Microbial Metabolite; Endogenous Metabolite; Phenylacetylglutamine targets multiple receptors and signaling pathways. It functions as a negative allosteric modulator (NAM) of the β2-adrenergic receptor (β2AR), but not β1AR, with an EC50 of 23 ± 3.5 μM for transient cAMP generation . Key residues on β2AR (E122 and V206) were identified as critical for PAGln-mediated NAM activity . PAGln also activates α2A, α2B, and β2-adrenergic receptors in platelets . In prostate cancer, PAGln targets the Wnt/β-catenin signaling pathway by upregulating CCNG2, promoting β-catenin phosphorylation and pathway inhibition .

Phenylacetylglutamine is a microbial metabolite found in the colon that is produced by fermenting amino acids. It is created through the nearly complete microbial conversion of phenylalanine, which leads to the glutamine conjugation of phenylacetic acid [1].

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Phenylacetylglutamine exhibits concentration-dependent activity in cellular models. In β2AR-overexpressing HEK293 cells, PAGln (10-267 μM, physiologically relevant levels) acts as a partial agonist, inducing transient cAMP production (EC50 = 23 ± 3.5 μM) after acute exposure (<10 min), but primarily functions as a negative allosteric modulator (NAM) following chronic exposure (≥15 min), shifting the isoproterenol EC50 from 0.6 ± 0.1 nM to 3.2 ± 0.4 nM (5.3-fold shift) . No such effects were observed on β1AR .
\ In prostate cancer research, PAGln significantly inhibits the proliferation, migration, and invasion of PC3 prostate cancer cells in a concentration-dependent manner . Mechanistic studies revealed that PAGln upregulates CCNG2 mRNA expression, which in turn promotes β-catenin phosphorylation, thereby suppressing the Wnt/β-catenin signaling pathway.

Colonic microbial metabolism substantially contributes to uremic solute production. p-Cresyl sulfate and indoxyl sulfate are the main representatives of solutes of microbial origin and also, protein-bound solutes, exhibiting high protein-binding affinity and dependence on tubular secretion. Phenylacetylglutamine is another microbial metabolite with high dependence on tubular secretion but low protein-binding affinity. The relevance of such solutes is unknown. Therefore, we prospectively followed 488 patients with CKD stages 1-5 and a measurement of serum phenylacetylglutamine by liquid chromatography-mass spectrometry. In a subgroup, we determined 24-hour urinary excretion as a surrogate of intestinal uptake as well as renal clearance of phenylacetylglutamine. We performed outcome analysis for mortality (51 events) and cardiovascular disease (75 events). Serum phenylacetylglutamine level correlated with 24-hour urinary excretion (rho=0.55; P<0.001) and clearance of phenylacetylglutamine (rho=-0.76; P<0.001). Phenylacetylglutamine clearance also correlated with eGFR (rho=0.84; P<0.001). Furthermore, serum phenylacetylglutamine level associated with mortality (hazard ratio per 1-SD increase, 1.77; 95% confidence interval, 1.22 to 2.57; P=0.003) and cardiovascular disease (hazard ratio, 1.79; 95% confidence interval, 1.32 to 2.41; P<0.001) after adjustment for age, sex, presence of diabetes mellitus, prior cardiovascular disease, and eGFR. Thus, serum phenylacetylglutamine level is elevated in patients with more advanced CKD and determined by intestinal uptake and renal clearance, and it is not fully accounted for by differences in eGFR. High serum phenylacetylglutamine level is a strong and independent risk factor for mortality and cardiovascular disease, suggesting the relevance of microbial metabolism and/or tubular dysfunction in CKD, irrespective of protein binding [1].

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Phenylacetylglutamine increases the susceptibility of ventricular arrhythmias in heart failure mice by exacerbated activation of the TLR4/AKT/mTOR signaling pathway. [2]
PAGln (Phenylacetylglutamine) increases the susceptibility to ventricular arrhythmias in heart failure mice. [2]
PAGln (Phenylacetylglutamine) deteriorates cardiac dysfunction in heart failure mice. [2]
PAGln worsens cardiac pathological structure remodeling and cardiac inflammation. [2]

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PAGln exacerbates maladaptive cardiac remodeling via enhanced activation of the TLR4/AKT/mTOR signaling pathway.[2]
In a prostate cancer xenograft mouse model (BALB/c nude mice bearing PC3 tumors), intraperitoneal administration of PAGln at 200 mg/kg daily for four weeks significantly suppressed tumor growth compared to saline controls. Additionally, in a lung metastasis model established via tail vein injection, PAGln treatment reduced metastatic tumor burden .
In a mouse model of thrombosis, PAGln enhanced platelet reactivity and promoted in vivo thrombus formation via activation of adrenergic receptors . In ex vivo studies using failing human heart left ventricle trabeculae, PAGln also promoted negative allosteric modulation, impacting contractile function under conditions of sympathetic tone .

Biochemical Measurements[1]
At inclusion, blood was taken by venous puncture for measurement of hemoglobin (grams per deciliter), albumin (grams per liter), C-reactive protein (milligrams per liter), cholesterol (milligrams per deciliter), calcium (milligrams per deciliter), phosphate (milligrams per deciliter), biointact parathyroid hormone (nanograms per liter), and creatinine (milligrams per deciliter), all measured using standard laboratory techniques. The eGFR was calculated using the CKD-EPI equation. We also had ancillary data available on free serum levels of p-cresyl sulfate determined as p-cresol with a dedicated gas chromatography-mass spectrometry method, allowing comparison with a protein-bound solute. Additionally, serum levels of Phenylacetylglutamine (PAG) were quantified by ultraperformance liquid chromatography-tandem mass spectrometry. For sample preparation, 50 μl serum or urine, 50 μl solution of milli-Q (MQ) water:MeOH:0.01 N sodium hydroxide (75:20:5 vol/vol/vol), 20 μl internal standard mixture (Phenylacetylglutamine (PAG)-d5), and 150 μl acetonitrile were thoroughly mixed in 96-well Ostro Plates (Waters). After separation by a positive pressure manifold, supernatants were collected in 2-ml collection plates. Subsequently, the organic phase was removed by a gentle stream of nitrogen for 30 minutes at 40°C. After dilution with 1000 μl MQ water, 5 μl final solution was injected on the ultraperformance liquid chromatography-tandem mass spectrometry system. Chromatographic separation was performed on an Acquity CSHFluoroPhenyl Column (50×2.5 mm; 1.7-μm particle size; Waters). The mobile phase, delivered at a flow rate of 0.5 ml/min at 40°C, consisted of a gradient of 0.1% formic acid in MQ water (A) and MeOH (B). The gradient was as follows: starting with 3% B, there was a subsequent increase to 16% B within 1 minute followed by an increase to 80% B within 3 minutes and thereafter, an increase to 95% B within 30 seconds for a duration of 1 minute, after which the initial 3% B was reintroduced with equilibration for a duration of 3.5 minutes before the next injection. Ionization of Phenylacetylglutamine (PAG) and the corresponding isotopologue (internal standard) was achieved in negative mode. The following multiple reaction monitoring transitions were used for quantification: Phenylacetylglutamine (PAG) 263→145 and Phenylacetylglutamine (PAG)-d5 268→145. Limit of detection and limit of quantification (LOQ) were 0.06 and 0.18 μM for Phenylacetylglutamine (PAG). For analysis, solute levels below the LOQ were treated as the average value of the limit of detection and the LOQ. The total, within–run, between–run, and between–day method imprecisions according to the National Committee for Clinical Laboratory Standards EP5-T guideline were 3.92%, 1.61%, 2.69%, and 2.02%, respectively, and the mean recovery was 97%. We also sampled 24-hour urinary collections when available at the time of inclusion to calculate renal clearance and 24-hour urinary excretion of Phenylacetylglutamine (PAG). Collections were considered complete when 24-hour urinary creatinine excretion was within 2 SDs (range =0.7–1.8 g) of the mean creatinine excretion for the geographic region of this study derived from the INTERSALT Study. Assuming steady-state conditions and negligible nonrenal clearance, 24-hour urinary excretion of Phenylacetylglutamine (PAG) was considered an indirect estimate of 24-hour intestinal uptake of Phenylacetylglutamine (PAG). Furthermore, protein intake was calculated according to the formula by Maroni et al. using 24-hour urinary urea nitrogen excretion and body weight.

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Using a cell-based functional assay, the activity of PAGln on β2AR was assessed by measuring cAMP production in HEK293 cells stably overexpressing β2AR. Following serum starvation, cells were pre-incubated with DMSO or varying doses of PAGln (within physiologically relevant ranges of 10-267 μM) for ≥15 minutes before stimulation with increasing concentrations of isoproterenol or norepinephrine. cAMP levels were then quantified using a homogeneous time-resolved fluorescence (HTRF) assay. For G-protein coupled receptor (GPCR) signaling studies, β-arrestin2 recruitment was monitored using an enzyme complementation assay (PathHunter) in β2AR-expressing cells. In silico docking studies coupled with site-directed mutagenesis identified that residues E122 and V206 on β2AR are critical for PAGln-elicited NAM activity .

H9C2 cell culture and Ang II-induced hypertrophy[2]
H9C2 cell were plated in 6 well plates at a 1 × 106 /ml density and cultured in Dulbecco's Modified Eagle Medium, supplemented with penicillin, streptomycin, and 10 % FBS. Ang II was dissolved in PBS and stimulation of Ang II at the concentration of 1 μM induced H9C2 hypertrophy. CCK-8 kits detected H9C2 cell viability at variable concentrations of 0, 25,50,100,200,400 μM Phenylacetylglutamine (PAGIn). In addition, TAK-242, a TLR4 inhibitor, was dissolved in 10 % dimethyl sulfoxide (DMSO) and added to cells at the concentration of 10 μM and Phenylacetylglutamine (PAGIn) at the concentration of 100 μM intervened H9C2 cell. Subsequent experiments were carried out 24 h later.

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The anti-cancer activity of PAGln was evaluated using PC3 human prostate cancer cells. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in 5% CO₂ .
For cell proliferation assays, cells were seeded in 96-well plates and treated with varying concentrations of PAGln. Cell viability was assessed using the CCK-8 assay, EdU incorporation, and colony formation assays .
Cell migration and invasion capabilities were evaluated using wound healing assays (scratch method) and Transwell assays (with or without Matrigel coating). Cells were treated with PAGln, and the number of migrated or invaded cells was quantified after 24-48 hours .
For mechanistic studies, total RNA was extracted for qRT-PCR analysis of CCNG2 mRNA expression. Protein levels of β-catenin and phosphorylated β-catenin were analyzed by Western blot. CCNG2 knockdown experiments using siRNA were performed to confirm target specificity .

Male 8 weeks old C57Bl/6 mice were housed in the laboratory animal center of the cardiovascular research institute of Wuhan University, at 12 h light/dark cycles at 22 ± 2 °C, with free access to food and water. Animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health and were approved by the appropriate authorities. The mice were randomly divided into 4 groups (24 per group) for treatment, namely, the Sham group, sham operation without phenylacetylglutamine (PAGln) intervention; Sham +phenylacetylglutamine (PAGln) group, sham operation with phenylacetylglutamine (PAGln) intervention; HF group, TAC surgery without phenylacetylglutamine (PAGln) intervention; and HF + phenylacetylglutamine (PAGln) group, TAC surgery with phenylacetylglutamine (PAGln) intervention. TAC surgery created overpressure-induced HF mice, as described previously. Briefly, after anesthetizing the mice with 3 % pentobarbital sodium at 40 mg/kg, a 27G needle was used to ligate the thoracic aorta. The sham surgery was only operated through thoracotomy without ligation. The mice, after the operation, start to intervene within 24 h through intraperitoneal injectionphenylacetylglutamine (PAGln) solution (100 mg/kg/d) for consecutive 4 weeks. [2]

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In a prostate cancer xenograft model, 6-week-old male BALB/c nude mice were injected subcutaneously with 1 × 10⁷ PC3 cells suspended in PBS. One week after tumor cell inoculation, mice were randomly assigned to groups (n=5 per group). The treatment group received daily intraperitoneal injections of PAGln at 200 mg/kg dissolved in physiological saline for four consecutive weeks, while the control group received an equal volume of saline daily. All mice were euthanized under general anesthesia (sodium pentobarbital, 150 mg/kg intraperitoneal), and tumors were excised for analysis .
In a lung metastasis model, PC3 cells were injected via the tail vein, followed by the same PAGln treatment protocol to evaluate metastatic burden .
For the cardiovascular studies, isolated mouse cardiomyocytes and failing human heart left ventricle trabeculae were used to assess the negative inotropic functional effects of PAGln .

Phenylacetylglutamine exhibits nonlinear pharmacokinetics. Following administration of its prodrug ornithine phenylacetate (OPA), plasma PAGln concentrations increase with increasing OPA infusion rates .
Renal function plays a critical role in PAGln elimination. Urinary PAGln clearance is linearly related to creatinine clearance (r = 0.831, P < 0.0001) . In patients with severe renal impairment, PAGln renal clearance decreases by five-fold compared to subjects with normal renal function, leading to significantly elevated PAGln plasma concentrations .
Hepatic function also affects PAA exposure, which in turn influences PAGln production. Phenylacetic acid exposure is 35% higher in Child-Pugh C patients than in Child-Pugh B patients . Dose adjustment should be considered for patients with low body weight and severely impaired hepatic function .

According to the Material Safety Data Sheet, Phenylacetylglutamine (CAS: 28047-15-6) is classified as "not a hazardous substance or mixture" under GHS criteria, with no known hazards and no special precautionary statements required .
In clinical studies of ornithine phenylacetate (where PAGln is the excretory product) in patients with acute liver injury/failure, the study drug was well-tolerated, and no safety signals were identified. Of the reported serious adverse events including 11 deaths, none were attributable to the study medication. Non-serious adverse events possibly related to the study drug were limited to headache and nausea/vomiting .
In a separate analysis, no correlation was observed between phenylacetic acid (PAA) plasma exposure and neurologic adverse events in patients with stable cirrhosis or acute hepatic encephalopathy .

[1]. Microbiota-Derived Phenylacetylglutamine Associates with Overall Mortality and Cardiovascular Disease in Patients with CKD. J Am Soc Nephrol. 2016 Nov;27(11):3479-3487.
[2]. Phenylacetylglutamine increases the susceptibility of ventricular arrhythmias in heart failure mice by exacerbated activation of the TLR4/AKT/mTOR signaling pathway. Int Immunopharmacol. 2023 Mar:116:109795.

N(2)-Phenylacetyl-L-glutamine is an L-configuration N(2)-phenylacetylglutamine. It is a human metabolite and the conjugate acid of N(2)-phenylacetyl-L-glutamic acid. There are reports on the existence of phenylacetylglutamine in humans, and relevant data are available.
Phenylacetylglutamine is the product of phenylacetate metabolism in primates and humans and is excreted in urine . It has been used as a noninvasive probe to study liver citric acid cycle intermediate labeling patterns using ¹³C tracers such as [3-¹³C]lactate or [3-¹³C]pyruvate. The labeling pattern of PAGln's glutamine moiety reflects that of liver α-ketoglutarate and glutamate . NMR assays have been developed to quantify urinary PAGln concentration and its ¹³C-labeling pattern, with a detection limit of 13 μmol for unlabeled PAGln .
The gut microbiota-dependent production of PAGln involves two distinct microbial pathways: one catalyzed by phenylpyruvate:ferredoxin oxidoreductase (PPFOR) and the other by phenylpyruvate decarboxylase (PPDC). Fecal levels of both enzymes are associated with atherosclerotic cardiovascular disease .
PAGln is supplied as a research-use-only compound, typically as a powder stored at -20°C, soluble in DMSO and other organic solvents .

C13H16N2O4

264.2771

281.089

C, 59.08; H, 6.10; N, 10.60; O, 24.22

28047-15-6

28047-15-6 (free acid) ; 104771-87-1 (sodium)

92258

White to off-white solid powder

1.4±0.1 g/cm3

646.6±55.0 °C at 760 mmHg

85-87?C

344.9±31.5 °C

0.0±2.0 mmHg at 25°C

1.595

-1.38

3

4

7

19

338

1

O([H])C([C@]([H])(C([H])([H])C([H])([H])C(N([H])[H])=O)N([H])C(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)=O

JFLIEFSWGNOPJJ-JTQLQIEISA-N

InChI=1S/C13H16N2O4/c14-11(16)7-6-10(13(18)19)15-12(17)8-9-4-2-1-3-5-9/h1-5,10H,6-8H2,(H2,14,16)(H,15,17)(H,18,19)/t10-/m0/s1

(2-phenylacetyl)-L-glutamine

Phenylacetyl-L-glutamine; NSC 203800; NSC-203800; Phenylacetylglutamine; 28047-15-6; PHENYLAC-GLN-OH; Phenylacetyl L-Glutamine; (S)-5-Amino-5-oxo-2-(2-phenylacetamido)pentanoic acid; Phenylacetyl-L-glutamine; alpha-N-Phenylacetyl-L-glutamine; L-Glutamine, N2-(phenylacetyl)-; NSC203800 PA-L-Glutamine;

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~378.39 mM)
H2O : ~25 mg/mL (~94.60 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 50 mg/mL (189.19 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)