Other

The Salk Institute Biotechnology Industrial Associates, Inc.

Siska Diagnostics, Inc.

University Patents, Inc.

The Salk Institute for Biological Studies

Emil T Kaiser

Gary F Musso

Soumitra S Ghosh

Geoffrey M Wahl

Leslie E Orgel

Gonul Velicelebi

Gregory R Moe

Nucleic acid hybridization probes are provided which comprise an N.sup.4 -(substituted amino)cytosine moiety, wherein the substituted amino group comprises a tag moiety, whereby the probe is detected. Methods of preparing probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in hybridization assays are also provided. Typical tag moieties employed with the probes of the invention are biotinyl, aminothiadiazole and fluorescein derivatives, connected to N.sup.4 -amino groups of modified cytosines of the probe through linker moieties. Probes tagged with biotin are typically detected by binding to the biotinyl moieties, through a streptavidin or avidin molecule, a reporter group which includes streptavidin or avidin and then detecting a signal due to the reporter group. Probes tagged with aminothiadiazole derivatives are typically detected by essentially the same method as those tagged with biotinyl but employing as reporter group one which binds to the derivative through a carbonic anhydrase molecule. Probes tagged with fluorescein derivatives are detected by a fluorescence spectroscopic method without binding of a reporter group to the tag.

Sulfonamide-derivative compounds for tagging nucleic acid probes

Fluorescent linker moieties are provided which comprise a fluorescent compound such as fluorescein attached to a linker moiety such that a functional group of the linker is available for attachment to an affinity molecule such as a nucleic acid which has an N.sup.4 (substituted amino) cytosine moiety. Probes tagged with fluorescent derivatives such as fluorescein, tetramethyrhodamine or tetraethylrhodamine may be detected by fluorescence spectroscopic methods.

Fluorescent compounds for tagging nucleic acid probes

Novel, biologically active, 28-amino acid analogs of human vasoactive intestinal peptide are provided.

Vasoactive intestinal peptide analogs

Novel biologically active vasoactive intestinal peptide (VIP) analogues are provided.

Compounds for tagging nucleic acid probes

Nucleic acid hybridization probes are provided which comprise nucleoside bases or terminal nucleotide phosphates chemically linked to aromatic sulfonamide inhibitors of carbonic anhydrase. Methods of preparing probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in hybridization assays are also provided. A probe of the invention is detected by binding to it a reporter group, such as a homopolymer or heteropolymer of enzymes, which includes a carbonic anhydrase which binds to the inhibitor linked to the probe, and then detecting the bound reporter group, as by production of a fluorescent or colored product in a reaction catalyzed by an enzyme component of the reporter group. Also provided are enzyme immunoassays wherein detection of antibody is by a process which comprises a chemical reaction catalyzed by a carbonic anhydrase.

Carbonic anhydrase inhibitor-tagged nucleic acid probes

Reaction of a ketoxime-derivatized resin with a strong acid salt of aspartic anhydride or glutamic anhydride yields a novel aspartyl or glutamyl ketoxime ester-derivatized resin, wherein the aspartyl or glutamyl groups are esterified predominantly at the .alpha.-carboxyl group and wherein the aspartyl or glutamyl groups are not covalently protected at the amino group or the carboxyl group that is not esterified. Aminolysis in the presence of a weak acid of the novel aspartyl or glutamyl ketoxime ester-derivatized resin, wherein the aspartyl or glutamyl groups remain as the strong acid salt, with a salt of an amino acid with a base or an amino acid ester yields the corresponding dipeptide or dipeptide ester. After aminoylsis, the ketoxime-derivatized resin can be reused. An advantageous solid-phase method is thus provided for making .alpha.-L-aspartyl dipeptide ester sweeteners, including aspartame, and the immunopotentiating dipeptide, .alpha.-L-glutamyl-L-asparagine.

Process and composition for amino-terminal, .alpha.-aspartyl and

Human pancreatic GRF and the hypothalamic GRFs for the human and several other mammalian species were earlier characterized and synthesized. The invention provides synthetic peptides which are potent stimulators of the release of pituitary GH in animals, including humans, and which have the formula: R.sub.1 -R.sub.2 R.sub.3 -Ala-R.sub.5 -R.sub.6 -R.sub.7 -R.sub.8 -R.sub.9 -R.sub.10 -R.sub.11 -R.sub.12 -R.sub.13 -Leu-R.sub.15 -Gln-Leu-R.sub.18 -R.sub.19 -R.sub.20 -R.sub.21 -Leu-Leu-Gln-Glu-R.sub.26 -R.sub.27 -R.sub.28 -Arg-Y wherein R.sub.1 is Tyr, D-Tyr, Met, Phe, D-Phe, Leu, His or D-His, which has either a C.sup.a Me or N.sup.a Me substitution or is unsubstituted; R.sub.2 is Ala or D-Ala; R.sub.3 is Asp or D-Asp; R.sub.5 is Ile or Leu; R.sub.6 is Phe or Tyr; R.sub.7 is Ser or Thr; R.sub.8 is Ser, Asn, Thr or Gln; R.sub.9 is Ala or Ser; R.sub.10 is Tyr, Phe or Leu; R.sub.11 in Arg, Orn or Lys; R.sub.12 is Arg, Orn or Lys; R.sub.13 is Ile, Leu, Phe or Val; R.sub. 15 is Gly or Ala; R.sub.18 is Ala or Ser; R.sub.19 is Ser or Ala; R.sub.20 is Arg, Orn or Lys; R.sub.21 is Arg, Orn or Lys; R.sub.26 is Leu, Ile, Val or Phe; R.sub.27 is Nle, Nva or a natural amino acid; R.sub.28 is Ala, Leu, Asn, Gln, or Ser; and Y is OH or NH.sub.2 ; provided however that at least four of the residues constituting R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, R.sub.13, R.sub.15, R.sub.18, R.sub.19, R.sub.20, R.sub.21 and R.sub.26 are different from the residues appearing in that respective position in the native molecule. These peptides as well as nontoxic salts thereof may be administered to animals, including humans and cold-booded animals, to stimulate the release of GH and may be used diagnostically.

GRF analogs

Novel peptide hormones with calcitonin-like activity

Reaction of a ketoxime-derivatized resin with a strong acid salt of aspartic anhydride or glutamic anhydride yields a novel aspartyl or glutamyl ketoxime ester-derivatized resin, wherein the aspartyl or glutamyl groups are esterified predominantly at the .alpha.-carboxyl group and wherein the aspartyl or glutamyl groups are not covalently protected at the amino group or the carboxyl group that is not esterified. Aminolysis in the presence of a weak acid of the novel aspartyl or glutamyl ketoxime ester-derivatized resin, wherein the aspartyl or glutamyl groups remain as the strong acid salt, with a salt of an amino acid with a base or an amino acid ester yields the corresponding dipeptide or dipeptide ester. After aminolysis, the ketoxime-derivatized resin can be reused. An advantageous solid-phase method is thus provided for making .alpha.-L-aspartyl dipeptide ester sweeteners, including aspartame, and the immunopotentiating dipeptide, .alpha.-L-glutamyl-L-asparagine.

Compounds of the formula: ##STR1## wherein R.sub.1 is a moiety selected from the group consisting of ##STR2## R.sub.2 -R.sub.22 are amino acid moieties wherein R.sub.2 is an optional moiety which when present is selected from the group consisting of Ser and Gly,

Peptide hormones with calcitonin-like activity

This invention relates to the synthesis of steroids which are useful for their biological activity or which may be converted to steroids which have such activity. These syntheses include the steroid compounds and processes for their preparation. An object of the invention is to provide syntheses which are applicable to materials which are readily available in good supply for converting such materials to steroids which are useful and desirable in the manufacture of pharmaceutical products. More particularly, I have sought to discover processes and intermediate compounds useful in the synthesis of 24, 25-dihydroxycholesterol from hyodeoxycholic acid or lithocholic acid which are constituents of, and readily available from, animal bile.

Synthesis of steroids

This invention relates to the synthesis of steroids which are useful for their biological activity or which may be converted to steroids which have such activity. More particularly, the invention pertains to the synthesis of 24,25-dihydroxycholesterol. It includes intermediate sterols of this synthesis and processes for their preparation.

Synthesis of 24,25-dihydroxycholesterol

The synthesis of 1.alpha.,25-dihydroxycholesterol, 1.alpha.,25-dihydroxy-7-dehydrocholesterol and 1.alpha.,25-dihydroxycholecalciferol and other sterol derivatives from bile acids. Also the synthesis of 3,6-diketo steroids useful in the production of 1.alpha.,25-dihydroxycholesterol and other sterols which are biologically active or can be converted to biologically active sterols. The invention involves the sterols so produced and the processes by which they are prepared.

The synthesis of 25-hydroxycholesterol and 1.alpha.,25-dihydroxycholesterol in which the sterol nucleus of an ester of hyodeoxycholic acid is stabilized by protection of the 3 and 6.alpha.-hydroxyl groups with alkyl groups, alkyl ether groups, preferably with the 3.beta.-methoxyethoxymethyl group or with the heterocyclic 2-tetrahydropyran group, then extending the chain from the carbon at the 24-position to a cyanide group at the 25-position and then subjecting the sterol so formed to a series of reactions by which it is transformed into 1.alpha.,25-dihydroxycholesterol or by a modified series of reactions to 25-hydroxycholesterol.

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New York, NY, United States of America

Emil T Kaiser