An orthogonal aminoacyl tRNA synthetase/tRNA pair has been evolved that allows the incorporation of the photoisomerizable amino acid phenylalanine-4'-azobenzene (AzoPhe) into proteins in E. coli in response to the amber nonsense codon. Further, we show that AzoPhe can be used to photoregulate the binding affinity of catabolite activator protein to its promoter. The ability to selectively incorporate AzoPhe into proteins at defined sites should make it possible to regulate a variety of biological processes with light, including enzyme, receptor, and ion channel activity.

Reported is a systematic study of the "fitness" (in terms of kcat/Km) of a series of phosphonate mimics of glucose 6-phosphate (G6P) as unnatural substrates for G6P dehydrogenase from Leuconostoc mesenteroides. The four G6P analogues (9, 10, 15a, and 15b) differ only in the degree of fluorination at the "bridging" phosphonate carbon. All have been synthesized from benzyl 6-O-trifluoromethanesulfonyl-2,3,4-tri-O-benzyl beta-D-glucopyranoside (6). The phosphonates with bridging CH2 (9) and CF2 (10) groups are cleanly obtained by direct displacements with the appropriate LiX2CP(O)(OEt)2 reagents (X = H, F) in 15 min at -78 degrees C. For the (alpha-monofluoro)alkylphosphonates (15a/b), homologation of 6 is achieved via lithiodithiane-mediated triflate displacement, followed by aldehyde unmasking [CaCO3, Hg(ClO4)2, H2O]. Addition of diethyl phosphite anion produces diastereomeric, (alpha-hydroxy)phosphonates 13a/b (1.4:1 ratio) which may be readily separated by chromatography. The stereochemistry of the minor diastereomer was established as 7(S) via X-ray crystallographic structure determination of its p-bromobenzoate derivative, 16b. Treatment of the major 7(R) diastereomer with DAST produces alpha-fluorinated phosphonate 14a, in modest yield, with inversion of configuration, as established, again, by X-ray crystallography. To our knowledge, this is first example of DAST-mediated fluorination of a (nonbenzylic, nonpropargylic) secondary (alpha-hydroxy)phosphonate and thus establishes the stereochemical course of this transformation. alpha-Deprotonation/kinetic quenching of 14a provides access to the 7(R)-epimer (14b). For all four protected phosphonates (7, 8, 14a, and 14b), diethyl phosphonate ester deprotection was carried out with TMSBr, followed by global hydrogenolytic debenzylation to produce the free phosphonates, as alpha/beta anomeric mixtures. Titrations of G6P itself and the free phosphonic acids provides second pKa values of 6.5 (1, bridging-O), 5.4 (10, bridging-CF2), 6.2 (14a, bridging-CHF), and 7.6 (9, bridging-CH2). Leuconostoc mesenteroides G6PDH-mediated oxidation and Lineweaver-Burk analysis yields normalized kcat/Km values of 0.043 (14b, bridging-7(R)-CHF), 0.11 (10, bridging-CF2), 0.23 (14b, bridging-CH2), and 0.46 (14a, bridging-7(S)-CHF) relative to G6P itself, largely reflecting differences in Km. The fact that kcat/Km increases by more than an order of magnitude in going from the 7(R)-alpha-monofluoroalkyl phosphonate (worst substrate) to the 7(S)-diastereomer (best substrate) is especially notable and is discussed in the context of the known phosphate binding pocket of this enzyme as revealed by X-ray crystallography (Adams, M. J. et al. Structure 1994, 2, 1073-1087).

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[reaction: see text] Treatment of primary alkyl triflates or iodides with the potassium salt of diethyl (alpha-fluoro-alpha-phenylsulfonylmethyl)phosphonate yields (alpha-fluoro-alpha-phenylsulfonylalkyl)phosphonates. These can be cleanly desulfonated, in a matter of minutes, with Na(Hg) in MeOH/THF/NaH(2)PO(4). This two-step procedure complements previously reported triflate displacement approaches to alpha-nonfluorinated and alpha,alpha-difluorinated phosphonates.

Can enzymes help organic chemists identify new transition‐metal catalysts? The first example of the in situ enzymatic monitoring of an organic transformation is described. Thus, a transition‐metal‐mediated allylic‐amination reaction in the organic layer (see scheme; R=para‐methoxyphenyl, tosyl) leads to release of ethanol which causes the spectroscopically observable formation of NADH in the aqueous phase. This approach uncovers a new Ni0‐promoted route to β,γ‐unsaturated amino acids. HMDS=1,1,1,3,3,3‐hexamethyldisilazane.

Dedicated to Professor Samuel J. Danishefsky on the occasion of his 65th birthdayThe move from the more deliberate, traditional approach to catalyst discovery to combinatorial approaches, has spurred great interest in the development of parallel-screening methods. As Crabtree and Loch recently put it, ideally one seeks ™an appropriate chemical sensor in a rapid parallel assay to detect rate and perhaps selectivity∫. [1] Herein, we describe the use of enzymes to report rapidly on reaction rate, for a set of parallel reactions. Seto and Abato recently described the use of enzymes to report on (enantio)selectivity. [2] From a broader perspective, these developments may be regarded as adding to the versatility of enzymes as tools for the organic chemist, an area that has seen remarkable expansion from asymmetric processing of unnatural substrates [3±5] and protecting-group cleavage under mild conditions, [6] to the creation of artificial enzymatic pathways. [7,8] There is currently great interest in ™combinatorial cataly-sis∫, [9] especially in transition-metal (TM) catalyzed reactions, for which reaction discovery and optimization often involve varying 1) the metal, 2) the ligand (type, structure, and stoichiometry), and 3) the substrate structure. By choosing such a model reaction, we sought both to establish proof of principle and to assess the ability of the screen to evaluate such variables one at a time.In our approach, the organic reaction under study is coupled, in situ, with an enzymatic reaction that permits continuous UV spectroscopic monitoring of the reaction. We term this approach ×in situ enzymatic screening× (ISES). This method is complementary to the previously communicated screening techniques, [10±19] in that it provides 1) evidence of product formation (not directly available using the elegant IR-thermography method of the Morken and Reetz groups) [11] and 2) relative rate profiles (not easily available with time-point detection systems employing gas [12] or liquid chromatography [10,13] or mass spectrometry), [14] 3) without the need to alter the substrate, by installing a chromophore, [15] a fluorophore, [16] , or an azo-dye precursor. [17] Consonant with our interest in developing synthetic methodology toward densely functionalized b,g-unsaturated amino acids [20] as inhibitors of PLP-dependent (PLP pyridoxal phosphate) enzymes, [21] we chose a TM-mediated allylicamination reaction as our model reaction. We were influenced, in this regard, by an important precedent from Trost et al.; [22a] scalemic vinylglycinol had been synthesized through Pd 0 -mediated allylic amination. [22] We set out to use ISES to identify other TMs, including less expensive ones, which were capable of catalyzing the intramolecular allylic-amination reaction illustrated in Scheme 1. [23] Success here would, in principle, validate the use of ISES in screening other variants of the allylic-displacement reaction. [24] Scheme 1. ISES of catalysts for allylic displacement. PMP para-methoxyphenyl.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.