兴奋剂重组人促红细胞生成素及其类似物检测方法的研究进展

重组人促红细胞生成素(rhEPO)是一种激素类兴奋剂,近年来被滥用在一些耐力性比赛项目中。由于重组与内源性EPO的氨基酸序列相同,区别很小,并且在尿样或血样中的浓度低,代谢快,给检测带来了很大的难度。本文从直接方法和间接方法两个方面综述了近几年来兴奋剂rhEPO及其类似物检测的研究进展,并结合本小组的工作展望了rhEPO检测的发展方向。     

Mass spectrometric detection of siRNA in plasma samples for doping control purposes

Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25–1 nmol mL⁻¹) and performance (precision 11–21%, recovery 23–67%) for typical antisense oligonucleotides currently used in clinical studies.     

Total synthesis of 4-F3t-neuroprostane and its 4-epimer

The first synthesis of 4-F_3t-Neuroprostane 1a and its 4-epimer is described. This molecule presents an important contribution to the study of neuronal oxidative stress in DHA-ω3 depleted brain. The key step involves the introduction of two unsaturated side chains into the chiral polyfunctional cyclopentane 4 via E selective HWE reaction and Z selective Wittig olefination for α and ω chains, respectively.     

Synthesis of 3-deoxy-3,3-difluoroshikimic acid and its 4-epimer from quinic acid

3-Deoxy-3,3-difluoroshikimic acid 2 and its 4-epimer 3 as new analogues of shikimic acid have been synthesised from quinic acid in overall yields of 30% and 12%, respectively.     

A total synthesis of (+)-preussin and its 5-epimer

(+)-Preussin (1) and its 5-epimer were synthesized from the divmylcarbmol (3) with Sharpless asymmetric epoxidation of 3 and the oxidative cyclization of 9 with PDC as the key steps     

Total synthesis of mycestericin A and its 14-epimer

The total synthesis of mycestericin A (1) and its 14-epimer 34 is described herein. The Overman rearrangement of an allylic trichloroacetimidate derived from l-tartrate generated a tetra-substituted carbon with nitrogen and subsequent stereoselective transformations afforded the highly functionalized left-half segment, vinyl iodide. Cross-coupling of the vinyl iodide with a chiral organometallic species synthesized from d-tartrate under the Negishi or Suzuki-Miyaura coupling conditions, followed by deprotection, completed the total synthesis of 1. The 14-epimer of mycestericin A was also synthesized, and a comparison of [α]_D values of peracetyl γ-lactone derivatives of mycestericin A and its 14-epimer as well as degradation studies of 1 and 34 fully confirmed the proposed absolute structure of mycestericin A.     

Stereoselective synthesis of (+)-boronolide and its 8-epimer

Starting from readily available carbohydrates the synthesis of 8-epi-(+)-boronolide 19 and (+)-boronolide was achieved with diastereoselective propargylation of alpha-oxygenated aldehyde as a key step.     

A simple synthesis of 6-deoxoteasterone and its 20-epimer

6-Deoxoteasterone, a brassinolide biosynthetic intermediate, and its 20-epimer were synthesized from steroidal 17-olefin and chiral α-alkoxyaldehyde using a Lewis acid mediated carbonyl-ene reaction as the key step. In this reaction, unusual stereoselectivity was observed.     

Asymmetric synthesis of (+)-passifloricin A and its 6-epimer

Stereoselective total syntheses of the antiprotozoal natural product (+)-passifloricin A and its C-6 epimer have been achieved in ～5% overall yield. The strategy is based on Jacobsen epoxidation, Grubbs’ metathesis and an Evans’ intramolecular oxa-Michael reaction.     

Total synthesis of lycoperdic acid and its C4-epimer

Efficient stereodivergent syntheses of (+)-lycoperdic acid (LPA) and 4-epi-LPA have been achieved based on asymmetric hydrogenation (H₂, Rh/(R,S)-MeBoPhoz) of racemic enamide as a key step.     

6alpha-Methylandrostenedione: gas chromatographic mass spectrometric detection in doping control

In recent years products containing 6alpha-methylandrost-4-ene-3,17-dione have appeared on the sport supplement market. Scientific studies have proven aromatase inhibition and anabolic and mild androgenic properties; however, no preparation has been approved for medical use up to now. In sports 6alpha-methylandrost-4-ene-3,17-dione has to be classified as a prohibited substance according to the regulations of the World Anti-Doping Agency (WADA). For the detection of its misuse the metabolism was studied following the administration of two preparations obtained from the Internet (Formadrol and Methyl-1-Pro). Several metabolites as well as the parent compounds were synthesized and the structures of 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, 6alpha-methylandrost-4-ene-3,17-dione, and 5beta-dihydromedroxyprogesterone were confirmed by nuclear magnetic resonance (NMR) spectroscopy. The main metabolite, 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, was found to be excreted as glucuronide and was still detectable in microg/mL amounts until urine collection was terminated (after 25 h). Additionally, samples from routine human sports doping control had already tested positive for the presence of metabolites of 6alpha-methylandrost-4-ene-3,17-dione. Screening analysis can be easily performed by the existing screening procedure for anabolic steroids using 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one as target substance (limit of detection <10 ng/mL). Its discrimination from the closely eluting drostanolone metabolite, 3alpha-hydroxy-2alpha-methyl-5alpha-androstan-17-one, is possible as the mono-TMS derivative.     

Total synthesis of natural (+)-membrenone C and its 7-epimer

Highly stereoselective asymmetric total syntheses of the polypropionate marine defense substance (+)-membrenone C and its 7-epimer have been achieved. Highlights of the strategy include the utilization of a desymmetrization technique to create five contiguous chiral centres from a single bicyclic precursor.     

First total synthesis of parvistone C and its C8-epimer

Diastereoselective first total synthesis of parvistone C 1 and C8-epimer 1a are described. The key features of our synthesis include Sharpless asymmetric dihydroxylation, stereoselective aryl Grignard reactions, Still–Gennari olefination, and intramolecular cyclization.     

Diastereoselective syntheses of deoxydysibetaine, dysibetaine, and its 4-epimer

(+/-)-Deoxydysibetaine 2 and 4-epi-dysibetaine 3 were prepared in a few steps from methyl pyroglutamate through a regioselective Mannich reaction at C-2. Natural (2S,4S)-dysibetaine 1, a sponge metabolite isolated from Dysidea herbacea, and (2S)-2 were synthesized from enantiopure (S)-pyroglutaminol with very high stereoselectivity. The key steps were an original formation of stereogenic quaternary center C-2 and the diastereoselective hydroxylation at C-4.     

基因兴奋剂检测的现状与策略

本文综述了基因兴奋剂检测的现状和反基因兴奋剂的策略。归纳了可能被运动员滥用的基因兴奋剂,分析了由这些基因表达的促红细胞生成素(EPO)、人生长激素(hGH)等蛋白的检测进展,讨论了未来检测基因兴奋剂的可能策略。     

Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.     

Potential parameters for the detection of hGH doping

The aim of our hGH application study with non-competitive athletes was the investigation of selected serum parameters from different processes affected by hGH. Fifteen athletes (age 21-33, mean 24) were treated with 0.06 IU hGH/kg BW per day or placebo (10 hGH, 5 placebo) respectively for 14 days. Blood samples were taken prior to, during and until 10 weeks after treatment.The concentrations of the following markers were determined in relevant serum samples: IGF-I, IGFBP-3, ALS, PIIINP, PINP, osteocalcin, and leptin. The IGF-I concentration increased rapidly within the hGH treatment group and showed significantly higher levels compared to baseline even 3 days after application. The response of the IGFBP-3 to the hGH applications was lower in comparison to IGF-I. The hGH group showed an increasing IGFBP-3 compared to baseline from day 4 till day 15. The response of PIIINP to hGH is clearly delayed compared to the IGF-I axis, but the PIIINP concentration remains on an increased level for a longer period (from day 4 until day 21). The time course and the extent of response varied strongly interindividually. PINP and osteocalcin showed only a small response to hGH applications. These parameters are characterised by a strong scattering of base values compared with the small response. In the hGH treatment group very different leptin concentrations were found at the beginning of the study, but after treatment decreasing leptin levels were observed in all cases.The determination of only one parameter will not be sufficient for detection of hGH abuse. A combination of markers by mathematical methods can be helpful to distinguish between placebo and hGH-treated athletes. By using the suggested discriminant function the data sets of hGH and placebo-treated athletes could be separated without false positive results.     

Procedures for monitoring recombinant erythropoietin and analogues in doping control

The present report summarizes the main analytical strategies developed to identify the presence of recombinant erythropoietin (EPO) administered as a doping agent. Indirect evidence is based on the analysis of blood parameters (haemoglobin, haematocrit, reticulocytes, macrocytes, etc.) and serum markers (concentration of EPO and serum transferrin receptors, etc.). The problem of intertechnique comparison for reliable results evaluation is emphasized, especially for serum markers. Charge differences between isoforms of recombinant EPO and native urinary EPO are the grounds for the isoelectric focusing–double blotting–chemiluminescence detection method presently approved for doping control. Works addressing its advantages and limitations are presented and commented on. The chemical bases of the differential detection are highlighted and some future approaches for detection are also presented. The appearance and detectability of EPO analogues and mimetics susceptible for abuse are also addressed.