechain protected by the 4,4'-dimethoxybenzhydryl group enabled successful incorporation of this residue onto a hydroxymethyl linkage agent without apparent side reaction.

Inhibition of human choriotropin binding to receptor by human choriotropin alpha peptides. A comprehensive synthetic approach.

Synthetic overlapping peptides of the alpha-subunit of human chorionic gonadotropin (hCG) were made by solid-phase peptide synthesis employing a comprehensive synthetic approach. The entire primary structure of the alpha-subunit was synthesized as a series of nine consecutive peptides, each 15 residues in length, and overlapping with its two adjacent neighbors by 5 residues on each side. Receptor binding activity of each synthetic peptide was measured by the inhibition of binding of 125I-labeled hCG to rat ovarian receptor. Peptides alpha 21-35, alpha 31-45, alpha 71-85, and alpha 81-92 were shown to compete for binding with native hCG, thus demonstrating that at least two regions on the alpha-subunit may be part of the binding site(s) of the hormone. The low affinity of the peptides (10(-5)-10(-6) M) compared to native hormone (10(-10) M) for receptor is not unexpected due to the probability of discontinuous and multiple sites involved in receptor binding. An ultrapure preparation of hCG alpha-subunit also had low affinity (10(-5), suggesting that conformational changes upon combination with beta-subunit to form dimer or changes in conformation after binding are necessary for high affinity interaction. These results correlate with previous predictions of binding sites based on studies employing chemical and enzymatic modifications of intact hormone and show that synthetic peptide strategies are helpful in the elucidation of protein structure and function.

Synthesis of a heptasaccharide hapten related to a biantennary glycan chain of human chorionic gonadotropin of a choriocarcinoma patient. A convergent approach.

Synthesis of the heptasaccharide hapten 8-methoxycarbonyloctyl O-beta-D-galactopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----2)-O-[beta-D-galactopyranosyl-(1----4)-O-(2-acetam ido-2- deoxy-beta-D-glucopyranosyl)-(1----4)]-O-alpha-D-mannopyranosyl-(1----3) -O- [alpha-D-mannopyranosyl-(1----6)]-beta-D-mannopyranoside is described, by use of the known, protected glycosyl acceptor 8-ethoxy-carbonyloctyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-(1----6)-2,4-di-O-benz yl-beta - D-mannopyranoside, and the key glycopentaosyl donors O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----4)-O-(2-acetami do-3,6- di-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-(1----2)-O-[(2,3,4,6-tetra-O- acetyl-beta-D-galactopyranosyl)-(1----4)-O-(2-acetamido-3,6-di-O-acetyl- 2- deoxy-beta-D-glucopyranosyl)-(1----4)]-3,6-di-O-benzyl-alpha-D-mannopyra nosyl trichloroacetimidate (5) and the corresponding fluoride 7, which, in turn, were prepared in 5 steps from allyl 3,6-di-O-benzyl-alpha-D-mannopyranoside in 35 and 22% overall yields, respectively. In model experiments, the key glycosyl donors 5 and 7 were also treated with the simple glycosyl acceptor 8-ethoxycarbonyloctanol, to give 8-methoxycarbonyloctyl O-beta-D-galactopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----2)-O-[beta-D-galactopyranosyl-(1----4)-O-(2-acetam ido-2- deoxy-beta-D-glucopyranosyl)-(1----4)]-alpha (and beta)-D-mannopyranoside.

Synthesis of a pentasaccharide hapten related to a monoantennary glycan chain of human chorionic gonadotropin.

A stereocontrolled synthesis of a pentasaccharide hapten, namely, 8-ethoxy-carbonyloctyl O-beta-D-galactopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D-gluc opy ranosyl) -(1----2)-O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl- (1----6)]-alpha-D-mannopyranoside, is described employing a trihexosyl glycosyl-donor, O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----4)-O-(2-acet amido-3,6- di-O-acetyl-2-deoxy-beta- D-glucopyranosyl)-(1----2)-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl trichloroacetimidate, and a mannobiosyl glycosyl-acceptor, 8-ethoxycarbonyl-octyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-(1----6)-2,4-di-O-benz yl-beta - D-mannopyranoside, as the key intermediates.

Synthesis of a heptasaccharide hapten related to an anomalous biantennary glycan-chain of human chorionic gonadotropin of a patient with choriocarcinoma. A stepwise approach.

A stereocontrolled synthesis of a heptasaccharide hapten, 8-methoxycarbonyloctyl 3-O-[2,4-di-O-(2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-beta-D- glucopyranosyl)-alpha-D-mannopyranosyl]-6-O-alpha-D-mannopyranosyl-beta- D-mannopyranoside (2), is described employing the lactosaminyl donor 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4, 6-tetra-O-acetyl-beta-D-galactopyranosyl)-beta-D-glucopyranosyl bromide and the mannotriosyl glycosyl acceptor 8-ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O-(3,6-di-O-benzyl-alpha-D-mannopyranosyl)-6-O-(2,3, 4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-beta-D-mannopyranoside, the reaction of which gave the biantennary structure 8-ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O-(3,6- di-O-benzyl-2,4-di-O-[3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3, 4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-beta-D-mannopyranoside (9), as well as a monoglycosylated product, 8-ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O-(3,6-di-O-benzyl-4-O-[3,6- di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6- tetra-O-benzyl-alpha-D-mannopyranosyl)-beta-D-mannopyranoside. The diglycosylated product 9 was transformed into 2, and the structure was confirmed by 1H-n.m.r. data.

Preparation and properties of human chorionic gonadotropin antagonist for biological studies: antifertility effects in the female rat.

We compared the effects of treatment of clinical grade hCG powders with anhydrous HF (hydrogen fluoride) or TFMS (trifluoromethane sulfonic acid). HF treatment yielded stable product (DG-hCG), which had desirable antagonistic activity in mouse Leydig cells. Binding of HF-hCG to ovarian granulosa cell FSH receptors was less than 5% as compared to purified ovine FSH. As LH/hCG receptor specificity was not significantly compromised crude hCG could be directly used in obtaining large amounts of the antagonist. The effects of antagonist (DG-hCG) and agonists crude hCG and purified hCG were evaluated in pregnant rats. When administered between days 1 to 5 of pregnancy, crude DG-hCG inhibited serum progesterone and oestradiol levels and implantation. The effect was dose-dependent. However, both crude hCG and purified hCG elevated progesterone level and partially inhibited implantation (up to about 40%). In the post-implantation period (days 8-11) crude DG-hCG treatment induced abortion due to a decrease in circulating progesterone and oestradiol levels. The agonists, crude hCG and purified hCG, on the other hand, elevated both steroid levels in serum and induced partial termination of pregnancy (up to 50%). During the second half of pregnancy, when luteotropic support of LH becomes unnecessary in the rat, crude DG-hCG (antagonist) had no effect on parturition. However, crude or purified hCG caused delay in parturition by sustaining high level of progesterone in circulation. Our data demonstrate that the antifertility effects of crude DG-hCG are more potent and consistent than the administration of either crude or purified hCG in the pregnant rat.

Chemical synthesis and immunological properties of a cyclic eicosapeptide (Gly88,90) 82-101 of the beta subunit of human chorionic gonadotrophin.

The eicosapeptide (Gly88,90) 82-101 hCG-beta was synthesized by the fragment condensation of the nonapeptide (Gly88,90) 82-90 and the undecapeptide 91-101 followed by iodine oxidation to make the disulfide loop. Intramolecularity of the disulfide linking cysteines at 93 and 100 was confirmed. Antipeptide antibodies were elicited in rabbits upon immunization with a conjugate of the peptide with tetanus toxoid. The repertoire of these antibodies was directed solely against the undecapeptide 91-101. These antibodies showed no recognition for hCG, hCG-beta or ARCM hCG-beta, suggesting that the conformational epitopes of hCG-beta in the region 82-101 may not be accessible to antibodies.

Synthesis and in vitro activity of a hormone-diphtheria toxin fragment A hybrid.

The synthesis and in vitro biological activity of a hybrid protein composed of intact human chorionic gonadotropin and fragment A of diphtheria toxin in a disulfide conjugate is reported. This hybrid retained greater than 90% of the binding ability of uncoupled hCG and was shown to be specifically toxic to a mouse Leydig cell tumor which binds hCG while being non-toxic towards cells which lack receptors for hCG.

Synthesis of carboxyl terminal segments of beta-subunit of human chorionic gonadotropin. Application of the two-phase method.

The synthesis in solution of carboxyl terminal peptide segments of the beta-subunit of human chorionic gonadotropin is described. The protected segments include sequences 119-131, 132-137, and 138-145. The syntheses were based on a standardized liquid-liquid extraction program for routine purification of intermediates (two-phase method). Condensation of terminally deblocked segments afforded protection peptides 132-145, 126-145, 120-145, and 119-145. Protected peptides 126-145 and 120-145 were deprotected in liquid hydrogen fluoride and used in conjugated form for immunization of rabbits. Data on the specificity of the antibody response are reported.