G. I. Tesser

A CONVENIENT SYNTHESIS OF AMINO ACID P-NITROANILIDES ; SYNTHONS IN THE SYNTHESIS OF PROTEASE SUBSTRATES

A method is described for the synthesis of Nα-protected bi- and trifunctional amino acid p-nitroanilides. The reaction uses phosphorus oxychloride as the condensing agent. The synthesis is simple, rapid, free of racemization and affords yields between 70–90%. The synthesis can be performed not only with amino acid derivatives of the urethane type including acid-labile (Z. Boc) and base-labile (Fmoc, Msc) Nα-proteclivc functions or allyl-derived protections, but also with Nα-trilyl amino acids, albeit in lower yield. The reaction runs in pyridine and its mechanism implies carboxyl activation by formation of a mixed anhydride with phosphorodichloridic acid (HOPOCl2).

Design and synthesis of thrombin substrates with modified kinetic parameters

For the continuous registration of thrombin formation in plasma (1), selective thrombin substrates are required, that show moderate binding affinities (high Km) and low turnover numbers (low kcat). Previously we have used SQ68 (CH3O-CO-CH2-CO-Aib-Arg-pNA) for this purpose. In order to find more substrates suitable for this application, we synthesized a series of 25 peptide p-nitroanilides. As lead structures SQ68 and S2238 (H-D-Phe-Pip-Arg-pNA) were used. By introduction of specific structure modifications we tried to alter the kinetic data in the required direction. The modifications were designed on basis of existing knowledge on the structure of the thrombin active-site and its surroundings. We indeed obtained a number of substrates with the kinetic constants in the desired range.

NMR studies of the interaction of gene-V protein of bacteriophage M13 with oligonucleotides

This paper describes the preparation of deuterated phenylalanine ([2H7]-phenylalanine) and the isolation of phage M13 encoded gene-V protein in which this deuterated amino acid was incorporated. Using this protein spectral assignments of resonances in the aromatic region of the 1H-NMR spectrum of the gene-V protein have been made. Furthermore the interaction of the gene-V protein with the tetranucleotide d(pC-G-C-G) and the hexanucleotide d(pC-G-C-G-C-G) was investigated. From the changes in the aromatic region of the NMR spectrum occurring after binding, it is concluded that at least one phenylalanine and one tyrosine is involved in the interaction with the oligonucleotides via stacking.

Preparation and characterization of polyclonal and monoclonal antibodies to polyamines.

In order to develop an immunocytochemical method suitable for the study of the cellular localization and intracellular distribution of polyamines we have prepared and characterized antibodies to polyamines. Artificial immunogens were prepared by coupling putrescine, spermidine and spermine to a carrier protein. Immunogens containing bovine serum albumin as a carrier protein were used to immunize rabbits (polyclonal antibodies) and mice (for the production of Mabs). The specificity of the antibodies was tested in an ELISA system utilizing antigens synthesized from thyroglobulin and one of the polyamines. Polyclonal antibodies to putrescine, spermidine and spermine were obtained. However, these antibodies showed a variable degree of cross-reactivity to the polyamines not used for immunization. Two hybridoma cell lines were developed. The first, MPut88, selectively produces a Mab to putrescine, the second, MSpm/d88 produces a Mab which recognizes spermine and spermidine but does not react with putrescine.

Role of the tryptophan residue in the interaction of pancreozymin with its receptor

1. Analogues of the C-terminal octapeptide and tetrapeptide of pancreozymin with a modified tryptophan residue have been tested on the rat pancreas adenylate cyclase activity, on the enzyme and fluid secretion of the rat pancreas in vivo and on the amylase release from rabbit pancreatic fragments. 2. Fluorination of the tryptophan residue in position 5 or 6 does not influence the effect of the peptides on any of the measured parameters. 3. Methylation of the nitrogen atom in the indolyl ring, which eliminates hydrogen bond formation, markedly reduces the affinity of the peptides for the adenylate cyclase activity and for the amylase release in rabbit pancreatic fragments. The effects on fluid and enzyme secretion in the rat pancreas in vivo are reduced nearly as much. 4. Tetrafluorination of the tryptophan residue, which reduces its charge donor capacity, causes a still larger reduction in activity and affinity of the octapeptide. 5. The tetrafluorinated tetrapeptide stimulates the adenylate cyclase activity and the enzyme and fluid secretion in vivo more than the unmodified tetrapeptide, which may be due to its increased hydrophobicity. 6. Replacement of the nitrogen atom in the indolyl ring of tryptophan by a sulfur or an oxygen atom, which also reduces the charge donor capacity, leads again to a large reduction in the affinity and activity of both the octapeptide and the tetrapeptide. 7. These findings suggest that the charge donor capacity of the tryptophan residue is of primary importance for the biologic activity of pancreozymin, while hydrogen bond formation and hydrophobicity are of secondary importance.

Synthesis of Penicillin N and Isopenicillin N

Abstract Enantiomeric 2-(N-allyloxycarbonyl)aminoadipic acid 1-allyl esters were obtained from the corresponding 2-(N-trityl)aminoadipic acid diallyl esters following selective hydrolysis of the allyl 6-ester group and subsequent exchange of the trityl group by the allyloxycarbonyl function. The resulting monoacids were used to acylate 6-aminopenicillanic acid allyl ester using a carbodiimide-mediated coupling. The products, the l - and d -isomers of 6-[6-(2-(N-allyloxycarbonyl) aminoadipyl)]aminopenicillanic acid diallyl ester were deprotected in one step by catalytic allyl transfer using tetrakis-(triphenylphosphine)palladium(0), to afford isopenicillin N and penicillin N, respectively. The presented straightforward route to penicillin N and isopenicillin N is uniquely compatible with the sensitive nature of the condensation products and gives entry to a new and high yielding procedure that is superior to existing approaches.

Preparation and characterization of monoclonal antibodies against ornithine decarboxylase

In order to develop a method for the immunocytochemical detection of ornithine decarboxylase (ODC), EC 4.1.1.17, we have prepared and characterized monoclonal antibodies (MAbs) against ODC. The primary structure of rat ODC (Rattus Norvegicus) was used for the selection of an epitope by computer calculations. The epitope (P16), a hexadecapeptide representing ODC-(345-360), was synthesized by means of solid phase peptide synthesis and coupled to a carrier protein. A bovine serum albumin conjugate of the P16 peptide was used as the immunogen for the production of MAbs in mice. Hybridoma clones were screened and the specificity of the monoclonal antibodies was tested in an ELISA utilizing a thyroglobulin conjugate of the hexadecapeptide. Two hybridoma cell lines were developed, i.e., MP16-2 and MP16-3. The epitope specificity of the MAbs produced by these cell lines was characterized in an ELISA using a set of small peptides representing parts of the P16 hexadecapeptide chain. MP16-2 recognized the ODC-(355-360) portion whereas MP16-3 reacted with the ODC-(345-350) part of the hexadecapeptide. Further studies showed that both MAbs also recognized native ODC but not the inhibited (i.e., ODC labelled with 3H-DFMO) enzyme indicating that the selected epitope was associated with the active site of ODC or a locus in its direct vicinity.

Interaction of tryptophan-modified analogues of cholecystokinin-octapeptide with cholecystokinin receptors on pancreatic acini

None of six different tryptophan-modified analogues of the C-terminal octapeptide of cholecystokinin differed from the unaltered peptide in terms of their efficacies for stimulating amylase secretion from dispersed acini prepared from guinea-pig pancreas. Replacement of hydrogen with fluorine in position 5 or 6 on the indole ring of the tryptophan residue did not alter the potency with which the peptide stimulated amylase secretion; however, replacement of hydrogen by fluorine in positions 4, 5, 6 and 7 of the indole ring, or modifying or replacing the indole nitrogen caused a 30- to 300-fold decrease in potency. Changes in the ability of the peptide to inhibit binding of 125I-labeled cholecystokinin. Our findings indicate that reducing the ability of the tryptophan residue to donate electrons produced a greater decrease in the affinity of the peptide for cholecystokinin receptors than did abolishing the ability of tryptophan to form hydrogen bonds, and modification that altered both abilities caused a greater decrease in affinity than did modification of only one ability. Finally, in the tryptophan residues of cholecystokinin octapeptide, tetrafluorination of the indole ring or replacing the indole nitrogen by oxygen reduced the ability of the peptide to cause residual stimulation of enzyme secretion, probably by accelerating the rate at which bound peptide dissociated from its receptors when the acini were washed and resuspended in fresh incubation solution.

The role of 152Val of the fibrinogen Aα-chain in the fibrin-induced rate enhancement of the plasminogen activation by t-PA

Abstract Fibrin enhances the rate of plasminogen activation by t-PA. We have described a site in fibrin(ogen), i.e. Aα-(148–160), which can mimic part of the rate enhancement induced by fibrin. During the fibrinogen-to-fibrin conversion, Aα-(148–160) appears to become accessible to proteins, since monoclonal antibodies against synthetic Aα-(148–160) react with fibrin, but not with fibrinogen. In previous publications we have reported on the role of position Aα-157. In this study we investigated the role of 152 Val on the rate-enhancing effect of Aα-(148–160). 152 Val was replaced by charged residues (Arg, Lys, Glu). Also incorporated were some uncharged polar residues (Ser, Tyr), uncharged nonpolar residues (Ala, Nle), and Gly and Pro. The results clearly indicate that, to maintain stimulatory activity, Val (which possesses an uncharged nonpolar side chain) at position Aα-152 may be exchanged by another uncharged nonpolar residue, e.g. Ala or Me or polar residues e.g. Tyr (with its aromatic, though polar side chain) or Ser. With the amino acids Gly, Glu, Arg, Lys or Pro at position Aα-152 the peptide becomes virtually inactive. Our results indicate that the amino acid residue at position Aα-152 is important for (inducibility of) a three-dimensional structure in Aα-(148–160) which is required for stimulatory activity of the peptide.

Synthesis and Properties of L-α-aming-γ-nitroguanidinobutyric acid

Abstract We wish to report the synthesis of L-α-amino-γ-nitroguanidinobutyric acid, a hitherto unkonwn amino acid derivative. This derivative can be the base for preparing peptides (for instance hormonse) contairing Lα-amono-γ-guanidinobutyric acid, the lower homologue of L-arginine. The higher homologue, L-homoarginine, has already been built in peptides through its nitroderivative, as was the case with angiotensin IIanalogues1and Har-Bradykinin2.