Search

β-Tryptase, a homotetrameric serine protease, has four identical active sites facing a central pore, presenting an optimized setting for the rational design of bivalent inhibitors that bridge two adjacent sites. Using diol, hydroxymethyl phenols or benzoyl methyl hydroxamates, and boronic acid chemistries to reversibly join two [3-(1-acylpiperidin-4-yl)phenyl]methanamine core ligands, we have successfully produced a series of self-assembling heterodimeric inhibitors. These heterodimeric tryptase inhibitors demonstrate superior activity compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and compounds demonstrated high selectivity against related proteases, good target engagement, and tryptase inhibition in HMC1 xenograft models. Screening 3872 possible combinations from 44 boronic acid and 88 diol derivatives revealed several combinations that produced nanomolar inhibition, and seven unique pairs produced greater than 100-fold improvement in potency over monomeric inhibition. These heterodimeric tryptase inhibitors demonstrate the power of target-driven combinatorial chemistry to deliver bivalent drugs in a small molecule form.

Tryptase, a serine protease released from mast cells, is implicated in many allergic and inflammatory disorders. Human tryptase is a donut-shaped tetramer with the active sites facing inward forming a central pore. Bivalent ligands spanning two active sites potently inhibit this configuration, but these large compounds have poor drug-like properties. To overcome some of these challenges, we developed self-assembling molecules, called coferons, which deliver a larger compound in two parts. Using a pharmacophoric core and reversibly binding linkers to span two active sites, we have successfully produced three novel homodimeric tryptase inhibitors. Upon binding to tryptase, compounds reassembled into flexible homodimers, with significant improvements in IC50 (0.19 ± 0.08 μM) over controls (5.50 ± 0.09 μM), and demonstrate good activity in mast cell lines. These studies provide validation for this innovative technology that is especially well-suited for the delivery of dimeric drugs to modulate intracellular macromolecular targets.

β-Tryptase is released from mast cells upon degranulation in response to allergic and inflammatory stimuli. Human tryptase is a homotetrameric serine protease with 4 identical active sites directed toward a central pore. These active sites present an optimized scenario for the rational design of bivalent inhibitors, which bridge 2 adjacent active sites. Using (3-[1-acylpiperidin-4-yl]phenyl)methanamine as the pharmacophoric core and a disiloxane linker to span 2 active sites we have successfully produced a novel bivalent tryptase inhibitor, compound 1a, with a comparable profile to previously described inhibitors. Pharmacological properties of compound 1a were studied in a range of in vitro enzymic and cellular screening assays, and in vivo xenograft models. This non-peptide inhibitor of tryptase demonstrated superior activity (IC50 at 100 pmol/L tryptase = 1.82 nmol/L) compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and 1a demonstrated good oral bioavailability and efficacy in HMC-1 xenograft models. Furthermore, compound 1a demonstrated extremely slow off rates and high selectivity against-related proteases. This highly potent, orally bioavailable and selective inhibitor of human tryptase will be an invaluable tool in future studies to explore the therapeutic potential of attenuating the activity of this elusive target.

Starting from cyclopentadiene, two racemic mixtures of 4-aminocyclopentane-1,3-diols were prepared in 8 steps and characterized. Structure determination proved the anticipated trans-orientation of the two oxygen atoms with respect to the plane of the ring. The fragment-like new compounds are small and hydrophilic, devoid of rotatable bonds, and offer stereochemically defined attachment points for substituents. Thus, these platforms for diversity are suitable starting points for the construction of combinatorial libraries of lead-like 4-amidocyclopentane-1,3-diols or natural product analogs. As a proof of concept, cyclopentanoid anandamide analogs were prepared using these molecular platforms and evaluated as tools for the investigation of unresolved issues in the molecular biology of anandamide.

Despite their simplicity, relatively few examples of 1,2,4 (1,3,4)-amino-, azido-, and hydroxy-substituted cyclopentanes are reported in the literature. We found that cyclopent-3-en-1-ol can be transformed into a significant variety of compounds of this class by relatively common and efficient synthetic procedures. Stereochemical control of epoxidation of the cyclopentene double bond can be achieved by varying the substitutents at C4. The C4 substituent and epoxide functional group can be converted into a variety of intermediates with differential protection designed for use in applications requiring regiospecific control for further elaboration of the cyclopentane scaffold.

We have synthesized a series of short, self-complementary oligonucleotide sequences modified at their 5'- and/or 3'- termini with a lipophilic dodecane (C12); these systems serve as models to assess the biophysical properties of double-stranded DNA (dsDNA) equipped with potentially stabilizing lipophilic substituents. Addition of C12 to the 5'-termini of self-complementary 10 nucleotide sequences increased their duplex melting temperatures (T(m)) by approximately 4-8 degrees C over their corresponding unmodified sequences. C12 functionalities added to both the 3'- and 5'-termini increased T(m) values by approximately 10-12 degrees C. The observed increases in T(m) correlated with greater duplex stabilities as determined by the free energy values (DeltaG) derived from T(m) plots. There is a greater degree of stabilization when C12 is positioned with a C.G base pair at the termini, and the stabilizing effect of lipophilic groups far exceeds the effect seen in adding an additional base pair to both ends of DNA. Stable, short dsDNA sequences are of potential interest in the development of transcription factor decoy oligonucleotides as possible therapeutic agents and/or biological tools. These results suggest that the stability of short dsDNA sequences are improved by lipophilic substituents and can be used as the basis for the design of dsDNAs with improved biological stabilities and function under physiological conditions.

A series of aliphatic and aromatic spacer molecules designed to cap the ends of DNA duplexes have been synthesized. The spacers were converted into dimethoxytrityl-protected phosphoramidites as synthons for oligonucleotides synthesis. The effect of the spacers on the stability of short DNA duplexes was assessed by melting temperature studies. End-caps containing amide groups were found to be less stabilizing than the hexaethylene glycol spacer. End-caps containing either a terthiophene or a naphthalene tetracarboxylic acid diimide were found to be significantly more stabilizing. The former showed a preference for stacking above an A*T base pair. Spacers containing only methylene (-CH(2)-) and amide (-CONH-) groups interact weakly with DNA and consequently may be optimal for applications that require minimal influence on DNA structure but require a way to hold the ends of double-stranded DNA together.

Improving the performance and lowering the analyte detection limits of optical and electronic biosensors is essential for advancing wide ranging applications in diagnostics and drug discovery. Most sensing methods require direct linkage of a recognition element and a sensor, which is commonly accomplished through an organic monolayer interface. Alkoxyorganosilanes are typically used to prepare sensor surfaces on dielectric oxides. However, many silanes lead to roughened or thick interfaces that degrade device sensitivity. Here, controlled vapor phase deposition of monoalkoxysilanes is found to lead to monolayers resistant to elevated temperatures and extreme pH conditions. The formation of high density, subnanometer monolayers is demonstrated by ellipsometry, XPS, and AFM. The uniform attachment of these monofunctional silanes to such biosensing platforms as microarrays, field effect devices, and the formation of surface enhanced Raman spectroscopy substrates is demonstrated. The advantages of using this silane deposition protocol for the above technologies are also discussed.

( E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine was biotinylated through a diisopropylsilylacetal linkage and attached to the surface of hydrophilic streptavidin-coated microspheres through the high-affinity noncovalent interaction between biotin and streptavidin. The functionalized microspheres form a stable suspension in water. Upon UV irradiation, the nonfluorescent ( E)-5-[2-(methoxycarbonyl)ethenyl]cytidine on the microspheres undergoes photocyclization to produce highly fluorescent 3-beta-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine. The fluorescence intensity of the microspheres can be correlated to the particle-specific UV doses applied at different suspension concentrations. The microspheres allow one to measure the UV dose (fluence) distribution in high-throughput water disinfection systems.

We have developed novel fluorogenic transformations based on formation of CC bonds catalyzed by palladium using iodocoumarin 1 as a model aryl iodide, where fluorescence is quenched completely due to effects of the heavy, polarizable iodine atom. Substitution of the iodine atom for the carbon using Sonogashira, Suzuki-Miyaura and Heck couplings results in a dramatic fluorescence en- hancement. This approach has been used successfully for the optimization of reaction conditions and kinet- ic studies in high throughput format.

Lagrangian actinometry represents a new method of photochemical reactor characterization. The method is based on an application of dyed microspheres, which were developed by attachment of (E)-5-[2-(methoxycarbonyl)ethenyl]cytidine (hereafter referred to as S) to polystyrene microspheres. S is a nonfluorescent molecule that when subjected to ultraviolet (UV) irradiation yields a single product, 3-beta-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine (hereafter referred to as P), which displays a strong fluorescence signal. Dyed microspheres were subjected to UV irradiation under a collimated beam and using a single-lamp, monochromatic (low pressure Hg), continuous-flow reactor. In parallel with these experiments, a biodosimetry experiment was conducted using Bacillus subtilis spores as the challenge organism. Particle-specific fluorescence intensity measurements were conducted on samples from the collimated-beam experiments and the flow-through reactor experiments by flow cytometry. Estimates of the dose distribution delivered by the flow-through reactor for each operating condition were developed by deconvolution of data resulting from flow cytometry analysis of these samples. In conjunction with these experiments, a numerical model was developed to simulate the behavior of the reactor system. A commercially available computational fluid dynamics package was used to simulate the flow field, while line-source integration was used to simulate the irradiance field. A particle-tracking algorithm was employed to interrogate the flow and irradiance field simulations for purposes of developing particle-specific (Lagrangian) estimates of dose delivery. Dose distribution estimates from the microspheres assays and the numerical simulations were combined with the measured dose-response behavior of B. subtilis spores to yield estimates of spore inactivation in the flow-through experiments. For the range of operating conditions used in these experiments, predictions of spore inactivation based on dose distribution estimates from both methods were in good agreement with each other, and with the measured spore inactivation behavior. Lagrangian actinometry is capable of yielding accurate, detailed measurements of dose delivery by continuous-flow UV systems. This method represents a substantial improvement over existing experiment-based methods of UV reactor characterization (e.g., biodosimetry) in that it yields a measurement of the dose distribution for a given operating condition. This method also represents an improvement over existing methods for validation of numerical simulations. Specifically, because this method yields a measurement of the dose distribution, it is possible to compare these measurements with predicted dose distributions from the numerical simulation. The combined application of biodosimetry, numerical modeling, and Lagrangian actinometry represents an extremely robust approach to reactor characterization and validation.

By connecting six terminal olefins sequentially in one molecule under metathesis conditions, three macrocycles were formed efficiently in one pot yielding a novel cryptand with tetrahedral connectivity.

A reversible biotinylation phosphoramidite was synthesized and incorporated onto the 5′-end of an oligoribonucleotide on a solid phase synthesizer. After cleavage and deprotection, the crude synthetic oligomer mixture was incubated with NeutrAvidin® coated microspheres, and the failure sequences removed by washing with a buffer followed by treating the microspheres with tetrabutylammonium fluoride to give a high quality unmodified full-length oligoribonucleotide.

The concept of using a dynamic base-pairing nucleobase as a mode for degenerate recognition presents a unique challenge to analysis of DNA structure. Proton and phosphorus NMR studies are reported for two nine-residue DNA oligodeoxyribonucleotides, d(CATGGGTAC).d(GTACNCATG) (1) and d(CATGTGTAC).(GTACNCATG) (2), which contained 1-(2'-deoxy-beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (N) in the center of the helix at position 14. The duplexes were compared to the canonical Watson-Crick duplexes, d(CATGGGTAC).d(GTACCCATG) (3) and d(CATGTGTAC).d(GTACACATG) (4). Two-dimensional NOESY spectra of 1-4 in H(2)O and D(2)O solutions collected at 5 degrees C allowed assignment of the exchangeable and nonexchangeable protons for all four oligodeoxyribonucleotides. Thermodynamic and circular dichroism data indicated that 1-4 formed stable, B-form duplexes at 5 degrees C. Two-dimensional (1)H-(31)P correlation spectra indicated that there were minor perturbations in the backbone only near the site of the triazole base. Strong NOESY cross-peaks were observed between the H5 and H1' of N14 in 1 and, unexpectedly, 2, which indicated that, in both duplexes, N14 was in the syn(chi)() conformation about the glycosidic bond. NOESY spectra of 1 and 2 recorded in 95% H(2)O, 5% D(2)O indicated that the imino proton of the base opposite N14, G5, or T5, formed a weak hydrogen bond with N14. These conformations place the polar carboxamide functional group in the major groove with motional averaging on the intermediate time scale.

A series of platinum(II) complexes have been prepared using cis-1,2-diaminocyclopentane and cis-3,4-diaminocyclopentanols; specifically, cis-[PtCl2(cis-1,2-diaminocyclopentane)] (A), cis-[PtCl2(cis,syn-3,4-diaminocyclopentanol)] (B), cis-[PtCl2(cis,anti-3,4-diaminocyclopentanol)] (C), and bis(cis,anti-3,4-diaminocyclopentanol)platinum(II) tetrachloroplatinate (D). The complexes were synthesized from K2PtCl4 and the corresponding cis-diamines. Crystal structures of the Pt(II) complexes have been determined by X-ray single crystal diffraction. Parameters are as follows: space group P21/n (no. 14), a=7.8133(4), b=13.5959(7), c=9.8561(7) A, β=113.201(4)°, V=962.33(19) A3, Z=4 for A; space group P21/n (no. 14), a=6.6076(2), b=11.3481(4), c=11.9739(4) A, β=98.364(2)°, V=888.30(9) A3, Z=4 for B; space group P21/c (no. 14), a=12.7358(2), b=20.8072(5), c=12.1253(3) A, β=112.6139(13)°, V=2966.1(2) A3, Z=12 for C; space group P21/c (no. 14), a=10.0248(3), b=8.9029(5), c=11.1253(6) A, β=109.174(3)°, V=937.85(15) A3, Z=2 for D. The coordination spheres about the Pt atoms are slightly distorted square planar geometries owing to the presence of the geometrically strained five-membered PtN2C2 ring.

A high sensitivity method for detecting low level mutations is under development. A PCR reaction is performed in which a restriction site is introduced in wild-type DNA by alteration of specific bases. Digestion of wild-type DNA by the cognate restriction endonuclease (RE) enriches for products with mutations within the recognition site. After reamplification, mutations are identified by a ligation detection reaction (LDR). This PCR/RE/LDR assay was initially used to detect PCR error in known wild-type samples. PCR error was measured in low |Deltap K a| buffers containing tricine, EPPS and citrate, as well as otherwise identical buffers containing Tris. PCR conditions were optimized to minimize PCR error using perfect match primers at the Msp I site in the p53 tumor suppressor gene at codon 248. However, since mutations do not always occur within pre-existing restriction sites, a generalized PCR/RE/LDR method requires the introduction of a new restriction site. In principle, PCR with mismatch primers can alter specific bases in a sequence and generate a new restriction site. However, extension from 3' mismatch primers may generate misextension products. We tested conversion of the Msp I (CCGG) site to a Taq I site (TCGA). Conversion was unsuccessful using a natural base T mismatch primer set. Conversion was successful when modified primers containing the 6 H,8 H -3, 4-dihydropyrimido[4,5- c ][1,2]oxazine-7-one (Q6) base at 3'-ends were used in three cycles of preconversion PCR prior to conversion PCR using the 3' natural base T primers. The ability of the pyrimidine analog Q6 to access both a T-like and C-like tautomer appears to greatly facilitate the conversion.

A convenient and efficient procedure for the synthesis of 13C methyl-labeled thymidine by way of lithiation of t-butyldimethylsilyl protected 2′-deoxyuridine is described.

DNA that encodes elements for degenerate replication events by use of artificial nucleobases offers a versatile approach to manipulating sequences for applications in biotechnology. We have designed a family of artificial nucleobases that are capable of assuming multiple hydrogen bonding orientations through internal bond rotations to provide a means for degenerate molecular recognition. Incorporation of these analogs into a single position of a PCR primer allowed for analysis of their template effects on DNA amplification catalyzed by Thermus aquaticus (Taq) DNA polymerase. All of the nucleobase surrogates have similar shapes but differ by structural alterations that influence their electronic character. These subtle distinctions were able to influence the Taq DNA polymerase dependent incorporation of the four natural deoxyribonucleotides and thus, significantly expand the molecular design possibilities for biochemically functional nucleic acid analogs.

The nucleoside analogs 1-(2'-deoxy-beta-D-ribofuranosyl)- 3-nitropyrrole (9), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitropyrazole (10), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitroimidazole (11) and 1-(2'-deoxy-beta-D-ribofuranosyl)-5-nitroindole (21) were incorporated into the oligonucleotide 5'-d(CGCXAATTYGCG)-3'in the fourth position from the 5'-end. Procedures for synthesis of two of the nitroazole nucleosides, 10 and 11, were developed for this study. Each of the nitroazoles was converted into a 3'-phosphoramidite for oligonucleotide synthesis by conventional automated protocols. Four oligonucleotides were synthesized for each modified nucleoside in order to obtain duplexes in which each of the four natural bases was placed opposite (position 9) the nitroazole. In order to assess the role of the nitro group on base stacking interaction, sequences were also synthesized in which the fourth base was 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole. Corresponding sequences containing an abasic site, as well as sequences containing inosine, were synthesized for comparison. Thermal melting studies yielded T m values and thermodynamic parameters. Each nucleoside analog displayed a unique pattern of base pairing preferences. The least discriminating analog was 3-nitropyrrole, for which T m values differed by 5 degrees C and Delta G 25 degrees C ranged from -6.1 to -6.5 kcal/mol. 5-Nitroindole gave duplexes with significantly higher thermal stability, with Tm values varying from 35.0 to 46.5 degrees C and -Delta G 25 degrees C ranging from 7.7 to 8.5 kcal/mol. Deoxyinosine (22), a natural analog which has found extensive use as a universal nucleoside, is far less non-discriminating than any of the nitroazole derivatives. Tm values ranged from 35.4 degrees C when paired with G to 62.3 degrees C when paired with C. The significance of the nitro substituent was determined by comparison of the base pairing properties of a simple azole nucleoside, 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole (12). The pyrazole-containing sequences melt at 10-20 degrees C lower than the corresponding nitropyrazole-containing sequences. On average, the pyrazole-containing sequences were equivalent in stability (average Delta G = -4.8 kcal/mol) to the sequences containing an abasic site (average Delta G = -4.7 kcal/mol).

Sonication with nucleic acids has become a standard method for obtaining aqueous dispersions of carbon nanotubes. On the basis of theoretical studies and scanning probe microscopy (SPM) imaging a widely accepted model for DNA association with SWCNT is one in which the DNA binds through non-covalent pi-stacking and hydrophobic interactions. Following the standard procedures established by others to prepare DNA associated single-wall carbon nanotubes (SWCNT), we have determined that sonication generates radical intermediates then form covalent anchors between the DNA and SWCNT. In light of this finding, results from studies on DNA associated carbon nanotubes, need to be more carefully interpreted. (C) 2012 Elsevier B.V. All rights reserved.

Highly efficient cleavage of the single-stranded oligonucleotide 5′-d(CTGGCTCACAAATACCAC TGAGATCTTTTTC)-3′ ( O1 ) at nucleotides G20, A21, and G22 was achieved by an iron-bipyridine complex linked to a targeting oligonucleotide 5′-d(AAA AAG ATC T ∗ CA)-3′ ( O2 ) through -SCH 2 CH 2 NHC(O)- at C5 of a 2′-deoxyuridine (T ∗ ).

A series of heterocyclic carboxamides have been designed as mimics for the natural nucleic acid bases. The nucleosides 1-(2′-deoxy-β-d-ribofuranosyl)imidazole-4-carboxamide (1), 1-(2′ -deoxy-β-d-ribofuranosyl)pyrazole-3-carboxamide (2), and 1-(2′ -deoxy-β-d-ribofuranosyl)pyrrole-3-carboxamide (3) were synthesized and their structures confirmed by spectroscopic and analytical means. This paper is dedicated to Dr. Yoshihisa Mizuno.

The syntheses of endcaps for covalently linking the 3′ and 5′ hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by Tm experiments on the self-complementary octamer.

The ability of packaging RNA (pRNA) from the phi29 DNA packaging motor to form nanoassemblies and nanostructures has been exploited for the development of the nascent field of RNA nanotechnology and subsequent applications in nanomedicine. For applications in nanomedicine, it is necessary to modify the pRNA structure for the conjugation of active molecules. We have investigated end-capped double-stranded DNA segments as reversible capture reagents for pRNA. These capture agents can be designed to allow the conjugation of any desired molecule for pRNA functionalization. The results of model studies presented in this report show that 5- to 7-nucleotide overhangs on a target RNA can provide efficient handles for the high-affinity association to capped double-stranded DNA.

(Pentacarbonyl)tungsten(0) nucleoside phosphite complexes were prepared by reaction of nucleoside methyl phosphites generated in situ with pentacarbonyl(η 2 -cis-cyclooctene)tungsten(0) (1)

A synthetic route to 2-carbamoyl-4-(2′-deoxy-β-D-ribofuranosyl)imidazole 3, starting from 2-deoxy-3,5-di-O-toluoyl-β-D-ribofuranosyl cyanide 4, was developed. The key steps are reduction of the cyano group of compound 4 to a formyl and subsequent condensation with tosylmethyl isocyanide to yield the formamido derivative 7, which was dehydrated to an isocyanide and ring closed with either ammonia or a primary amine to yield protected C-4 linked imidazolyl deoxyribosyl derivatives 9a–c. Ring closure with H2S followed by removal of the toluoyl protecting groups with ammonia gave 5-(2′-deoxy-β-D-ribofuranosyl)thiazole 11. A cyano group can be introduced at C-2 of the imidazole nucleosides by way of the reagent N-cyano-4-(dimethylamino)pyridinium bromide. Subsequent hydrolysis of the cyano functional group with alkaline hydrogen peroxide yields a carboxamide substituent. All of the transformations were able to be carried out without affecting the β-configuration at the anomeric carbon. A p-nitrophenylethyl protecting group was introduced at N-3 of the imidazole during ring closure in order to obtain a protected derivative that could be selectively modified at the deoxyribosyl (erythro-pentofuranosyl) hydroxy groups.

Synthesis of 2′-deoxyguanosine tethered through N-2 to an imidazole is described. The modified 2′-deoxyguanosine was converted to two different phosphoramidites, one with and the other without a 6- O -protecting group. The phosphoramidites were incorporated into oligonucleotide alone or together with a 2′-deoxyuridine tethered to a bipyridine. Protection and deprotection of the imidazole are also briefly described.

Synthesis of 2′-deoxyguanosine tethered at N-2 to a carboxypentyl group and 2′-deoxyuridine tethered through C-5 to a bipyridine is described. The modified nucleosides were converted to the corresponding phosphoramidites and incorporated into mono- and di-modified oligonucleotides. The carboxypentyl group was introduced in the ester form into oligonucleotides and recovered by hydrolysis with aqueous triethylamine.

W(CO)5(C8H14) and W(CO)4(C8H14)2 were synthesized photochemically from W(CO)6 andcis-cyclooctene with a respective yield of 71% and 18%. The compounds were characterized by NMR, IR and single X-ray crystallography. The IR spectra of the two compounds exhibited characteristic bands for the mono- and bis-substituted metal carbonyl complexes. In W(CO)4(C8H14)2, thecis-cyclooctene ligands are in atrans configuration with the double bonds perpendicular to each other. The two crystal structures were refined toR=0.040 for W(CO)5(C8H14) and 0.025 for W(CO)4(C8H14)2. Although the tungsten-carbon (of the carbonyl groups) distances are very similar in both structures, the distances between the tungsten atom and the olefinic carbons are shorter in W(CO)4(C8H14)2, 2.36 A, than in W(CO)5(C8H14), 2.51 A.

Synthesis of 2′-deoxyguanosine tethered at N-2 to a carboxypentyl group and 2′-deoxyuridine tethered through C-5 to a bipyridine is described. The modified nucleosides were converted to the corresponding phosphoramidites and incorporated into mono- and di-modified oligonucleotides. The carboxypentyl group was introduced in the ester form into oligonucleotides and recovered by hydrolysis with aqueous triethylamine.

A synthetic route to 2-carbamoyl-4-(2′-deoxy-β-D-ribofuranosyl)imidazole 3, starting from 2-deoxy-3,5-di-O-toluoyl-β-D-ribofuranosyl cyanide 4, was developed. The key steps are reduction of the cyano group of compound 4 to a formyl and subsequent condensation with tosylmethyl isocyanide to yield the formamido derivative 7, which was dehydrated to an isocyanide and ring closed with either ammonia or a primary amine to yield protected C-4 linked imidazolyl deoxyribosyl derivatives 9a–c. Ring closure with H2S followed by removal of the toluoyl protecting groups with ammonia gave 5-(2′-deoxy-β-D-ribofuranosyl)thiazole 11. A cyano group can be introduced at C-2 of the imidazole nucleosides by way of the reagent N-cyano-4-(dimethylamino)pyridinium bromide. Subsequent hydrolysis of the cyano functional group with alkaline hydrogen peroxide yields a carboxamide substituent. All of the transformations were able to be carried out without affecting the β-configuration at the anomeric carbon. A p-nitrophenylethyl protecting group was introduced at N-3 of the imidazole during ring closure in order to obtain a protected derivative that could be selectively modified at the deoxyribosyl (erythro-pentofuranosyl) hydroxy groups.

A convenient and efficient procedure for the synthesis of 13C methyl-labeled thymidine by way of lithiation of t-butyldimethylsilyl protected 2′-deoxyuridine is described.

We present an optimized synthetic strategy for the attachment of molecules to 5’-adenosine monophosphate (AMP), which can then be used to label the 5'-end of RNA by T7 RNA polymerase mediated in vitro transcription. Through the use of a boronate affinity gel, we have developed an efficient route to the preparation of folate conjugated AMP with high yields and purity. Affi-Gel boronate is an affinity resin that selectively binds nucleoside and nucleoside derivatives at pH > 7.5 and releases them at pH < 6.5. This resin is used to efficiently bind and purify ribonucleotides such as AMP. This allows for the addition of a large excess of reactants and reagents in order to drive the reaction to completion and then allow easy purification without HPLC. The synthesis can be successfully scaled up to produce large quantities of AMP conjugates.