328-38-1

Basic information

• Product Name: D-2-Amino-4-methylpentanoic acid
• Synonyms: D-LACTULOSE OCTAACETATE;D-LEUCINE extrapure;(2R)-2-Amino-4-methylpentanoic acid;D-Leucine, 99% 10GR;H-D-Leu-OH (D-Leucine);(2R)-2-Amino-4-methylpentanoic acid, (2R)-2-Amino-4-methylvaleric acid, H-D-Leu-OH;D-Leucine ReagentPlus(R), 99%;D-Leucine Vetec(TM) reagent grade, 97%
• CAS NO: 328-38-1
• Molecular Formula: C₆H₁₃NO₂
• Molecular Weight: 131.17
• EINECS: 206-327-7
• Product Categories: Leucine [Leu, L];Amino Acids and Derivatives;alpha-Amino Acids;Amino Acids;Biochemistry;Amino Acids;amnio acid;amino acid
• Mol File: 328-38-1.mol
• Article Data: 100

Chemical Properties

• Melting Point: >300 °C(lit.)
• Boiling Point: 225.8 ºC at 760 mmHg
• Flash Point: 90.3 ºC
• Appearance: White/Crystalline Powder, Crystals, or Flakes
• Density: 1.2930 (estimate)
• Vapor Pressure: 0.0309mmHg at 25°C
• Refractive Index: -15 ° (C=4, 6mol/L HCl)
• Storage Temp.: -15°C
• Solubility: Aqueous Acid (Sparingly), Water (Slightly)
• PKA: 2.55±0.21(Predicted)
• Water Solubility: 24 g/L (25 ºC)
• BRN: 1721721
• CAS DataBase Reference: D-2-Amino-4-methylpentanoic acid(CAS DataBase Reference)
• NIST Chemistry Reference: D-2-Amino-4-methylpentanoic acid(328-38-1)
• EPA Substance Registry System: D-2-Amino-4-methylpentanoic acid(328-38-1)

Safety Data

• Hazard Codes: Xi
• Statements: 36/37/38
• Safety Statements: 22-24/25-36-26
• WGK Germany: 3
• RTECS: OH2840000
• TSCA: Yes
• Hazardous Substances Data: 328-38-1(Hazardous Substances Data)

Usage

Chemical Properties

White powder

Uses

D-Leucine is used in the formation of sterols. It is also used as a selectable marker.

Definition

ChEBI: The D-enantiomer of leucine.

InChI:InChI=1/C6H13NO2/c1-4(2)3-5(7)6(8)9/h4-5H,3,7H2,1-2H3,(H,8,9)/t5-/m1/s1

Synthetic route

N-(9-fluorenylmethoxycarbonyl)-D-leucine (35661-60-0, 126727-03-5, 114360-54-2) → (R)-leucine (328-38-1)

Conditions	Yield
With piperidine In N,N-dimethyl-formamide at 20℃;	100%

(R)-N-benzylleucine (89384-51-0) → (R)-leucine (328-38-1)

Conditions	Yield
With hydrogen; palladium on activated charcoal In acetic acid for 24h;	98%

(R)-2-azido-4-methylpentanoic acid (126191-08-0) → (R)-leucine (328-38-1)

Conditions	Yield
With hydrogen; palladium on activated charcoal In water; acetic acid under 760 Torr; for 4h;	92%

D-α-Hydrazino-isocapronsaeure (24292-07-7) → (R)-leucine (328-38-1)

Conditions	Yield
With hydrogen; platinum(IV) oxide	91%

(R)-2-Amino-4-methyl-pentanoic acid ((1S,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl)-methyl-amide (170642-24-7) → (R)-leucine (328-38-1) + pseudoephedrine (90-82-4)

Conditions	Yield
With water for 10h; Heating;	A 86% B n/a

(R)-2-Amino-4-methyl-pentanoic acid ((1S,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl)-methyl-amide (170642-24-7) → (R)-leucine (328-38-1)

Conditions	Yield
In water for 10h; Heating; other reagent;	86%

4-methyl-2-oxopentanoic acid (816-66-0) → (R)-leucine (328-38-1)

Conditions	Yield
With D-glucose; Bacillus subtilis glucose dehydrogenase; Symbiobacterium thermophilum meso-Diaminopimelate dehydrogenase W121L/H227I mutant; NADP; ammonium chloride In aq. buffer at 37℃; for 24h; pH=8.5; Kinetics; Reagent/catalyst; Enzymatic reaction;	68%
With D-Alanine; meso-diaminopimelate dehydrogenase; pyridoxal 5'-phosphate; alcohol dehydrogenases from Bacillus stearothermophilus; alcohol dehydrogenases from Thermoanaerobacter brockii; NADH In isopropyl alcohol at 35℃; for 12h; pH=8; Reagent/catalyst; Enzymatic reaction; enantioselective reaction;	53.2%
With meso-2,6-D-diaminopimelic acid dehydrogenase mutant BC621; ammonium chloride; NADPH In various solvent(s) for 24h;
With D-amino acid transaminase; D-Alanine; R-selective ω-transaminase from Arthrobacter sp; (3-hydroxy-5-hydroxymethyl)-2-methylisonicotinic acid 5-phosphate; isopropylamine In aq. phosphate buffer for 7h; pH=7;	n/a
With D-Glucose; Bacillus megaterium glucose dehydrogenase; ammonia; Symbiobacterium thermophilum mesodiaminopimelate dehydrogenase Enzymatic reaction;	n/a

sodium 4-methyl-2-oxovalerate (4502-00-5) → (R)-leucine (328-38-1)

Conditions	Yield
With zinc(II) perchlorate; (S)-15-amino-methyl-14-hydroxy-5,5-dimethyl-2,8-dithia<9>(2,5)pyridinophane In methanol for 24h; Ambient temperature;	66%

LEUCINE (328-39-2) → (R)-leucine (328-38-1)

Conditions	Yield
With yeast; Sucrose
beim Durchgang durch den Organismus des Kaninchens;
With penicillium glaucum

DL-leucine ethyl ester (2899-43-6) → (R)-leucine (328-38-1)

Conditions	Yield
With (-)-(R,R)-dibenzoyltartaric acid

N-formyl-valine (5338-45-4) → (R)-leucine (328-38-1)

Conditions	Yield
With ethanol; brucine man filtriert das sich ausscheid.Brucinsalz des Formyl-d-leucins ab,zerlegt es durch Natronlauge u.verseift das erhalt.Formyl-d-leucin durch Kochen mit 10prozentig.Salzsaeure o.durch Schuetteln mit 30prozentig.Bromwasserstoffsaeure b.37grad;

N-formyl-D-leucine (5338-45-4, 6113-61-7, 89921-44-8, 44978-39-4) → (R)-leucine (328-38-1)

Conditions	Yield
Hydrolysis;

N-chloroacetyl-D-leucine (64830-83-7) → (R)-leucine (328-38-1)

Conditions	Yield
With hydrogenchloride

Z-D-Leu-OH (28862-79-5) → (R)-leucine (328-38-1)

Conditions	Yield
With hydrogenchloride; palladium Hydrogenation;

LEUCINE (328-39-2) → (R)-leucine (328-38-1) + L-leucine (61-90-5)

N-acetyl-D-leucine (19764-30-8) → (R)-leucine (328-38-1)

Conditions	Yield
In water at 37℃; for 0.166667h; Rate constant; D-aminocyclase from Alcaligenes denitrificans DA181, pH 7.8, bovine serum albumin;
With D-aminoacylase from Alcaligenes xylosoxydans Enzyme kinetics; Further Variations:; Reagents; Deacetylation;

isobutyl triflate (60306-25-4) + trans-(2S,5S)-N--2,5-bis(methoxymethoxymethyl)pyrrolidine (108437-90-7) → (R)-leucine (328-38-1) + L-leucine (61-90-5)

Conditions	Yield
Yield given. Multistep reaction. Yields of byproduct given;

glycyl-L-leucylglycylglycine (7325-21-5) → (R)-leucine (328-38-1) + [(S)-2-(2-Amino-acetylamino)-4-methyl-pentanoylamino]-acetic acid (2576-67-2) + glycyl-D-leucylglycine (131830-34-7) + glycyl-D-leucylglycylglycine (117760-61-9) + cyclo(glycyl-L-leucyl) (5815-67-8, 5845-67-0, 108315-75-9) + cyclo-(glycyl-D-leucyl) (108315-75-9)

Conditions	Yield
In various solvent(s) at 148.5℃; Product distribution; Rate constant; rate of racemization was investigated during decomposition;

Leucine p-nitrophenyl ester (17663-34-2, 66575-12-0) → (R)-leucine (328-38-1) + L-leucine (61-90-5)

Conditions	Yield
With (R)-<methyl>pyridine; copper(II) ion In various solvent(s) at 25℃; Rate constant; enantioselectivity of cleavage; other chiral ligands;

(2R,2'R)-N-(2'-amino-4'-methylpentanoyl)bornane-10,2-sultam (129568-83-8) → (R)-leucine (328-38-1)

Conditions	Yield
With lithium hydroxide; ion exchange 1.) THF; Yield given. Multistep reaction;
With lithium hydroxide; Amberlite IR-120 1) THF, room temperature, 2) H2O, room temperature, 15 h; Yield given. Multistep reaction;

theonellamine B (105091-14-3, 119816-11-4) → N-methylalanine (3913-67-5) + L-valine (72-18-4) + L-threonine (72-19-5) + N-methyl-L-valine (2480-23-1) + (R)-leucine (328-38-1) + 3-amino propanoic acid (107-95-9)

Conditions	Yield
With hydrogenchloride at 110℃; for 4h;

theonellamine B (105091-14-3, 119816-11-4) → L-valine (72-18-4) + N-methyl-L-valine (2480-23-1) + (R)-leucine (328-38-1) + 3-amino propanoic acid (107-95-9)

Conditions	Yield
With hydrogenchloride at 110℃; for 40h; Further byproducts given;

(R)-2-Azido-4-methyl-pentanoic acid benzyl ester → (R)-leucine (328-38-1) + L-leucine (61-90-5)

Conditions	Yield
With hydrogen; Pd-BaSO4 In ethanol under 760 Torr; Ambient temperature; Yield given. Yields of byproduct given;

ulithiacyclamide A (74847-09-9) → L-threonine (72-19-5) + (R)-leucine (328-38-1) + L-cystine (56-89-3)

Conditions	Yield
With triphenyl phosphite; oxygen In dichloromethane Product distribution; Ambient temperature; configuration;

L-leucine (phenylmethyl)ester (80089-22-1) → (R)-leucine (328-38-1) + L-leucine (61-90-5) + L-Leucine benzyl ester (1738-69-8) + D-leucine benzyl ester (46741-65-5)

Conditions	Yield
With sodium bicarbonate buffer; subtilisin 8350 at 35℃; for 1h; Product distribution; var. DL-amino acid esters;

9-fluorenylmethyloxycarbonyl-L-leucine anhydride (70626-37-8) → (R)-leucine (328-38-1) + L-leucine (61-90-5)

Conditions	Yield
With dmap; p-hydroxymethylphenoxyacetyl-polydimethylacrylamide resin; trifluoroacetic acid Product distribution; racemisation in dependence on sequence of addition of reagents; 1) dimethylacetamide, 3 h;

N-carbamyl-D-leucine (54896-73-0) → (R)-leucine (328-38-1)

Conditions	Yield
With potassium phosphate buffer; Pseudomonas sp. AJ-11220 In various solvent(s) at 30℃; for 2h;
With hydrogenchloride; sodium nitrite at 0℃;
With potassium phosphate buffer; Pseudomonas sp. AJ-11220 In various solvent(s) at 30℃; for 2h; pH 7.5; enzymatic reaction;

5-isobutyl-imidazolidine-2,4-dione (67337-73-9) → (R)-leucine (328-38-1)

Conditions	Yield
at 37℃; Pseudomonas Sp.;

diethylaluminium cyanide (5804-85-3) + 2-Methoxy-naphthalene-1-sulfinic acid [3-methyl-but-(E)-ylidene]-amide → (R)-leucine (328-38-1)

Conditions	Yield
With hydrogenchloride 1.) THF, from -78 to -40 degC; 2.) reflux; Yield given. Multistep reaction;

methanol (67-56-1) + (R)-leucine (328-38-1) → (R)-leucine methyl ester hydrochloride (5845-53-4, 6322-53-8, 7517-19-3)

Conditions	Yield
With sulfuryl dichloride at 0 - 20℃; for 16h; Inert atmosphere;	100%
With thionyl chloride at 20℃;	99%
With thionyl chloride for 12h; Ambient temperature;	94%

(R)-leucine (328-38-1) + benzyl chloroformate (501-53-1) → Z-D-Leu-OH (28862-79-5)

Conditions	Yield
With sodium hydroxide In water at 0 - 20℃;	100%
With sodium hydroxide In water at 20℃; for 2h;	90%
With sodium hydrogencarbonate	87%
Stage #1: (R)-leucine With sodium hydroxide In water at 0℃; Cooling with ice; Stage #2: benzyl chloroformate In 1,4-dioxane; water at 20℃;
With sodium hydroxide In 1,4-dioxane at 0 - 20℃;

ethanol (64-17-5) + (R)-leucine (328-38-1) → D-(-)-leucine ethyl ester hydrochloride (73913-65-2)

Conditions	Yield
With thionyl chloride for 4h; Reflux;	100%
With hydrogenchloride Heating;	87%

phthalic anhydride (85-44-9) + (R)-leucine (328-38-1) → (R)-2-(1,3-dioxoisoindolin-2-yl)-4-methylpentanoic acid (29588-87-2)

Conditions	Yield
for 0.0611111h; microwave irradiation;	98%
at 150℃;

(R)-leucine (328-38-1) → N-1-naphthalenesulfonyl-D-leucine (68305-80-6)

Conditions	Yield
With hydrogenchloride In 1,4-dioxane	98%

(R)-leucine (328-38-1) + 1-Naphthalenesulfonyl chloride (85-46-1) → N-1-naphthalenesulfonyl-D-leucine (202326-38-3)

Conditions	Yield
With sodium hydroxide In 1,4-dioxane at 0 - 20℃; for 4h; pH=9 - 10;	98%

(R)-leucine (328-38-1) + di-tert-butyl dicarbonate (24424-99-5) → N-Boc-D-Leu (16937-99-8)

Conditions	Yield
With sodium hydroxide In 1,4-dioxane at 0 - 20℃; Inert atmosphere;	97%
Stage #1: (R)-leucine With sodium hydroxide In tetrahydrofuran; water for 0.25h; Inert atmosphere; Cooling with ice; Stage #2: di-tert-butyl dicarbonate In tetrahydrofuran; water at 20℃; for 24h; pH=Ca. 10.5; Inert atmosphere; Cooling with ice;	97.2%
Stage #1: (R)-leucine With potassium carbonate In acetone at 20℃; for 0.0833333h; Stage #2: di-tert-butyl dicarbonate In acetone	95%

(R)-leucine (328-38-1) + tetrachlorophthalic anhydride (117-08-8) → TCP-D-Leu-OH

Conditions	Yield
In N,N-dimethyl-formamide Microwave irradiation;	97%

N-succinimidyl 3,5-dichlorobenzoate (143558-02-5) + (R)-leucine (328-38-1) → (R)-N-(3,5-dichlorobenzoyl)leucine

Conditions	Yield
With sodium hydrogencarbonate In tetrahydrofuran; water at 20℃; for 18h;	97%

(R)-leucine (328-38-1) + 4-bromobenzenesulfonyl chloride (98-58-8) → N-p-bromo-benzenesulfonyl-D-leucine (68305-78-2)

Conditions	Yield
With hydrogenchloride In 1,4-dioxane	96.4%

(R)-leucine (328-38-1) + 4-bromobenzenesulfonyl chloride (98-58-8) → N-p-bromobenzenesulfonyl-D-leucine (1308991-43-6)

Conditions	Yield
With sodium hydroxide In 1,4-dioxane at 0 - 20℃; for 4h; pH=9 - 10;	96.4%

(R)-leucine (328-38-1) + benzyl alcohol (100-51-6) → D-leucine benzyl ester (46741-65-5)

Conditions	Yield
With toluene-4-sulfonic acid In benzene for 16h; Heating;	95%

(R)-leucine (328-38-1) + cyclohexanylcarbonyl chloride (2719-27-9) → (R)-2-(cyclohexanecarboxamido)-4-methylpentanoic acid

Conditions	Yield
With potassium phosphate In tetrahydrofuran for 24h; Inert atmosphere;	94%
With sodium hydroxide for 2.5h; Ambient temperature;

(R)-leucine (328-38-1) + 4-Fluorobenzenesulfonyl chloride (349-88-2) → N-p-fluorobenzenesulfonyl-D-leucine

Conditions	Yield
With sodium hydroxide In 1,4-dioxane at 0 - 20℃; for 4h; pH=9 - 10;	94%
With sodium carbonate In water

(R)-leucine (328-38-1) + di(μ-chloro)bis[1-(phenylazo)phenyl-C(2)N]dipalladium(II) (14873-53-1) → Pd(C6H4NNC6H5)(NH2CH(CH2CH(CH3)2)CO2)

Conditions	Yield
With NaOMe In methanol N2-atmosphere; dropwise addn. of 1 equiv. NaOMe to aminoacid, gentle heating to dissoln., addn. of stoich. amt. Pd-complex, stirring for 15 h; solvent removal (vac.), dissoln. in CH2Cl2, centrifugation, solvent removal (vac.); elem. anal.;	94%

(R)-leucine (328-38-1) + 4-Fluorobenzenesulfonyl chloride (349-88-2) → N-p-fluoro-benzenesulfonyl-D-leucine

Conditions	Yield
With hydrogenchloride In 1,4-dioxane	94%

(R)-leucine (328-38-1) → (R)-2-hydroxy-4-methylpentanoic acid (20312-37-2)

Conditions	Yield
With sulfuric acid; sodium nitrite In water at 5 - 20℃;	93.8%
With sulfuric acid; sodium nitrite at 0℃;	79%
With sulfuric acid; water; sodium nitrite	76%

methanol (67-56-1) + (R)-leucine (328-38-1) → D-leucine methyl ester (23032-21-5)

Conditions	Yield
With thionyl chloride for 6h; Ambient temperature;	91%
With chlorosulfonic acid at 20℃; for 2h;	80%
With hydrogenchloride

(R)-leucine (328-38-1) + N-t-butoxycarbonyl-(R)-proline N-hydroxysuccuinimide ester (102185-34-2) → N-(tert-butoxycarbonyl)-(R)-prolyl-(R)-leucine (91711-95-4)

Conditions	Yield
With sodium hydroxide; potassium hydrogencarbonate In 1,4-dioxane 1.) 0 deg C, overnight; 2.) r.t., 24 h;	90%

(R)-leucine (328-38-1) + acetic anhydride (108-24-7) → N-acetyl-D-leucine (19764-30-8)

Conditions	Yield
Stage #1: (R)-leucine; acetic anhydride With sodium hydroxide In water at 5 - 15℃; for 4 - 5h; Stage #2: With hydrogenchloride In water at 5 - 15℃; for 2.5h;	89%
Stage #1: (R)-leucine; acetic anhydride With sodium hydroxide In water at 5 - 15℃; for 2h; pH=8-9; Inert atmosphere; Large scale; Stage #2: With hydrogenchloride In water at 5 - 15℃; for 2.5h; Inert atmosphere; Large scale;	89%
With sodium hydroxide In water at 20℃; pH=14;
In tetrahydrofuran; water

(R)-leucine (328-38-1) + 2,7-disulfo-9-fluorenylmethoxycarbonyl chloride → C21H21NO10S2(2-)*2Na(1+)

Conditions	Yield
With sodium hydrogencarbonate In water; acetonitrile at 20℃; for 0.5h; pH=8.5;	88.7%

(R)-leucine (328-38-1) + N-((1S,2R,4aS,5R,8aS)-2-hydroxy-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalen-1-yl)formamide → (S)-(1S,2R,4aS,5R,8aS)-1-formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalen-2-yl 2-amino-4-methylpentanoate

Conditions	Yield
Stage #1: (R)-leucine With dicyclohexyl-carbodiimide In dichloromethane for 0.333333h; Stage #2: N-((1S,2R,4aS,5R,8aS)-2-hydroxy-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalen-1-yl)formamide With dmap In dichloromethane at 20℃; for 2h;	88%

(R)-leucine (328-38-1) + (1R,2S,5R)-menthyl chloroformate (14602-86-9) → N-[(-)-menthoxylcarbonyl]-D-leucine (1627153-13-2)

Conditions	Yield
With sodium hydroxide In water at 0℃; for 0.5h; Schlenk technique; Inert atmosphere;	87%

(R)-leucine (328-38-1) → (R)-2-bromo-4-methylpentanoic acid (42990-28-3)

Conditions	Yield
With hydrogen bromide; sodium nitrite In water at 0℃; for 2.5h;	86%
With hydrogen bromide; sodium nitrite In water at 0℃; for 2.5h; Inert atmosphere;	85%
With potassium bromide; sodium nitrite In water	83%

Upstream product

• 328-39-2 LEUCINE 
• 64830-83-7 N-chloroacetyl-D-leucine 
• 19764-30-8 N-acetyl-D-leucine 
• 60306-25-4 isobutyl triflate 
• 108437-90-7 trans-(2S,5S)-N--2,5-bis(methoxymethoxymethyl)pyrrolidine 
• 7325-21-5 glycyl-L-leucylglycylglycine 
• 17663-34-2 Leucine p-nitrophenyl ester 
• 129568-83-8 (2R,2'R)-N-(2'-amino-4'-methylpentanoyl)bornane-10,2-sultam 
• 67337-73-9 5-isobutyl-imidazolidine-2,4-dione 
• 89384-51-0 (R)-N-benzylleucine 
• 7647-01-0 hydrogenchloride 
• 1466-83-7 N-benzoyl-DL-leucine 
• 13079-20-4 2-Amino-4-methyl-pentanoic acid amide 
• 91384-09-7 DL-leucine isobutyl ester; hydrochloride 

Downstream Products

• 29588-87-2 (R)-2-(1,3-dioxoisoindolin-2-yl)-4-methylpentanoic acid 
• 66789-78-4 N-[(2,4,5-trichloro-phenoxy)-acetyl]-D-leucine 
• 20312-37-2 (R)-2-hydroxy-4-methylpentanoic acid 
• 80913-68-4 N-[(2,4-Dichlor-phenoxy)-acetyl]-D-leucin 
• 117910-69-7 N-(4-nitro-benzoyl)-D-leucine 
• 31413-05-5 N-[(4-chloro-phenoxy)-acetyl]-D-leucine 
• 6297-56-9 N-benzylsulfonyl-D-leucine 
• 16937-99-8 N-Boc-D-Leu 
• 7533-40-6 2-amino-4-methyl-1-pentanol 
• 42990-28-3 (R)-2-bromo-4-methylpentanoic acid 
• 24365-47-7 Ac-D-Leu-L-Leu-L-Argal 
• 5845-53-4 (R)-leucine methyl ester hydrochloride 
• 37696-36-9 DNP-D-leucine 
• 85313-43-5 t-Bumeoc-D-Leu*DCHA 

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Three new cyclic peptide-polyketide hybrids (1-3) and two new chaetiacandin-type polyketides (4 and 5) along with nine known compounds were isolated from cultures of a halophyte-associated fungus, Colletotrichum gloeosporioides JS0417. Spectroscopic analysis revealed that 1-3 were cyclic depsipeptides where 3,5,11-trihydroxy-2,6-dimethyldodecanoic acid was linked to two amino acids through amide and ester bonds to form a 12-membered ring. Relative and absolute configurations for the peptides were determined with spectroscopic analysis and chemical reactions. The cyclic depsipeptides 2 and 6 were determined to act as strong adiponectin-secretion-promoting modulators with potential to treat metabolic diseases associated with hypoadiponectinemia. Notably, a known compound, tryptophol, significantly inhibited PGE2synthesis and also promoted adiponectin secretion, exhibiting a similar biological activity profile to aspirin, but with greater potency. The presence of an isoleucine moiety and non-glycosylation may be important for biological activity of the cyclic peptide-polyketide hybrids, and non-methoxylation of the side chain may influence activity of the indole derivatives.

Single-step fluorescent probes to detect decrotonylation activity of HDACs through intramolecular reactions

Lysine crotonylation plays vital roles in gene transcription and cellular metabolism. Nevertheless, methods for dissecting the molecular mechanisms of decrotonyaltion remains limited. So far, there is no single-step fluorescent method developed for enzymatic decrotonylation activity detection. The major difficulty is that the aliphatic crotonylated lysine doesn't allow π-conjugation to a fluorophore and decrotonylation can not modulate the electronic state directly. Herein, we have designed and synthesized two activity-based single-step fluorogenic probes KTcr-I and KTcr-II for detecting enzymatic decrotonylation activity. These two probes can be recognized by histone deacetylases and undergo intramolecular nucleophilic exchange reaction to generate fluorescence signal. Notably, peptide sequence-dependent effect was observed. KTcr-I can be recognized by Sirt2 more effectively, while KTcr-II with LGKcr peptide sequence preferentially reacted with HDAC3. Compared to other methods of studying enzymatic decrotonylation activity, our single-step fluorescent method has a number of advantages, such as facileness, high sensitivity, cheap facility and little material consumed. We envision that the probes developed in this study will provide useful tools to screen inhibitors which suppress the decrotonylation activity of HDACs. Such probes will be useful for further delineating the roles of decrotonylation enzyme and aid in biomarker identification and drug discovery.