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1.
Org Lett ; 25(27): 5001-5005, 2023 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-37382389

RESUMO

The solution-based gram-scale synthesis of complex and highly potent proprotein convertase subtilisin-like/kexin type 9 (PCSK9) inhibitor 1 is presented. Construction of Northern fragment 2, followed by stepwise installation of Eastern 3, Southern 4, and Western 5 fragments, provided macrocyclic precursor 19. This intermediate was cross-linked via an intramolecular azide-alkyne click reaction, which preceded macrolactamization to afford the core framework of compound 1. Finally, coupling with poly(ethylene glycol) side-chain-based 6 gave the PCSK9 inhibitor 1.


Assuntos
Pró-Proteína Convertase 9
2.
Carbohydr Polym ; 289: 119465, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35483863

RESUMO

Quantitation of isomeric pneumococcal polysaccharides in vaccines is a challenging task due to mixture complexity, their low quantities, and identical monosaccharide compositions. Differentiation and quantitation of isomeric pneumococcal polysaccharides were investigated here based on a partial chemical degradation mass spectrometry approach to generate an oligosaccharide marker for one isomer, and not the other. Mild base conditions were successful at generating unique ions for the isomers with the weakest glycosidic bonds, while strong base and acid conditions were successful at generating unique ions for the more stable isomers. Linear relationships between the ion abundance of the oligosaccharide marker and the starting pneumococcal polysaccharides concentration were established for all isomers. Furthermore, precision measurements for each method were below 12% demonstrating good robustness. Therefore, partial chemical degradation followed by mass spectrometry was successful at differentiating and quantifying isomeric pneumococcal polysaccharides and may be adopted for other bacterial types.


Assuntos
Oligossacarídeos , Vacinas Pneumocócicas , Íons , Isomerismo , Espectrometria de Massas/métodos , Oligossacarídeos/química
3.
Nature ; 603(7901): 439-444, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35296845

RESUMO

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.


Assuntos
Guanosina , Nucleotidiltransferases , Adenosina , Animais , Interferons , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais
4.
J Med Chem ; 65(1): 485-496, 2022 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-34931831

RESUMO

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Dor/tratamento farmacológico , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/farmacologia , Venenos de Aranha/síntese química , Animais , Degranulação Celular/efeitos dos fármacos , Cistina/química , Desenho de Fármacos , Temperatura Alta , Mastócitos/efeitos dos fármacos , Modelos Moleculares , Medição da Dor/efeitos dos fármacos , Ratos , Venenos de Aranha/farmacologia
5.
J Chromatogr A ; 1595: 190-198, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-30803788

RESUMO

The evolution of supercritical fluid chromatography (SFC) instrumentation, improved detection capability, and expanded modifier range has led to extending the reach of SFC to the analysis of a broader spectrum of analytes beyond enantioselective separations. However, preparative SFC has yet to see the same technological revitalization, especially in regards to the purification of highly polar analytes. Enhanced fluidity liquid chromatography (EFLC) has been demonstrated as one of the ways to extend the applicable range of SFC instrumentation to highly polar analytes such as proteins, carbohydrates, and nucleotides. Despite recent applications of EFLC for challenging mixtures of hydrophilic metabolites and analogs, its viability in preparative purification, which is of great importance to the pharmaceutical industry, remains unknown. Herein, multiple chromatographic parameters that are critical to achieve feasible EFLC purification methods were investigated, including system pressure as a function of modifier composition (for several MeOH:H2O ratios), effect of diluent injection conditions on peak shape, and optimization of mass load with diluent composition. The usage of 50% acetonitrile or methanol diluents provided the most volumetric loading capacity. In the case of sucrose, leveraging higher analyte solubility in water proved to be more favorable than the volumetric loading capacity of diluents with higher organic content. In fact, an 80 mg injection of sucrose was possible on a 2 cm preparative HILIC column with minimal peak shape degradation. The combined information led to the successful demonstration of EFLC for the preparative separation of sugars using readily available MS-directed SFC instrumentation.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia com Fluido Supercrítico , Acetonitrilas/química , Interações Hidrofóbicas e Hidrofílicas , Metanol/química , Nucleotídeos/química , Proteínas/química , Água/química
6.
Chem Sci ; 9(2): 415-424, 2018 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-29629112

RESUMO

This work describes the application of vibrational (VCD) and electronic (ECD) circular dichroism spectroscopy to solve the longstanding debate around the absolute configuration of (+)-frondosin B (1). The absolute configuration of (+)-1 could confidently be assigned as (R) using these spectroscopic techniques. The discrepancy in the optical rotation (OR) values obtained in previous studies can be attributed to an undetected minor impurity (ca. 7%) that arose unexpectedly in a key step late in the synthesis. Additionally, the conditions used in the final step of the previous reports for demethylation to form the natural product proceeded with significant loss of enantiopurity. The large OR measured for the impurity at its observed level, when compared to the small rotation for the less enantiopure natural product 1, led to a measured OR value for the synthetic material that had the opposite sign of the natural product.

7.
J Chromatogr A ; 1429: 374-9, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26747691

RESUMO

MISER chromatographic analysis (Multiple Injections in a Single Experimental Run) using supercritical fluid chromatography (SFC) with pressurized carbon dioxide-based eluents is well suited to the high throughput analysis of enantiopurity. SFC is currently the preferred method for fast enantiopurity analysis, with analysis times of only a few seconds achievable in some cases. Injector programming using both the Agilent Infinity and Shimadzu Nexera UC instruments permitted MISER SFC experiments to be performed. Several case studies are presented, showcasing the power and versatility of the technique, with 'plate analysis times' (the time required for analysis of enantiopurity of 96 samples) of less than 33-34 min achievable in the best cases.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia com Fluido Supercrítico , Dióxido de Carbono/química
8.
J Chromatogr A ; 1363: 250-6, 2014 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-25082525

RESUMO

Fast chiral supercritical fluid chromatography (SFC) separations have become important due to the increasing use of high-throughput experimentation (HTE) in organic synthesis. These HTE experiments can generate hundreds of samples for chiral analysis that need to be assayed in a short time. In general, chiral SFC can provide much faster analysis times compared to liquid chromatography (LC). Additionally, columns packed with smaller particles can provide faster and more efficient separations. In this study, the effect of the particle size on the speed and resolution of chiral separations by SFC was evaluated. The performance of Chiralcel OD columns packed with either 5 or 3 µm particles were compared using van Deemter or other kinetic plots. The benefits of using smaller particle columns for chiral SFC analysis are illustrated.


Assuntos
Cromatografia com Fluido Supercrítico/métodos , Cinética , Tamanho da Partícula
9.
J Pharm Biomed Anal ; 96: 54-7, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-24727281

RESUMO

A new core-shell particle column showed excellent performance and durability for separation of short (∼21-mer) ribonucleic acid (RNA) oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC). Previously investigated core-shell C18 columns showed excellent peak shapes and separations of closely eluting impurities by IP-RPLC. However, these columns showed only modest long-term stability at the neutral pH and elevated column temperatures of ≥60°C, typically used for IP-RPLC analysis of oligonucleotides. The newly introduced SunShell C18 column provided separations comparable to the previously evaluated core-shell columns, but with significantly improved long-term column stability when operated at neutral pH and elevated column temperature.


Assuntos
Cromatografia de Fase Reversa/métodos , Oligonucleotídeos/análise , Concentração de Íons de Hidrogênio , Oligonucleotídeos/química , Temperatura , Fatores de Tempo
10.
Chirality ; 25(11): 799-804, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23946148

RESUMO

In this study we describe the evaluation of a recently developed supercritical fluid chromatography (SFC) instrument for automated chiral SFC method development. The greatly improved gradient dwell volume and liquid flow control of the new instrument in combination with the use of shorter columns containing smaller stationary phase particles affords chiral SFC method development that is faster and more universal than previous systems.


Assuntos
Cromatografia/métodos , Estereoisomerismo , Estilbenos/química
11.
J Chromatogr A ; 1304: 69-77, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23859796

RESUMO

New mixed-mode columns consisting of reversed-phase and ion-exchange separation modes were evaluated for the analysis of short RNA oligonucleotides (∼20mers). Conventional analysis for these samples typically involves using two complementary methods: strong anion-exchange liquid chromatography (SAX-LC) for separation based on charge, and ion-pair reversed-phase liquid chromatography (IP-RPLC) for separation based on hydrophobicity. Recently introduced mixed-mode high performance liquid chromatography (HPLC) columns combine both reversed-phase and ion-exchange modes, potentially offering a simpler analysis by combining the benefits of both separation modes into a single method. Analysis of a variety of RNA oligonucleotide samples using three different mixed-mode stationary phases showed some distinct benefits for oligonucleotide separation and analysis. When using these mixed-mode columns with typical IP-RPLC mobile phase conditions, such as ammonium acetate or triethylammonium acetate as the primary ion-pair reagent, the separation was mainly based on the IP-RPLC mode. However, when changing the mobile phase conditions to those more typical for SAX-LC, such as salt gradients with NaCl or NaBr, very different separation patterns were observed due to mixed-mode interactions. In addition, the Scherzo SW-C18 and SM-C18 columns with sodium chloride or sodium bromide salt gradients also showed significant improvements in peak shape.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Oligonucleotídeos/isolamento & purificação , Sequência de Bases , Cromatografia por Troca Iônica/métodos , Oligonucleotídeos/química
12.
J Pharm Biomed Anal ; 72: 25-32, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23146223

RESUMO

An investigation into the use of core-shell particle columns for separation of short (∼21 base pairs) RNA oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC) showed improved resolution for a number of test analytes relative to conventional (fully-porous) reversed-phase columns. The best resolutions were obtained using columns packed with smaller sub-2µm core-shell particles.


Assuntos
Cromatografia de Fase Reversa/métodos , Oligonucleotídeos/análise , Oligonucleotídeos/química , Cromatografia de Fase Reversa/instrumentação , Tamanho da Partícula
13.
Chirality ; 23(2): 128-32, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20803492

RESUMO

Calculation of chromatographic enantioselectivity (α) is critically important in enantioselective chromatographic method development studies. The fact that α can only be calculated from isocratic elution conditions, whereas gradient elution conditions are predominantly used in method development screening, presents some problems for the use of α as a scoring indicator for automated, intelligent enantioselective chromatography method development systems. In this study, an empirical algorithm was developed to estimate α at isocratic conditions based upon information collected from a gradient elution. The algorithm was validated for SFC applications and has been shown to accurately predict enantioselectivity for a wide variety of racemic test analytes eluted on different chiral column and mobile phase conditions.


Assuntos
Algoritmos , Celulose/análogos & derivados , Técnicas de Química Analítica/métodos , Cromatografia Líquida/métodos , Cromatografia com Fluido Supercrítico/métodos , Bibliotecas de Moléculas Pequenas/química , Estereoisomerismo
14.
J Chromatogr A ; 1217(7): 1134-8, 2010 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-19850298

RESUMO

A systematic approach to the evaluation of new chiral stationary phases (CSPs) for supercritical fluid chromatography (SFC) using a standard library of racemic analytes is described. A standard library of racemic analytes representing a variety of functional group classes was assembled from a mixture of proprietary and commercial compounds. The library is dispensed and stored in a convenient 96-well microplate format to facilitate ease of use, and to minimize the amount of analyte required for analysis. Automated SFC screening was performed on both established CSPs in common use, as well as a group of six recently commercialized CSPs. Screening results were archived in a structure-searchable database that allows convenient comparison of performance data to determine which CSPs shows the best performance.


Assuntos
Cromatografia com Fluido Supercrítico/métodos , Bibliotecas de Moléculas Pequenas/química , Flavanonas/química , Estereoisomerismo
15.
J Pharm Biomed Anal ; 51(1): 153-7, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-19766423

RESUMO

Simultaneous enantioseparation of a basic API compound, (R)-2-Amino-N-[2-[1,2-dihydro-1-(methylsulfonyl) spiro [3H-indole-3,4'-piperidin]-1'-yl]-2-oxo-1-[(phenylmethyloxy) ethyl]-2-methylpropanamide monomethanesulfonate (compound-A) and its neutral penultimate intermediate, (R)-2-BOC-Amino-N-[2-[1,2-dihydro-1-(methylsulfonyl) spiro [3H-indole-3,4'-piperidin]-1'-yl]-2-oxo-1-[(phenylmethyloxy) ethyl]-2-methylpropanamide monomethanesulfonate (compound-B) was investigated using reversed phase (RPLC) and normal phase liquid chromatography (NPLC). After an initial screening, a Sepapak-4 column, a new type of polysaccharide chiral stationary phase (CSP), was selected for further method development based on hits on separation selectivity for both compounds under RPLC and NPLC. After comparing the pros and cons, a method utilizing the Sepapak-4 chiral column (150 mm x 4.6 mm, 3 microm particle size) in RPLC mode was finally developed. Separations were performed in gradient elution mode starting at 50% A (10 mM, NH(4)COOH at pH 6.5)/50% B (50/50 EtOH/MeCN) to 25% A (10 mM, NH(4)COOH at pH 6.5)/75% B (50/50 EtOH/MeCN). The flow rate was 1.0 mL/min; the column temperature was 50 degrees C; the UV wavelength was 220nm and the mass spectrometric detection was APCI in the positive ionization mode. The reaction mixture sample was directly diluted in ethanol. Baseline enantioseparation were achieved for both compound-A and its intermediate simultaneously with resolution greater than 2.0. The method was validated in terms of injection precision, linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, and ruggedness. The specificity of the method was further evaluated by spiking a mixture of enantiomers of compound-A and its intermediate into a reaction matrix containing all of the synthetic reagents. No matrix interference was observed across the elution windows of compound-A and its intermediate. Additionally, the peak purity of each enantiomer was evaluated by mass spectra, indicating the specificity of the method.


Assuntos
Ácidos Aminoisobutíricos/química , Cromatografia Líquida/métodos , Sulfonamidas/química , Ácidos Aminoisobutíricos/análise , Carbamatos/análise , Carbamatos/química , Espectrometria de Massas/métodos , Estereoisomerismo , Sulfonamidas/análise
16.
Chirality ; 21 Suppl 1: E105-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19885821

RESUMO

A novel family of atropisomers based on a conformationally constrained seven membered ring system is investigated using a combination of preparative chiral chromatography, circular dichroism, and other analytical techniques. The influence of structure on the rate of atropisomer interconversion was explored with a series of analogs showing a range of interconversion rates ranging from very fast (undetectable on the HPLC timescale) to very slow (half life of many days).


Assuntos
Antipsicóticos/química , Antipsicóticos/classificação , Sulfonamidas/química , Sulfonamidas/classificação , Cromatografia Líquida de Alta Pressão/métodos , Dicroísmo Circular/métodos , Meia-Vida , Conformação Molecular , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade
17.
Artigo em Inglês | MEDLINE | ID: mdl-18819851

RESUMO

Owing to slow rotation of a sterically constrained dimethylamide substituent, two slowly interconverting enantiomers of a preclinical candidate for pharmaceutical development, 1, (6-(3-Chloro-4-fluoro-benzyl)-4-hydroxy-2-methyl-3,5-dioxo-2,3,5,6,7,8-hexahydro-[2,6]naphthyridine-1-carboxylic acid dimethylamide) are observed by chiral chromatography. Isolation of pure enantiomer by preparative chiral chromatography followed by enantiopurity analysis over time allowed for a study of the kinetics of enantiomer interconversion under a variety of conditions. Relatively slow racemization was observed in alcohol solvents, with a half life on the order of 5-10 h. A dramatic influence of aqueous buffer pH on racemization was noted, with higher pH leading to rapid racemization within a few minutes, and lower pH leading to essentially no racemization for periods up to a week. A hypothesis explaining this unusual effect of pH on carboxamide bond rotation is offered, and some suggestions for potential utility of such a system are considered.


Assuntos
Amidas/química , Cromatografia Líquida de Alta Pressão/métodos , Dimetilaminas/química , Naftiridinas/química , Rotação Ocular , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Espectrofotometria Ultravioleta
18.
Org Lett ; 10(5): 741-4, 2008 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-18247494

RESUMO

Herein we report the asymmetric synthesis of 1,2-dipyridyl-1,2-diarylethanes via an unusual Cu(I)-catalyzed dimerization reaction. Subjection of a variety of enantioenriched substituted 2-pyridyl alcohols to a one-pot protocol generates the desired products in good yields and diastereoselectivities and with ee's up to >99%.


Assuntos
Cobre/química , Etano/análogos & derivados , Etano/síntese química , Hidrocarbonetos Bromados/síntese química , Catálise , Etano/química , Hidrocarbonetos Bromados/química , Estrutura Molecular , Estereoisomerismo
19.
Chirality ; 19(10): 787-92, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17722016

RESUMO

Despite its status as the preferred method for routine enantiopurity analysis in pharmaceutical research, supercritical fluid chromatography (SFC) has historically been unsuited for the accurate and precise measurements required for release testing of active pharmaceutical ingredients (APIs) under current good manufacturing processes (cGMPs). Insufficient signal to noise, as compared to HPLC, has heretofore been the major limitation of the chiral SFC approach. We herein describe an investigation into the fundamental limitations and sources of noise in the SFC approach, identifying thermal, electronic, and mechanical sources of noise within the flow cell as key parameters contributing to reduced sensitivity. A variety of instrument modifications are explored, ultimately leading to the development of a new and improved flow cell and other instrument modifications that allow suitable sensitivity and accuracy to carry out GMP release testing for enantiopurity analysis using SFC.


Assuntos
Cromatografia/métodos , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/química , Dióxido de Carbono , Cromatografia/instrumentação , Cromatografia/estatística & dados numéricos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/estatística & dados numéricos , Desenho de Equipamento , Sensibilidade e Especificidade , Estereoisomerismo
20.
Chirality ; 19(9): 693-700, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17354260

RESUMO

The modern use of preparative chromatography in pharmaceutical development is illustrated by the case of a recent preclinical candidate from these laboratories. The synthesis of the candidate employed a coupling of two enantiopure intermediates, each of which could be resolved using preparative chiral chromatography. SFC screening was employed to identify the enantioselective stationary phases, and semipreparative SFC methods derived from this screening were used to produce gram amounts of enantiopure intermediate for initial studies. However, initial larger scale resolution required the translation of the SFC methods to HPLC conditions. Preparative chiral HPLC on a 30-cm i.d. column was then used to produce enantiopure intermediates which were coupled to give 170 g of the preclinical candidate. Subsequent preparation of the candidate at larger scale for later-stage clinical evaluation employed an improved synthesis in which one component was constructed by asymmetric synthesis. Resolution of the other component, now a more advanced intermediate, was carried out using newly obtained large-scale SFC equipment. Some discussion is presented on the varying strategies whereby preparative chiral chromatography can be used to support either short-term or long-term synthetic goals in preclinical pharmaceutical development.


Assuntos
Química Farmacêutica/métodos , Cromatografia/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Desenho de Fármacos , Modelos Químicos , Preparações Farmacêuticas/química , Estereoisomerismo , Tecnologia Farmacêutica/métodos , Fatores de Tempo
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