Syntese av 1,2-Azaborines og utarbeidelse av deres protein komplekser med T4 Lysozyme Mutants
Summary
A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.
Abstract
We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.
Introduction
Boron-nitrogenholdig heterosykler (dvs. 1,2-azaborines) har nylig trukket betydelig oppmerksomhet som isosterer av Arenes. Dette isosterism kan føre til spredning av de eksisterende strukturelle motiver for å utvide den kjemiske rommet 2, 3, 4. Azaborines har potensiell anvendelse for anvendelse i biomedisinsk forskning 5, 6, 7, 8, spesielt i området medisinsk kjemi som kjemikere gjennomføre syntese av biblioteker av strukturelt og funksjonelt relevante molekyler. Signifikant, men mens det finnes mange godt utviklede synteseveier til tilgjengelige Arene-inneholdende molekyler, har bare et begrenset antall fremgangsmåter for syntese av azaborines blitt rapportert 9, 10, </sopp> 11, 12, 13. Dette er hovedsakelig på grunn av et begrenset antall alternativer for bor kilden og luft- og fukt sensitiv karakter av molekylet i den tidlige fasen av syntetisk sekvens.
I den første delen av denne artikkelen vil vi beskrive en fler gram skala syntese av N -TBS- B-Cl-1,2-azaborine (3) ved anvendelse av standard luftfrie teknikker. Denne forbindelsen virker som et allsidig mellomprodukt som kan bli ytterligere funksjonalisert for å strukturelt mer komplekse molekyler, 14 15. Å starte fra 3, vil syntese og rensing av N -H- B-etyl-1,2-azaborine (5) for bruk i proteinbindingsstudier beskrives. På grunn av volatiliteten av 5, krever sin effektiv isolering nøyaktig kontroll av reaksjonstemperatur, tid og distillation forhold.
I den andre delen, protokoller for protein ekspresjon og isolering av T4 lysozym mutanter (L99A og L99A / M102Q) 17, 18, 19, 20 vil bli presentert, etterfulgt av krystallisering protein og fremstilling av protein-ligand-krystall-komplekser. T4 lysozym mutanter L99A og L99A / M102Q ble valgt som biologiske modellsystemer for å undersøke hydrogenbinding evnen til NH inneholder azaborine molekyler 17. Ved hjelp av en standard molekylærbiologi-protokollen, er proteinet uttrykt i Escherichia coli RR1-stamme og indusert med isopropyl-β-D-1-tiogalaktopyranosid (IPTG). Proteinrensing utføres ved anvendelse av ionebytte-kolonnekromatografi. Protein krystallisering utføres med sterkt konsentrert renset proteinløsning (> 95% renhet ved gelelektroforese) ved hjelp av opphengsslippe damp diffusjon metode. På grunn av følsomheten av denne studien er ligander til oksygen, blir protein-ligand-kompleksene fremstilt i henhold til luftfrie betingelser.
Protocol
Representative Results
Discussion
I den første delen av denne protokollen, beskrev vi en modifisert syntese av 1,2-azaborines basert på tidligere rapporterte fremgangsmåter 12, 13. Triallylborane 22 ble brukt som en erstatning for de ruter ved hjelp av allyltriphenyl tinn eller kalium allyltrifluoroborate for å fremstille N -allyl- N -TBS- B -allyl kloridadduktet (1). Denne metoden gir en mer atom-økonomisk og miljøvennlig…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported by the National Institutes of Health NIGMS (R01-GM094541) and Boston College.
Materials
| Tetrahydrofuran (THF), inhibitor-free, for HPLC, ≥99.9% | Sigma Aldrich | 34865 | |
| Diethyl ether (Et2O), for HPLC, ≥99.9%, inhibitor-free | Sigma Aldrich | 309966 | |
| Methylene chloride (CH2Cl2), (Stabilized/Certified ACS) | Fisher | D37-20 | |
| Toluene | Fisher | T290-4 | |
| Pentane, HPLC | Fisher | P399-4 | |
| Acetonitrile | Fisher | A21-4 | |
| Calcium hydride (CaH2), reagent grade, 95% | Sigma Aldrich | 208027 | Pyrophoric |
| Palladium on activated carbon (Pd/C), 10 wt% Pd | Strem | 46-1900 | |
| 1.0 M Boron trichloride solution in hexane | Sigma Aldrich | 211249 | Highly toxic/ Pyrophoric |
| Triethylamine, ≥99.5% | Sigma Aldrich | 471283 | |
| Grubbs 1st generation catalyst | materia | C823 | |
| Acetamide | Sigma Aldrich | A0500 | |
| n-Butanol, anhydrous, 99.8% | Sigma Aldrich | 281549 | |
| Ethyllithium solution, 0.5 M in benzene/cyclohexane | Sigma Aldrich | 561452 | Highly toxic/ Pyrophoric |
| HCl solution, 2.0 M in Et2O | Sigma Aldrich | 455180 | |
| 2-Methylbutane, anhydrous, ≥99% | Sigma Aldrich | 277258 | |
| Escherichia coli, (Migula) Castellani and Chalmers (ATCC® 31343™) | ATCC | 31343 | |
| T4 lysozyme WT* (L99A) | Addgene | 18476 | |
| T4 lysozyme mutant (S38D L99A M102Q N144D) | Addgene | 18477 | |
| Ampicillin sodium salt | Sigma Aldrich | A0166 | |
| isopropyl-β-D-1-thiogalactopyranoside (IPTG) | Invitrogen | AM9464 | |
| Sodium phosphate monobasic anhydrous | Fisher | BP329 | |
| Sodium Phosphate dibasic anhydrous | Fisher | BP332 | |
| Sodium chloride | Fisher | S642212 | |
| Ethylenediaminetetraacetic acid | Fisher | BP118 | |
| Magnesium chloride | Sigma Aldrich | M4880 | Corrosive |
| Thermo scientific pierce DNaseI | Fisher | PI-90083 | |
| GE Healthcare Sepharose Fast Flow Cation Exchange Media | Fisher | 45-002-931 | |
| Tris-base | Fisher | BP152-500 | |
| Sodium azide | TCI | S0489 | Highly toxic |
| 2-Mercaptoethanol | Fisher | ICN806443 | |
| Sartorius Vivaspin 20 Centrifugal Concentrators | Fisher | 14-558-501 | |
| Potassium phosphate monobasic | Sigma Aldrich | P5379 | |
| 2-Hydroxyethyl disulfide | Sigma Aldrich | 380474 | |
| N-paratone | Hampton Research | HR2-643 | |
| 4 RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO | Fisher | 08-667E | |
| CryoLoop | Hampton Research | cryogenic tubing shaped into a loop | |
| CryoTong | Thermo Fisher | cryogenic tong | |
| Coot | Electron density images are generated from the software |
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