Tammy J. Dwyer

KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry

Many candidate unnatural DNA base pairs have been developed, but surprisingly, some of the best replicated adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replicating one of the more promising pairs, dNaM-d5SICS, and show that efficient replication results from the polymerase itself inducing the required natural-like structure.

Solution Structure, Mechanism of Replication, and Optimization of an Unnatural Base Pair

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge-on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivatives of the nucleotide analogue dMMO2, which forms an unnatural base pair with the nucleotide analogue d5SICS. Replacing the para-methyl substituent of dMMO2 with an annulated furan ring (yielding dFMO) has a dramatically negative effect on replication, while replacing it with a methoxy (dDMO) or with a thiomethyl group (dTMO) improves replication in both steady-state assays and during PCR amplification. Thus, dTMO-d5SICS, and especially dDMO-d5SICS, represent significant progress toward the expansion of the genetic alphabet. To elucidate the structure-activity relationships governing unnatural base pair replication, we determined the solution structure of duplex DNA containing the parental dMMO2-d5SICS pair, and also used this structure to generate models of the derivative base pairs. The results strongly support the intercalative mechanism of replication, reveal a surprisingly high level of specificity that may be achieved by optimizing packing interactions, and should prove invaluable for the further optimization of the unnatural base pair.

Enabling Bifunctionality and Hemilability of N‐Heteroaryl NHC Complexes

N-Heterocyclic carbene (NHC) complexes have been shown to be extremely versatile and stable catalysts for reactions as diverse as olefin metathesis, transfer hydrogenation, and C-C coupling reactions. NHCs are attractive ligACHTUNGTRENNUNGands due to their strong s-donating ability, poor back bondACHTUNGTRENNUNGing,[2a–h] and relative air, moisture, and thermal stability of their complexes. Electronic and steric optimization of the properties of NHC-metal complexes is possible through NHC ligand design. Further improvement of catalyst performance through NHC functionalization has been studied using a variety of pendant groups, including heterocycle, phosphine, amine, and imine donor functions. In many cases the added ligand creates a stable chelate or pincer, whereas in other cases a hemilabile system and its temporary ligand loss (as in 2) favor catalysis. In contrast, this paper introduces a fundamentally different approach, where a substituent on an NHC ligand will act as a hydrogen bond acceptor (3) or base (4), which could facilitate catalysis (Scheme 1). Some recent studies on NHC metal complexes have raised the possibility of pendant nitrogen involvement, though as far as we are aware, no direct evidence was presented in these studies. In particular, for 5 a, decoordination of the pyrimidine (cf. 1!2) would make a basic nitrogen available near the metal active site (cf. 2!3 or 4). However, the NMR spectrum of 5 a was reported to remain sharp even at 110 8C, suggesting that decoordination and rotation about the pyrimidine NHC bond is a process with a high activation barrier. Therefore, in order to fully realize the potential of NHC ligands with pendant heterocyclic bases their ability to chelate with a metal must be decreased (Scheme 1), and we hypothesized that this could be achieved by introducing a large substituent R. Here we report successful strategies toward achieving this aim, which should be of general application to NHC chemistry.

Electronic effects in a prototype push-pull ethylene: a study of rotational barriers in C4H4N4 isomers

Quantum mechanical calculations of the rotational barriers in the prototype push-pull ethylene 1,1-diamino-2,2-dicyanoethylene (DADCE) and its non-push-pull analogues diaminofumaronitrile (DAFN) and diaminomaleonitrile (DAMN) have been used quantitatively to assess the influence of the push-pull effect. Results for DADCE indicate an in vacuo barrier for CC rotation of ≈ 28 kcal mol−1 which decreases in solvents of increasing dielectric strength. The barrier for CN rotation in DADCE is found to be ≈ 8 kcal mol−1 and increases with solvent dielectric strength. The comparable CC barrier connecting DAMN and DAFN is calculated to be ∗> 20 kcal mol−1 higher than the DADCE barrier. Analysis of the conformational energies of DADCE, DAMN, and DAFN gives further evidence for the increased dipolar character and nitrogen lone pair conjugation in DADCE as compared with DAMN and DAFN.

Determination of the acid dissociation constants for WR-1065 by proton NMR spectroscopy.

The acid dissociation constants for N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065) were determined in D2O by 360- and 500-MHz NMR spectroscopy. Results obtained at 0.21 M initial ionic strength and 26 degrees C were corrected to 25 degrees C yielding pKD1 = 8.28 +/- 0.04, pKD2 = 9.88 +/- 0.07, and pKD3 = 11.58 +/- 0.03. Correction of these values for the effect of the deuterium isotope upon the ionization reaction yielded dissociation constants in water of pKH1 = 7.69 +/- 0.09, pKH2 = 9.35 +/- 0.09, and pKH3 = 11.10 +/- 0.08. Analysis of the changes in chemical shift with pD indicated that the first ionization occurs largely through ionization of the thiol group (approximately 67%) and to a lesser extent the secondary ammonium group (approximately 30%), whereas the third ionization involves mainly the secondary ammonium group (approximately 65%) and to a lesser extent the primary ammonium group (approximately 30%). Estimates of the microscopic pK values for WR-1065 were also obtained from the results.

Rotational Barriers in Push-Pull Ethylenes: An Advanced Physical-Organic Project Including 2D EXSY and Computational Chemistry

An integrated upper-division physical-organic experiment for chemistry majors has been developed. It involves the determination and mechanistic interpretation of the C=C and C-N rotational barriers in a push-pull ethylene. In the course of the experiment students will synthesize an organic compound, acquire variable temperature 1D and 2D NMR spectra, and use computational quantum chemistry to gain a deeper understanding of the unique electronic features of the molecule. Low temperature 2D EXchange SpectroscopY (EXSY) is used to quantitate the rotational barriers in a series of solvents. The quantum mechanical calculations provide a means to compare the properties of the push-pull ethylene with a similar non-push-pull system. Analysis of the experimental and theoretical results leads to a nearly complete picture of how substituent effects can influence bond lengths, rotational barriers, and the electronic distribution in these ethylenes.