JP6343604B2 - BCN基を含むアミノ酸をコードする直交性コドンと、直交性PylRS合成酵素とを使用して、BCN基を含むアミノ酸をポリペプチドに取り込む方法 - Google Patents
BCN基を含むアミノ酸をコードする直交性コドンと、直交性PylRS合成酵素とを使用して、BCN基を含むアミノ酸をポリペプチドに取り込む方法 Download PDFInfo
- Publication number
- JP6343604B2 JP6343604B2 JP2015512124A JP2015512124A JP6343604B2 JP 6343604 B2 JP6343604 B2 JP 6343604B2 JP 2015512124 A JP2015512124 A JP 2015512124A JP 2015512124 A JP2015512124 A JP 2015512124A JP 6343604 B2 JP6343604 B2 JP 6343604B2
- Authority
- JP
- Japan
- Prior art keywords
- bcn
- amino acid
- tetrazine
- polypeptide
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/13—Labelling of peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
- C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
- C12Y601/01026—Pyrrolysine-tRNAPyl ligase (6.1.1.26)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2602/00—Systems containing two condensed rings
- C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
- C07C2602/14—All rings being cycloaliphatic
- C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB1208875.3A GB201208875D0 (en) | 2012-05-18 | 2012-05-18 | Methods |
| GB1208875.3 | 2012-05-18 | ||
| GB1210303.2 | 2012-06-08 | ||
| GBGB1210303.2A GB201210303D0 (en) | 2012-06-08 | 2012-06-08 | Polypeptides and methods |
| PCT/GB2013/051249 WO2013171485A1 (en) | 2012-05-18 | 2013-05-15 | Methods of incorporating an amino acid comprising a bcn group into a polypeptide using an orthogonal codon encoding it and an orthorgonal pylrs synthase. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2015523965A JP2015523965A (ja) | 2015-08-20 |
| JP6343604B2 true JP6343604B2 (ja) | 2018-06-13 |
Family
ID=48748281
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2015512124A Active JP6343604B2 (ja) | 2012-05-18 | 2013-05-15 | BCN基を含むアミノ酸をコードする直交性コドンと、直交性PylRS合成酵素とを使用して、BCN基を含むアミノ酸をポリペプチドに取り込む方法 |
Country Status (8)
| Country | Link |
|---|---|
| US (3) | US20150148525A1 (esLanguage) |
| EP (1) | EP2852664B1 (esLanguage) |
| JP (1) | JP6343604B2 (esLanguage) |
| CN (2) | CN108410827B (esLanguage) |
| BR (1) | BR112014028588B1 (esLanguage) |
| CA (1) | CA2873793C (esLanguage) |
| IN (1) | IN2014DN09922A (esLanguage) |
| WO (1) | WO2013171485A1 (esLanguage) |
Families Citing this family (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2852664B1 (en) * | 2012-05-18 | 2017-09-06 | Medical Research Council | Methods of incorporating an amino acid comprising a bcn group into a polypeptide using an orthogonal codon encoding it and an orthorgonal pylrs synthase. |
| CN104744293B (zh) * | 2013-12-31 | 2016-08-17 | 深圳先进技术研究院 | 胆碱类似物及其制备方法和应用、由染料分子标记的四嗪探针及其制备方法和应用 |
| GB2528227A (en) | 2014-03-14 | 2016-01-20 | Medical Res Council | Cyclopropene amino acids and methods |
| US12091391B2 (en) | 2015-04-30 | 2024-09-17 | Oregon State University | Reagents and methods for bioorthogonal labeling of biomolecules in living cells |
| JP6967221B2 (ja) * | 2015-08-19 | 2021-11-17 | 国立研究開発法人理化学研究所 | 非天然アミノ酸導入抗体 |
| CN105254655B (zh) * | 2015-11-20 | 2017-03-22 | 江汉大学 | 一种基于bodipy的荧光氨基酸及其合成方法与应用 |
| CN106565762B (zh) * | 2016-11-17 | 2018-10-23 | 陕西师范大学 | 一种原位产生近红外荧光的bodipy类染料及其制备和应用 |
| JP7216006B2 (ja) | 2017-03-22 | 2023-01-31 | ジェネンテック, インコーポレイテッド | ヒドロゲル架橋ヒアルロン酸プロドラッグ組成物及び方法 |
| JOP20190245A1 (ar) | 2017-04-20 | 2019-10-15 | Novartis Ag | أنظمة توصيل إطلاق مستدام تتضمن روابط بلا أثر لنقطة الربط |
| US11560384B2 (en) | 2017-05-04 | 2023-01-24 | University Of Utah Research Foundation | Benzonorbornadiene derivatives and reactions thereof |
| AR111963A1 (es) * | 2017-05-26 | 2019-09-04 | Univ California | Método y moléculas |
| TW202027794A (zh) | 2018-10-03 | 2020-08-01 | 瑞士商諾華公司 | 血管生成素樣3多肽之持續遞送 |
| GB201817444D0 (en) | 2018-10-26 | 2018-12-12 | Res & Innovation Uk | Methods and compositions |
| CN111849929B (zh) * | 2019-04-30 | 2021-05-11 | 苏州鲲鹏生物技术有限公司 | 高效引入赖氨酸衍生物的氨酰基—tRNA合成酶 |
| GB201906775D0 (en) | 2019-05-14 | 2019-06-26 | Res & Innovation Uk | Synthetic genome |
| IL267744A (en) * | 2019-06-30 | 2019-08-29 | Nat Inst Biotechnology Negev Ltd | Labelling molecules and methods of use thereof |
| CN115701451B (zh) * | 2021-08-02 | 2023-08-01 | 宁波鲲鹏生物科技有限公司 | 高效引入赖氨酸衍生物的氨酰基-tRNA合成酶及其应用 |
| EP4399283A2 (en) * | 2021-09-06 | 2024-07-17 | Veraxa Biotech GmbH | Novel aminoacyl-trna synthetase variants for genetic code expansion in eukaryotes |
| JP2026519275A (ja) | 2023-04-28 | 2026-06-15 | ユナイテッド キングダム リサーチ アンド イノベーション | tRNAに基づく方法及び関連組成物 |
| GB202307998D0 (en) | 2023-05-29 | 2023-07-12 | Res & Innovation Uk | Aminoacyl-irna synthetase sets |
| CN119613545B (zh) * | 2024-12-16 | 2025-09-16 | 天津大学 | 非天然氨基酸修饰的抗ccl5纳米抗体 |
Family Cites Families (33)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040029129A1 (en) * | 2001-10-25 | 2004-02-12 | Liangsu Wang | Identification of essential genes in microorganisms |
| JP5275566B2 (ja) | 2003-06-18 | 2013-08-28 | ザ スクリプス リサーチ インスティチュート | 反応性非天然アミノ酸遺伝コード付加 |
| WO2005019415A2 (en) * | 2003-07-07 | 2005-03-03 | The Scripps Research Institute | Compositions of orthogonal lysyl-trna and aminoacyl-trna synthetase pairs and uses thereof |
| CA2549830A1 (en) | 2003-12-18 | 2006-01-05 | The Scripps Research Institute | Selective incorporation of 5-hyroxytryptophan into proteins in mammalian cells |
| MX2007003142A (es) | 2004-09-21 | 2007-07-04 | Scripps Research Inst | Incorporacion in vivo de alquinil aminoacidos a proteinas en eubacterias. |
| MX2007004892A (es) | 2004-10-27 | 2007-08-16 | Scripps Research Inst | Componentes de traduccion ortogonales para la incorporacion in vivo de aminoacidos no naturales. |
| US8431558B2 (en) * | 2004-11-01 | 2013-04-30 | The Regents Of The University Of California | Compositions and methods for modification of biomolecules |
| JP2007037445A (ja) | 2005-08-02 | 2007-02-15 | Institute Of Physical & Chemical Research | tRNA合成方法、核酸、アミノアシルtRNA合成方法及び非天然型アミノ酸組み込み蛋白質の製造方法 |
| WO2007090198A2 (en) | 2006-02-01 | 2007-08-09 | Encode Bio, Inc. | Fluorescent assays using orthogonal trna - aminoacyl synthetase pairs |
| US20080096819A1 (en) | 2006-05-02 | 2008-04-24 | Allozyne, Inc. | Amino acid substituted molecules |
| US20090110662A1 (en) | 2007-04-30 | 2009-04-30 | Intezyne Technologies, Inc. | Modification of biological targeting groups for the treatment of cancer |
| CN101952428B (zh) * | 2007-09-20 | 2013-10-23 | 独立行政法人理化学研究所 | 突变体吡咯赖氨酰-tRNA合成酶以及使用该酶的非天然氨基酸整合蛋白质的制造方法 |
| GB0721291D0 (en) * | 2007-10-30 | 2007-12-12 | Medical Res Council | Methods and compositions |
| KR20110118176A (ko) * | 2009-02-25 | 2011-10-28 | 빅텍 프라이빗 리미티드 | 치쿤군야의 검출을 위한 프로브 및 프라이머 |
| GB2470770A (en) | 2009-06-04 | 2010-12-08 | Medical Res Council | Incorporation of unnatural amino acids having an orthogonal functional group into polypeptides using orthogonal tRNA/tRNA synthetase pairs |
| GB0917240D0 (en) * | 2009-10-01 | 2009-11-18 | Medical Res Council | Incorporation of methyl lysine into poiypeptide |
| GB0917242D0 (en) * | 2009-10-01 | 2009-11-18 | Medical Res Council | Acetyl lysine incorporation with tRNA synthetase |
| MX368648B (es) | 2010-04-15 | 2019-10-09 | Seattle Genetics Inc | Pirrolobenzodiazepinas usadas para tratar enfermedades proliferativas. |
| US8859629B2 (en) * | 2010-04-27 | 2014-10-14 | Synaffix B.V. | Fused cyclooctyne compounds and their use in metal-free click reactions |
| US20120077186A1 (en) | 2010-05-07 | 2012-03-29 | Oregon Health & Science University | Use of cysteine-derived suppressor trnas for non-native amino acid incorporation |
| WO2011156686A2 (en) | 2010-06-11 | 2011-12-15 | The Regents Of The University Of Colorado, A Body Corporate | Method for synthesizing a cyclic multivalent peptide using a thiol-mediated reaction |
| ES2661095T3 (es) | 2011-02-03 | 2018-03-27 | European Molecular Biology Laboratory | Aminoácidos no naturales que comprenden un grupo análogo de ciclooctinilo o trans-ciclooctenilo y usos de los mismos |
| GB201110815D0 (en) * | 2011-06-24 | 2011-08-10 | Medical Res Council | Incorporation of subtituted lysines into polypeptides |
| US20130066063A1 (en) * | 2011-09-09 | 2013-03-14 | John Cooke Hodges | Bicyclo[6.1.0]non-4-yne regents for chemical modification of oligonucleotides |
| GB201201100D0 (en) | 2012-01-20 | 2012-03-07 | Medical Res Council | Polypeptides and methods |
| EP2852664B1 (en) | 2012-05-18 | 2017-09-06 | Medical Research Council | Methods of incorporating an amino acid comprising a bcn group into a polypeptide using an orthogonal codon encoding it and an orthorgonal pylrs synthase. |
| SI2890402T1 (sl) | 2012-08-31 | 2019-07-31 | Sutro Biopharma, Inc. | Modificirane aminokisline vsebujoče acido skupino |
| US9670521B2 (en) | 2012-09-24 | 2017-06-06 | Medimmune Limited | Amino acid derivatives |
| GB2528227A (en) | 2014-03-14 | 2016-01-20 | Medical Res Council | Cyclopropene amino acids and methods |
| GB201419109D0 (en) | 2014-10-27 | 2014-12-10 | Medical Res Council | Incorporation of unnatural amino acids into proteins |
| US20180171321A1 (en) * | 2015-03-27 | 2018-06-21 | The University Of Queensland | Platform for a non-natural amino acid incorporation into proteins |
| GB201817444D0 (en) | 2018-10-26 | 2018-12-12 | Res & Innovation Uk | Methods and compositions |
| EP3868882A1 (en) * | 2020-02-21 | 2021-08-25 | European Molecular Biology Laboratory | Archaeal pyrrolysyl trna synthetases for orthogonal use |
-
2013
- 2013-05-15 EP EP13734818.1A patent/EP2852664B1/en active Active
- 2013-05-15 IN IN9922DEN2014 patent/IN2014DN09922A/en unknown
- 2013-05-15 US US14/401,803 patent/US20150148525A1/en not_active Abandoned
- 2013-05-15 CN CN201810228028.9A patent/CN108410827B/zh active Active
- 2013-05-15 BR BR112014028588-8A patent/BR112014028588B1/pt active IP Right Grant
- 2013-05-15 WO PCT/GB2013/051249 patent/WO2013171485A1/en not_active Ceased
- 2013-05-15 JP JP2015512124A patent/JP6343604B2/ja active Active
- 2013-05-15 CN CN201380037290.0A patent/CN104718285A/zh active Pending
- 2013-05-15 CA CA2873793A patent/CA2873793C/en active Active
-
2020
- 2020-05-12 US US16/872,826 patent/US11732001B2/en active Active
-
2023
- 2023-05-16 US US18/198,205 patent/US20230340013A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| IN2014DN09922A (esLanguage) | 2015-08-14 |
| BR112014028588B1 (pt) | 2023-03-21 |
| EP2852664A1 (en) | 2015-04-01 |
| CA2873793C (en) | 2022-02-22 |
| US20230340013A1 (en) | 2023-10-26 |
| US20150148525A1 (en) | 2015-05-28 |
| WO2013171485A1 (en) | 2013-11-21 |
| CA2873793A1 (en) | 2013-11-21 |
| CN108410827A (zh) | 2018-08-17 |
| EP2852664B1 (en) | 2017-09-06 |
| HK1208704A1 (en) | 2016-03-11 |
| US11732001B2 (en) | 2023-08-22 |
| BR112014028588A8 (pt) | 2021-06-29 |
| BR112014028588A2 (pt) | 2017-06-27 |
| CN108410827B (zh) | 2023-05-09 |
| JP2015523965A (ja) | 2015-08-20 |
| CN104718285A (zh) | 2015-06-17 |
| US20200271658A1 (en) | 2020-08-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11732001B2 (en) | Methods of incorporating an amino acid comprising a BCN group into a polypeptide using an orthogonal codon encoding it and an orthogonal pylrs synthase | |
| US9968690B2 (en) | Norbornene modified peptides and their labelling with tetrazine compounds | |
| Miyake-Stoner et al. | Generating permissive site-specific unnatural aminoacyl-tRNA synthetases | |
| US20170152287A1 (en) | Methods and compositions for site-specific labeling of peptides and proteins | |
| WO2013148189A1 (en) | Probe incorporation mediated by enzymes | |
| US12091391B2 (en) | Reagents and methods for bioorthogonal labeling of biomolecules in living cells | |
| US10774039B2 (en) | Cyclopropene amino acids and methods | |
| Aphicho et al. | Visualizing the complexity of proteins in living cells with genetic code expansion | |
| HK1208704B (en) | Methods of incorporating an amino acid comprising a bcn group into a polypeptide using an orthogonal codon encoding it and an orthorgonal pylrs synthase | |
| US20250243238A1 (en) | Tetrazine amino acids and methods for their production and use | |
| EP4725952A1 (en) | Compounds comprising non-canonical amino acids and uses thereof | |
| Scinto | Dual-reactivity trans-cyclooctenol probes for sulfenylation in live cells and affinity bioorthogonal chemistry tags for site-selective synthesis of protein-protein and protein-material conjugates | |
| CA2941069C (en) | Cyclopropene amino acids and methods | |
| Michels | Precision crosslinking and active-site modifications via site-specific protein engineering | |
| Galbada Liyanage | Synthesis and the Genetic Encoding of Novel Lysine Post-Translational Modifications | |
| Lee | Expanding the Genetic Code for Synthesis of Proteins with Native Biological Modifications and Novel Chemical, Biophysical Probes | |
| Chen | Adding new chemistry to proteins via genetic incorporation | |
| HK1231504B (en) | Cyclopropene amino acids and methods |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20160229 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20161219 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20170309 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20170517 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20171003 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20171214 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20180426 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20180521 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6343604 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |