Trace analysis of dimethoxytrityl alcohol (DMT-OH) in oligonucleotide matrices using liquid chromatography coupled with tandem mass spectrometry.

A liquid chromatography (LC) method with ultraviolet (UV) and tandem mass spectrometry (MS/MS) detection was developed to quantify dimethoxytrityl alcohol (DMT-OH), a small molecule byproduct generated during the detritylation reaction in oligonucleotide synthesis. The pros and cons of quantification via multiple analytical methods including LC coupled with UV, selected ion monitoring (SIM), and multiple reaction monitoring (MRM) were evaluated. The MRM method was ultimately selected for further qualification and exhibited good linearity (R2 = 0.997 from 0.5 to 64 ng mL-1), accuracy (recoveries ranging 75-90% with ≤ 2% RSD), repeatability (<5% RSD), and sensitivity (LOQ of 1.6 ng mL-1). The MRM method was further applied to analyze DMT-OH in various oligonucleotide intermediates and drug substances. Similar MRM methods for six other small molecule impurities (aniline, benzamide, isobutyramide, 2-phenylacetamide, succinamide, and uny-CTP) as well as their application are also presented.

Development of Kilogram-Scale Convergent Liquid-Phase Synthesis of Oligonucleotides.

Oligonucleotide drugs show promise to treat diseases afflicting millions of people. To address the need to manufacture large quantities of oligonucleotide therapeutics, the novel convergent liquid-phase synthesis has been developed for an 18-mer oligonucleotide drug candidate. Fragments containing tetra- and pentamers were synthesized and assembled into the 18-mer without column chromatography, which had a similar impurity profile to material made by standard solid-phase oligonucleotide synthesis. Two of the fragments have been synthesized at ∼3 kg/batch sizes and four additional tetra- and pentamer fragments were synthesized at >300-g scale, and a 34-mer was assembled from the fragments. Critical impurities are controlled in the fragment syntheses to provide oligonucleotides of purities suitable for clinical use after applying standard full-length product purification process. Impurity control in the assembly steps demonstrated the potential to eliminate chromatography of full-length oligonucleotides, which should enhance scalability and reduce the environmental impact of the process. The convergent assembly and telescoping of reactions made the long synthesis (>60 reactions) practical by reducing production time, material loss, and chances for impurity generation.

Selective activation/coupling of polyhalogenated nucleophiles in ni/cr-mediated reactions: synthesis of c1-c19 building block of halichondrin bs.

The C1-C19 building block 46 of halichondrin Bs was synthesized via a selective activation/coupling of ß-bromoenone 34 with aldehyde 35 in a Ni/Cr-mediated reaction. The first phase of study was a method development to effect a coupling of a "naked" vinylogous anion with an aldehyde. The study with the coupling of 9 + 10 → 11 revealed: (1) ß-bromoenone 9b is a better nucleophile than the corresponding ß-iodo- and ß-chloroenones 9a,c; (2) (Me)2Phen(OMe)2·NiCl2 13b is a better Ni-catalyst than (Me)2Phen(H)2·NiCl2 13a; and (3) a low Ni-catalyst loading, for example, 0.05-0.1 mol % Ni-catalyst against 10 mol % Cr-catalyst, is crucial for an effective coupling. The second phase of study was a method development to realize a selective activation/coupling of polyhalogenated nucleophiles such as 34. The competition experiment of 10 + 9b over 10 + 31a-c revealed: (1) (Me)2Phen(OMe)2·NiCl2 13b is more effective than (Me)2Phen(H)2·NiCl2 13a for the required selective activation/coupling; (2) a low Ni-catalyst loading, for example, 0.05-0.1 mol % Ni-catalyst against 10 mol % Cr-catalyst, is crucial for discriminating ß-bromoenone 9b from the three types of vinyl iodides 31a-c. The third phase of study was an application of the developed method to execute the proposed coupling of 34 + 35 → 36. For this application, a polyether-type Ni-catalyst 37c, readily soluble in the reaction medium, was introduced to achieve the selective activation/coupling with higher efficiency. With use of ion-exchange resin-based device, the coupling product 36 was transformed to the C1-C19 building block 46 of halichondrin Bs without purification/separation of the intermediates.

Unified Synthesis of C1-C19 Building Blocks of Halichondrins via Selective Activation/Coupling of Polyhalogenated Nucleophiles in (Ni)/Cr-Mediated Reactions.

A unified synthesis of the C1-C19 building blocks 8-10 of halichondrins A-C was developed from the common synthetic intermediates 26a,b. Acetylenic ketones 26a,b were in turn synthesized via selective activation/coupling of polyhalogenated nucleophiles 23a,b with aldehyde 11 in a (Ni)/Cr-mediated coupling reaction. Compared with Ni/Cr-mediated couplings of vinyl iodides and aldehydes, this (Ni)/Cr-mediated coupling exhibited two unique features. First, the coupling was found to proceed with a trace amount or no added Ni-catalyst. Second, TES-Cl, a dissociating agent to regenerate the Cr-catalyst, was found to give a better yield than Zr(Cp)2Cl2. An adjustment of the oxidation state was required to transform acetylenic ketones 26a,b into C1-C19 building blocks 8 and 9 of halichondrins A and B, respectively. In the halichondrin B series, a hydroxyl-directed (Me)4NBH(OAc)3 reduction of E- and Z-ß-alkoxy-enones 30 was found cleanly to achieve the required transformation, whereas a DMDO oxidation of E-vinylogous ester 27 allowed to introduce the C13 hydroxyl group with a high stereoselectivity in the halichondrin A series. In the halichondrin C series, Hf(OTf)4 was used to convert the double oxy-Michael product 28 into C1-C19 building block 10.

Facile synthesis of fluorescent active triazapentalenes through gold-catalyzed triazole-alkyne cyclization.

Fluorescent active triazapentalene zwitterions (TAPZs) were prepared through Au(I) catalyzed triazole-alkyne 5-endo-dig cyclization. While an effective gold catalyst turnover (0.5% loading, up to 96% yield) was achieved, the stability of these new 10-π-electron bicyclic structures was also significantly improved, which warranted future applications of these fluorescent dyes.

A pillared metal-organic framework incorporated with 1,2,3-triazole moieties exhibiting remarkable enhancement of CO2 uptake.

The replacement of the pillar ligand, 4,4'-bipyridine, in the prototypal pillared MOF, MOF-508, with the custom-designed ligand, 4,4'-(2H-1,2,3-triazole-2,4-diyl)dipyridine, affords a porous metal-triazolate framework, MTAF-3, which demonstrates remarkable enhancement of CO(2) uptake capacity by a factor of ~3 compared to the parent MOF-508.

1,2,3-triazole: unique ligand in promoting iron-catalyzed propargyl alcohol dehydration.

A 1,2,3-traizole-promoted iron(III)-catalyzed propargyl alcohol dehydration was developed for the synthesis of conjugated enynes. The desired conjugated enynes were prepared in good to excellent yields (up to 95%) with a large substrate scope and excellent stereoselectivity (only Z-isomers).

Porous double-walled metal triazolate framework based upon a bifunctional ligand and a pentanuclear zinc cluster exhibiting selective CO2 uptake.

The self-assembly of a custom-designed bifunctional ligand featuring both 1,2,3-triazolate and carboxylate donor groups with a pentanuclear zinc cluster generated in situ affords a double-walled metal triazolate framework (MTAF) material, MTAF-1 (Zn(5)(µ(3)-O)(2)(C(9)N(3)H(5)O(2))(5)(H(+))(4)(H(2)O)(17)(C(3)H(7)NO)(10)), which exhibits a surface area of 2300 m(2)/g and demonstrates interesting selective CO(2) uptake performances.

Synthesis of allene triazole through iron catalyzed regioselective addition to propargyl alcohols.

Allene triazole derivatives were successfully synthesized for the first time through iron catalyzed regioselective triazole addition to tertiary propargyl alcohols. The reaction proceeds under mild conditions, giving the desired allene-triazoles in good to excellent yields (up to 96%). The resulting allene-triazoles were confirmed by X-ray crystallography and indicated improved stability.

N-2-aryl-1,2,3-triazoles: a novel class of UV/blue-light-emitting fluorophores with tunable optical properties.

The N-2-aryl-1,2,3-triazole derivatives (NATs) were developed as a new class of UV/blue-light-emitting fluorophores. Though both N-1-aryl-1,2,3-triazoles and N-2-aryl-1,2,3-triazoles gave strong photo absorption under excitation at 330 nm, only the N-2-analogous showed strong fluorescence emission in the UV/blue range with high efficiency in various solvents (quantum yield Φ around 0.3-0.5). Significant substituted group effects were observed, allowing tunable optical properties with emission (λ(max)) from 350-400 nm and Stokes shift from 38-93 nm. The computational studies along with X-ray crystal structures indicated the significance of the effective conjugation between triazole ring and aryl groups on the N-2 position. The planar intramolecular charge transfer (PICT) mechanism was proposed, which was supported by solvent effect studies. Simple derivatizations gave NAT-modified lysine and strong UV/blue emitting bis-NAT (Φ=0.76, λ(max)=390), which suggested the great potential of this new class of fluorophores in biological and material science research.

Iron-catalyzed C-O bond activation for the synthesis of propargyl-1,2,3-triazoles and 1,1-bis-triazoles.

The FeCl(3)-catalyzed triazole propargylation was developed. This transformation was suitable for a large scope of substituted propargyl alcohols, giving the corresponding propargyl triazoles in excellent yields (up to 96%). Further derivatization produced the 1,1-bis-triazoles in excellent yields and regioselectivity, which could be applied as potential transition metal ligands or new reagents.

1,2,3-Triazole as a special "X-factor" in promoting hashmi phenol synthesis.

(31)P NMR experiments revealed rapid decomposition of the Ph(3)PAu(+)TfO(-) complex in the presence of the furan-yne, which resulted in poor reactivity as reported in the literature. Therefore, instead of tuning different ligands (PR(3)), the 1,2,3-triazole was applied as a special X-factor to stabilize the catalyst. The desired phenol products were prepared in excellent yields (1% cat. up to 95% yield) and chemoselectivity (>20:1).

Highly efficient synthesis of vinyl substituted triazoles by Au(I) catalyzed alkyne activation.

Au(I) catalyzed 1,2,3-triazole addition to non-activated alkyne was reported. A large group of substituted NH-1,2,3-triazoles were suitable for this transformation along with both internal and terminal alkynes. The N-1 and N-2 vinyl substituted 1,2,3-triazoles were prepared in up to 98% yield with as low as 0.2% catalyst loading, thereby providing a new protocol for the synthesis of potentially biological-active vinyl-triazole building blocks.

Efficient synthesis of N-2-aryl-1,2,3-triazole fluorophores via post-triazole arylation.

Efficient post-triazole regioselective N-2 arylation was developed from C-4, C-5 disubstituted-1,2,3-NH-triazoles. Three different approaches had been investigated, including S(N)Ar, Cu(I) catalyzed aryl amidation and Cu(II) mediated boronic acid coupling. The N-2-aryl triazoles were successfully synthesized with excellent yields. The structures were characterized by X-ray crystallography and some N-2-triazole products gave strong fluorescence with various emission controlled by the C-5 groups.