US4800166A
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Method and apparatus for monitoring the automated synthesis of peptides
Assignee: APPLIED PROTEIN TECHNOLOGIES Inc 103 BROOKLINE STREET CAMBRIDGE MASSACHUSETTS 02139
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Filing: 1986-06-06
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Granted: 1989-01-24
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Expires: 2006-06-06
Abstract
Abstract A method for monitoring a solid-phase peptide synthesis for free amino ends of unreacted peptide chains, the monitoring occurring at the end of each addition of a blocked amino acid to the peptide chains which are anchored to the solid phase. The method includes reacting the solid phase with a monitoring agent that forms a covalent bond with unblocked amino groups. The solid phase is then washed to remove the unreacted monitoring agent. A cleaving reagent is then used to selectively remove the covalently bound monitoring reagent from the ends of the unblocked peptide chains, while leaving the blocked chains intact. The amount of monitoring agent thus removed is then quantitatively measured to determine what proportion of the initial peptide chains failed to react with the blocked amino acid. Preferred monitoring agents for use in this process include trityl (triphenylmethyl) -based groups, and particularly preferred agents are trityl, monomethoxytrityl, dimethoxytrityl, and trimethoxytrityl chlorides.
Inventors
- 👤 Marcus J. Horn
- 👤 Joanne Recchia
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 04:03
CPC Classifications
G; G01; G01N; G01N1/00; G01N1/28; G01N1/30; G01N1/31; B; B01; B01J; B01J19/00; B01J19/0046; C; C07; C07K; C07K1/00; C07K1/04; C07K1/045; G01N33/00; G01N33/48; G01N33/50; G01N33/68; G01N33/6803; G01N33/6818; B01J2219/00; B01J2219/00274; B01J2219/00277; B01J2219/00279; B01J2219/00281; B01J2219/00283; B01J2219/00351; B01J2219/00353; B01J2219/00414; B01J2219/00416; B01J2219/00418; B01J2219/00423; B01J2219/00479; B01J2219/00488; B01J2219/00497; B01J2219/005; B01J2219/00583; B01J2219/0059; B01J2219/00596; B01J2219/0068; B01J2219/00686; B01J2219/00718; B01J2219/0072; B01J2219/00725; C40; C40B; C40B40/00; C40B40/04; C40B40/10; C40B60/00; C40B60/14; Y; Y10; Y10T; Y10T436/00; Y10T436/12
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
193 words
A process for monitoring a solid-phase peptide synthesis for free amino ends of unreacted peptide chains, comprising: A. synthesizing a peptide chain by adding a blocked amino acid, comprising an amino acid bound to a blocking agent, to a peptide chain that is anchored to the solid-phase; B. reacting the solid phase with a monitoring agent that forms a covalent bond with amino groups under reaction conditions that promote such covalent bonding. C. washing the solid phase with a washing solution to remove non-covalently bound monitoring agent from the solid phase; D. reacting the solid phase with a cleaving agent that selectively removes the covalently bound monitoring agent from the amino ends of the unreacted peptide chains while leaving the blocked amino termini and the peptide chains intact, said monitoring agent not being the same as or similar to said blocking agent of the first step, such that said cleaving agent will cleave said monitoring agent but not said blocking agent; E. measuring quantitatively the amount of the monitoring agent removed; and F. depending upon the number of unreacted peptide chains, as determined by the quantitative measurement of said removed monitoring agent, either
Claim 2
Dependent Claim
193 words
A process for monitoring a solid-phase peptide synthesis for free amino ends of unreacted peptide chains, comprising: A. synthesizing a peptide chain by adding a blocked amino acid, comprising an amino acid bound to a blocking agent, to a peptide chain that is anchored to the solid-phase; B. reacting the solid phase with a monitoring agent that forms a covalent bond with amino groups under reaction conditions that promote such covalent bonding. C. washing the solid phase with a washing solution to remove non-covalently bound monitoring agent from the solid phase; D. reacting the solid phase with a cleaving agent that selectively removes the covalently bound monitoring agent from the amino ends of the unreacted peptide chains while leaving the blocked amino termini and the peptide chains intact, said monitoring agent not being the same as or similar to said blocking agent of the first step, such that said cleaving agent will cleave said monitoring agent but not said blocking agent; E. measuring quantitatively the amount of the monitoring agent removed; and F. depending upon the number of unreacted peptide chains, as determined by the quantitative measurement of said removed monitoring agent, either
Claim 3
Dependent Claim
34 words
repeating the process with the same blocked amino acid or; 2. removing the blocking agent from the terminal amino group and repeating the process with a subsequent blocked amino acid in the peptide sequence.
Claim 4
Dependent Claim
34 words
repeating the process with the same blocked amino acid or; 2. removing the blocking agent from the terminal amino group and repeating the process with a subsequent blocked amino acid in the peptide sequence.
Claim 5
Dependent Claim
16 words
The method according to claim 1 wherein the monitoring agent is a trityl (triphenylmethyl) -based groups.
Claim 6
Dependent Claim
18 words
The method according to claim 2 wherein the trityl-based group is a trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl group.
Claim 7
Dependent Claim
30 words
The method according to claim 2 wherein the trityl-based group is generated from a trityl-based compound selected from the group consisting of trityl-based halides, trityl-based carboxylates, and trityl-based acid anions.
Claim 8
Dependent Claim
31 words
The method according to claim 2 wherein the trityl-based group is generated from a trityl-based compound selected from the group consisting of trityl chloride, monomethoxytrityl chloride, dimethoxytrityl chloride, and trimethoxytrityl chloride.
Claim 9
Dependent Claim
25 words
The method according to claim 3 wherein the monitoring agent is dissolved in a basic reaction solution and the cleaving reagent is an acidic solution.
Claim 10
Dependent Claim
20 words
The method according to claim 6 wherein the basic reaction solution comprises diisopropylethylamine; and the cleaving reagent comprises trichloroacetic acid.
Claim 11
Dependent Claim
42 words
The method according to claim 1 wherein A. the monitoring agent is dimethoxytrityl chloride dissolved in a reaction solution comprising diisopropylethylamine; B. the washing solution comprises hydroxybenzotriazole and trifluoroethanol; and C. the cleaving reagent comprises trichloroacetic acid and a carbonium ion scavenger.
Claim 12
Dependent Claim
13 words
The method according to claim 8 wherein the carbonium ion scavenger is anisole.
Claim 13
Dependent Claim
42 words
The method according to claim 1 wherein A. the monitoring agent is monomethoxytrityl chloride dissolved in a reaction solution comprising diisopropylethylamine; B. the washing solution comprises hydroxybenzotriazole and trifluoroethanol; and C. the cleaving reagent comprises trichloroacetic acid and a carbonium ion scavenger.
Claim 14
Dependent Claim
13 words
The method according to claim 10 wherein the carbonium ion scavenger is anisole.
Claim 15
Dependent Claim
41 words
The method according to claim 1 wherein A. the monitoring agent is trimethoxytrityl chloride dissolved in a reaction solution comprising diisopropylethylamine; B. the washing solution comprises methylene chloride; and C. the cleaving reagent comprises trichloroacetic acid and a carbonium ion scavenger.
Claim 16
Dependent Claim
13 words
The method according to claim 12 wherein the carbonium ion scavenger is anisole.
Claim 17
Dependent Claim
35 words
The method according to claim 1 wherein A. the monitoring agent is trityl chloride dissolved in a reaction solution comprising diisopropylethylamine; B. the washing solution comprises dimethylformamide; and C. the cleaving reagent comprises trichloroacetic acid.
Claim 18
Dependent Claim
29 words
A process as defined in claim 1 wherein step F.2. further includes the addition of a capping agent prior to removing said blocking agent from the terminal amino group.
Claim 19
Dependent Claim
253 words
A process for monitoring a solid-phase peptide synthesis for free amino ends of unreacted peptide chains, comprising: A. synthesizing a peptide chain by adding a blocked amino acid, comprising an amino acid bound to a blocking agent, to a peptide chain that is anchored to the solid-phase; B. reacting the solid phase with a monitoring agent that forms a covalent bond with amino groups under reaction conditions that promote such covalent bonding. C. washing the solid phase with a washing solution to remove non-covalently bound monitoring agent from the solid phase; D. reacting the solid phase with a cleaving agent that selectively removes the covalently bound monitoring agent from the amino ends of the unreacted peptide chains while leaving the blocked amino termini and the peptide chains intact, said monitoring agent not being the same as or similar to said blocking agent of the first step, such that said cleaving agent will cleave said monitoring agent but not said blocking agent; E. measuring quantitatively the amount of the monitoring agent removed; and F. depending upon the number of unreacted peptide chains, as determined by the quantitative measurement of said removed monitoring agent, either 1. repeating the process with the same blocked amino acid or; 2. removing the blocking agent from the terminal amino group and repeating the process with a subsequent blocked amino acid in the peptide sequence or; 3. adding a capping agent to prevent the subsequent elongation of the unblocked chains and thereby terminate their growth and then performing step F.2.
Claim 20
Dependent Claim
253 words
A process for monitoring a solid-phase peptide synthesis for free amino ends of unreacted peptide chains, comprising: A. synthesizing a peptide chain by adding a blocked amino acid, comprising an amino acid bound to a blocking agent, to a peptide chain that is anchored to the solid-phase; B. reacting the solid phase with a monitoring agent that forms a covalent bond with amino groups under reaction conditions that promote such covalent bonding. C. washing the solid phase with a washing solution to remove non-covalently bound monitoring agent from the solid phase; D. reacting the solid phase with a cleaving agent that selectively removes the covalently bound monitoring agent from the amino ends of the unreacted peptide chains while leaving the blocked amino termini and the peptide chains intact, said monitoring agent not being the same as or similar to said blocking agent of the first step, such that said cleaving agent will cleave said monitoring agent but not said blocking agent; E. measuring quantitatively the amount of the monitoring agent removed; and F. depending upon the number of unreacted peptide chains, as determined by the quantitative measurement of said removed monitoring agent, either 1. repeating the process with the same blocked amino acid or; 2. removing the blocking agent from the terminal amino group and repeating the process with a subsequent blocked amino acid in the peptide sequence or; 3. adding a capping agent to prevent the subsequent elongation of the unblocked chains and thereby terminate their growth and then performing step F.2.
References Cited (Backward Citations)
US3862113A
Cited
US3997516A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-01-24 | 1992-07-25 | 1993-01-24 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-01-24 | 1996-07-25 | 1997-01-24 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-01-24 | 2000-07-25 | 2001-01-24 | $7700.00 | Window Closed / Lapsed |