Recent advances in racemic protein crystallography

Yan, Bingjia; Ye, Linzhi; Xu, Weiliang; Liu, Lei

doi:10.1016/j.bmc.2017.05.020

“…(5a, 6a, 8, and 10). The relative integrated peak area of ligated products are shown at various time points relative to TCEP addition (1,20,50,110,230,350,470,1430, and 2870 min). CAN ligations were fit to first-order rate constants.…”

Section: Methodsmentioning

confidence: 99%

“…NCL has been extended beyond Cys junctions via free-radical desulfurization, a robust technique that allows the more common Ala, as well as other amino acids amenable to thiol surrogates, to be used as ligation junctions. [4][5] NCL has produced many useful synthetic proteins, [6][7] including targets for mirror-image drug discovery, [8][9][10][11][12] functional native and mirror-image enzymes, [13][14][15][16][17][18] mirror-image proteins for racemic crystallography, [19][20][21][22] and proteins containing complex post-translational modifications. [23][24][25][26] However, NCL has several challenges that limit its scope in chemical protein synthesis (CPS).…”

Section: Introductionmentioning

confidence: 99%

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Erickson¹,

Fulcher²,

Kay³

2020

Preprint

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<div><div><div><p>Chemoselective ligation reactions, such as native chemical ligation (NCL), enable the assembly of synthetic peptides into proteins. However, the scope of proteins accessible to total chemical synthesis is limited by ligation efficiency. Sterically hindered thioesters and poorly soluble peptides can undergo incomplete ligations, leading to challenging purifications with low yields. This work describes a new method, ClickAssisted NCL (CAN), which overcomes these barriers. In CAN, peptides are modified with traceless “helping hand” lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles for strain-promoted alkyne-azide cycloaddition (SPAAC) reactions. This cycloaddition templates the peptides to increase their effective concentration and greatly accelerate ligation kinetics. After ligation, mild hydroxylamine treatment tracelessly removes the linkers to afford the native ligated peptide. Although DBCO is incompatible with standard Fmoc solid-phase peptide synthesis (SPPS) due to an acid-mediated rearrangement that occurs during peptide cleavage, we demonstrate that copper(I) protects DBCO from this side reaction, enabling direct production of DBCO-containing synthetic peptides. Excitingly, low concentrations of triazole-linked model peptides reacted ~1,200-fold faster than predicted for non-templated control ligations, which also accumulated many side products due to the long reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that the SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in a one-pot fashion. CAN will be useful for overcoming ligation challenges to expand the reach of chemical protein synthesis.</p></div></div></div>

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“…NCL or CAN was performed using the STEVE and KENT model peptides (5a, 6a, 8, and 10). The relative integrated peak area of ligated products are shown at various time points relative to TCEP addition (1,20,50,110,230,350,470,1430, and 2870 min). CAN ligations were fit to first-order rate constants.…”

Section: Figurementioning

confidence: 99%

“…NCL has been extended beyond Cys junctions via free-radical desulfurization, a robust technique that allows the more common Ala, as well as other amino acids amenable to thiol surrogates, to be used as ligation junctions. [4][5] NCL has produced many useful synthetic proteins, [6][7] including targets for mirror-image drug discovery, [8][9][10][11][12] functional native and mirror-image enzymes, [13][14][15][16][17][18] mirror-image proteins for racemic crystallography, [19][20][21][22] and proteins containing complex post-translational modifications. [23][24][25][26] However, NCL has several challenges that limit its scope in chemical protein synthesis (CPS).…”

Section: Introductionmentioning

confidence: 99%

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Erickson¹,

Fulcher²,

Kay

³

2020

Preprint

View full text Add to dashboard Cite

<div><div><div><p>Chemoselective ligation reactions, such as native chemical ligation (NCL), enable the assembly of synthetic peptides into proteins. However, the scope of proteins accessible to total chemical synthesis is limited by ligation efficiency. Sterically hindered thioesters and poorly soluble peptides can undergo incomplete ligations, leading to challenging purifications with low yields. This work describes a new method, ClickAssisted NCL (CAN), which overcomes these barriers. In CAN, peptides are modified with traceless “helping hand” lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles for strain-promoted alkyne-azide cycloaddition (SPAAC) reactions. This cycloaddition templates the peptides to increase their effective concentration and greatly accelerate ligation kinetics. After ligation, mild hydroxylamine treatment tracelessly removes the linkers to afford the native ligated peptide. Although DBCO is incompatible with standard Fmoc solid-phase peptide synthesis (SPPS) due to an acid-mediated rearrangement that occurs during peptide cleavage, we demonstrate that copper(I) protects DBCO from this side reaction, enabling direct production of DBCO-containing synthetic peptides. Excitingly, low concentrations of triazole-linked model peptides reacted ~1,200-fold faster than predicted for non-templated control ligations, which also accumulated many side products due to the long reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that the SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in a one-pot fashion. CAN will be useful for overcoming ligation challenges to expand the reach of chemical protein synthesis.</p></div></div></div>

show abstract

“…(1) With full atomistic control, the chemical route offers access to proteins that are otherwise difficult to prepare via the recombinant route. For example, enantiomeric proteins can be prepared using D-amino acids as building blocks, (2,3) and their preparation has facilitated studies that generate critical structural (4) and mechanistic (5) insights. To this end, synthetic routes to many potent antibacterial peptides need to be established.…”

Section: Introductionmentioning

confidence: 99%

Total Chemical Synthesis of Aureocin A53, Lacticin Q and Their Enantiomeric Counterparts

Lander¹,

Li²,

Jiang³

et al. 2020

Preprint

1

0

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<div> <p><a>Aureocin A53 (AucA) and lacticin Q (LnqQ) are class IId bacteriocins that display broad-spectrum activity against Gram-positive bacteria in the nanomolar range, of which their modes of action is unclear and their synthesis has not been reported. Here, we reported the chemical synthetic routes of AucA and LnqQ, enabled through the technique of native chemical ligation. To demonstrate the versatility of the syntheses, we prepared their enantiomeric counterparts, </a>D-AucA and D-LnqQ, comprised of entirely D-amino acids. X-ray crystal structure of AucA obtained through racemic protein crystallography at 1.13 Å indicates unique Lys-Trp-sulfate network. This work lays foundations for gaining further mechanistic insights into these potent bacteriocins through total chemical synthesis. </p> </div> <br>

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Recent advances in racemic protein crystallography

Cited by 23 publications

References 219 publications

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Total Chemical Synthesis of Aureocin A53, Lacticin Q and Their Enantiomeric Counterparts

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