Research Summary                        List of Publications

Research Overview

   In Situ Enzymatic Screening (ISES): A New Tool for Catalyst Development

    Our ISES work provides a fundamentally new approach to catalyst screening (CEN blurb). For reactions to which it optimally applies, ISES offers an information rich screen, in real time, without the need to alter the substrate (e.g. install a chromophore, mass tag or isotopic label). In our lab, this tool has already been used to develop the first asymmetric, Ni(0)-mediated allylic amination reaction, and to uncover several interesting combinatorial hits for Co(III)-based HKR chemistry.

  Detailed Description

     Unnatural Amino Acids as New Mechanism-Based Inhibitors: Design, Synthesis      and Evaluation

    Our unnatural amino acids project centers around the quaternary a-vinyl amino acid substructure. These compounds have proven to act as suicide substrates for vitamin B 6-enzymes, and to serve as vehicles for the exploration of new PLP-enzyme inactivation trigger space. And we continue to examine multiple approaches (chiral auxiliary-mediated, self-reproduction of chirality, enzymatic resolution, asymmetric Ni-catalysis) to their synthesis in enantioenriched form.    

Detailed Description

    Chemoenzymatic and Ring E-Modular Entry into the (-)-Podophyllotoxin &     Etoposide Drug Families

       We have developed the first catalytic asymmetric synthesis of (-)-podophyllotoxin. (link) Key contributions include the enzymatic desymmetrization of an advanced meso intermediate, the invention of a &reverse-Kahne* glycosylation reaction to install the sugar, (link) and the establishment of a truly ring E-modular route. This synthesis has served us as a tool to define better the structural features that are important for the inhibition of both tubulin polymerization (with R. Himes-Kansas) and of cancer cell growth.

     Detailed Description

    Surrogates for Biological Phosphates: Synthesis and Biochemical Study

      We have a long-standing interest in hydrolytically stable, phosphatase resistant surrogates for the phosphate ester functionality. Our triflate displacement approach provides and convenient synthetic entry into to phosphonates bearing 0-2 a-fluorine atoms. In our lab, this has led to phosphonates that (a) bind to an important glycoprotein receptor (cation independent mannose phosphate receptor) (b) are pseudo-substrates for the enzyme catalyzing the committing step in the pentose phosphate shunt (glucose 6-phosphate dehydrogenase), or (c) interact with a key enzyme in bacterial cell wall biosynthesis (glucosamine 6-phosphate synthase). One of the most elegant applications of this bioorganic tool in the community is the use of our difluorinated phosphonate analogue of phosphoserine by Ettore Appella at the NIH to study the phosphorylation state of the important tumor suppressor protein, p53.

         Detailed Description

Detailed Research Description

    In Situ Enzymatic Screening (ISES): A New Tool for Catalyst Development

       Parallel to the rapid expansion of combinatorial approaches to catalyst discovery has come an interest in the development of affiliated screening methods. The ideal screen would be convenient, rapid, information-rich, high throughput, and in real time. Most methods are aliquot-based. A number of creative, real time approaches have been delineated, including the use of IR thermography (Reetz and Morken groups), the application of pH sensors (Miller group), and the introduction of chromophore-based methods (Crabtree, Hartwig, Morken, Sames). Several elegant enantiomer competition methods have been communicated [e.g. IR thermography (Reetz), fluorescence (Miller, Kazlauskas, Reymond, Bornscheuer), UV/vis (Kazlauskas). However, for de novo introduction of chirality, in situ screening methods are lacking. To our knowledge, the only such screen described heretofore that provides information on both relative rate and enantioselectivity involves the use of a stereo- and isotopically-enriched 13C-probe (Morken and coworkers J. Am. Chem. Soc.2002, 124, 9020-9021).

scheme1

     The approach that we are investigating involves coupling an organic reaction/catalyst under study, usually in an organic layer, with an enzymatic reporting reaction, usually in an adjacent aqueous layer. For this technique to apply, one must study a reaction for which the product or byproduct is an enzymatic substrate. If done in a bilayer, this (by)product must have some water solubility. And finally, the enzyme(s) chosen must lead to a spectroscopic signal. Advantages of this technique include the possibility for in situ monitoring, without the need to take aliquots and without the need to alter the substrate. Furthermore, if one could take advantage of the chirality of the enzymatic reporter to provide information about the handedness of the product, one could potentially rank catalysts both in terms of relative rates and relative enantioselectivities.

       In its first iteration, we successfully applied this technique to a model intramolecular allylic amination reaction, initially finding conditions under which Ni(0) could promote this chemistry, (link) then developing the first examples of asymmetric Ni(0)-mediated allylic amination, with P,N-ligands, (link) and P,P-ligands (link). A Ni(0)-mediated entry into the PLP-enzyme inhibitor, L-vinylglycine, resulted. This provided an interesting link to our vitamin B 6-dependent enzyme work (vide infra).

       We are now exploring a second iteration, in which enzymatic sensors report back to the synthetic chemist on sense and magnitude of enantioselection, in addition to rate. A double-cuvet ISES technique has been chosen and hydrolytic kinetic resolution of epoxides, a reaction of importance, and known to be catalyzed by metal-salen complexes has been chosen as the model reaction. The actual experimental configuration for this example of &Double-Cuvette ISES* is illustrated in Scheme 1. (link) Note that we find it convenient to use an inverted bilayer here, with the substrate, racemic propylene oxide, and the catalyst in a lower organic layer. In enabling work, we successfully identified dehydrogenases that show preferences for (R)- and (S)- 1,2-propanediol. By comparing the rates at which the reduced nicotinamide cofactor is generated in the two reporting cuvets, knowing the inherent (R):(S) preferences of each reporting enzyme, we are able to empirically estimate the ee of the propanediol that has been formed. This is done, in situ, without the need to draw aliquots. We then compare our results with the ee determined by chiral HPLC.

     As is illustrated in Scheme 2, we have assembled a novel 7 x 7 matrix of chiral salen-type ligands. Note that the chiral scaffold resides in the 1,2-diamine moiety and is derived from amino acid, carbohydrate and terpenoid sources. Similarly, a rather diverse array of o-hydroxy aromatic aldehydes has been selected. The Co(III)-salen complexes derived from these salens were then screened for their ability to catalyze the hydrolytic kinetic resolution (HKR) of propylene oxide ( Jacobsen and coworkers, J. Am. Chem. Soc.2002, 124, 1307-1315). A bar graph comparing the ISES-predicted ee under biphasic conditions, with that observed by chiral HPLC under typical hydrolytic kinetic resolution (HKR) conditions is presented in Scheme 3. One can see that for most reasonably selective catalysts, ISES makes qualitatitvely good predictions of the sense and magnitude of enantioselectivity.

                             Scheme 3

table

  • Broussy, Sylvain, Cheloha, Ross W., and Berkowitz, David B.. "Enantioselective, Ketoreductase-Based Entry into Pharmaceutical Building Blocks: Ethanol as Tunable Nicotinamide Reductant." Organic Letters 2009, 11(2), 305-308. pdf
  • Sangeeta Dey, Douglas R. Powell, Chunhua Hu, and David B. Berkowitz,* "Cassette"-ISES (In Situ Enzymatic Screening) Identifies Complementary Chiral Scaffolds for Hydrolytic Kinetic Resolution Across a Range of Epoxides. Angew. Chem. Int. Ed. 2007, 46, 7010-7014.pdf

  • David B. Berkowitz, Sangeeta Dey, Kannan R. Karukurichi and Weijun Shen ※A Method for Measuring the Stereoselectivity and Relative Rate for Organic Chemical Reactions.§ U.S. Patent Application, Serial No. 11/434,247, filed May 16, 2006.

  • Sangeeta Dey, Kannan R. Karukurichi, Weijun Shen, and David B. Berkowitz,* Double-Cuvette ISES: In Situ Estimation of Enantioselectivity and Relative Rate for Catalyst Screening. J. Am. Chem. Soc. 2005, 127, 8610-8611.pdf
  • David B. Berkowitz* and Gourhari Maiti Following an ISES Lead: The First Examples of Asymmetric Ni(0)-Mediated Allylic Amination. Org. Lett. 2004, 6, 2661-2664.pdf
  • David B. Berkowitz,* Weijun Shen and Gourhari Maiti In Situ Enzymatic Screening (ISES) of P,N-Ligands for Ni(0)-Mediated Asymmetric, Intramolecular Allylic Amination. Tetrahedron: Asymmetry 2004 15, 2845-2851.pdf
  • David B. Berkowitz,* Mohua Bose and Sungjo Choi In Situ Enzymatic Screening (ISES): A Tool for Catalyst Discovery and Reaction Development. Angewandte Chemie International Edition, English 2002, 41, 1603-1607.pdf  For the Chemical and Engineering News feature on this work, see: jpg

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Unnatural Amino Acids as New Mechanism-Based Inhibitors: Design, Synthesis and Evaluation

  Amino acid decarboxylases (AADC's) represent an enzymatic class, from the point of view of rational drug design [e.g. DOPA DC (Parkinson's disease); ornithine DC (cancer)]. Mechanistically, these enzymes require an electron sink. This is typically a PLP cofactor but, in some cases, a pyruvamide cofactor is used. This project concerns the design, synthesis and evaluation of new classes of a-branched amino acids, as potential AADC inhibitors. Inhibitors have been designed wherein the a-carboxyl, a-amino and side chain functionalities are retained, but the a-proton is replaced with a ※Trojan horse§ functional group, such as a vinyl or halovinyl group. These designs resulted from an analysis of available mechanistic and inhibitory data for this class of enzymes. It is also worthy of note that in addition to their direct application as AADC inhibitors, the novel alpha-branched amino acids being synthesized here will almost certainly be useful as building blocks for: (i) de novo peptide design, (ii) combinatorial chemistry, or (iii) unnatural amino acid mutagenesis (Schultz, Hecht).

scheme4

     Another attractive feature of this project design is illustrated in Scheme 4. Namely, as we have now demonstrated, a-vinyl amino acids, potential DC inhibitors in and of themselves, are synthetic precursors to alpha-chloro- or alpha-bromovinyl amino acids (link) and to alpha-oxiranyl amino acids. (link) Moreover, we have recently found that one can stereoselectively install the interesting (2*Z-fluoro)vinyl trigger from the a-vinyl AA precursor. (link), and have demonstrated its function as a suicide trigger in PLP-dependent lysine decarboxylase from Hafnia alvei (link)

     Thus, the synthetic problem reduces to the development of efficient, and stereo-controlled routes to the vinyl AA &central players,* as it were. Our initial contribution was to develop a general synthesis of racemic alpha-vinyl amino acids via the formal vinylation of amino acid-derived dianions (Scheme 5a). Ethylene oxide serves as the vinyl cation equivalent, and a nice range of side chain functionality-suitably protected-can be carried in these dianions. (link) This may be coupled with an enzymatic resolution, though thus far, this approach requires two enzymatic cycles, and only provides the L-antipodes. (link) More exploration is probably warranted.

    Parallel to the enzymatic resolution studies, we sought to develop asymmetric versions of our dianion alkylation chemistry. As is depicted in Scheme 5b, a chiral alpha-vinylglycine-derived dianion dienolate leads preferentially to L-alpha-vinyl AAs upon introduction of the AA side chain by alkylation. The sense of diastereoselection is in accord with a chain-extended, chelate model for the reactive conformation of these dianions (Scheme 5b). (link) This work grew out of earlier efforts in the group to provide access to L- a-methyl AA*s, by a similar approach. (link)

       More recently, we have found that one can formally transfer chirality from L-alpha-vinylglycine to higher L-alpha-vinyl amino acids utilizing selenium chemistry to mask the double bond as a directing group (Scheme 6). (link) The unmasking procedure involves two new transformations. Exposure of the phenylselenomethyl-substituted oxazolines to KOt-Bu results in an efficient eliminative ring-opening to provide enantiomerically enriched, a-E-(2-phenylseleno)vinylamino acids. The (E)-vinyl selenides are cleanly transformed into the corresponding (E)-vinyl stannanes via treatment with Bu3SnH. To our knowledge, this is the first report of a vinyl selenide a-vinyl stannane transformation, and this move may be of synthetic utility in other contexts. We have further demonstrated that the alpha-(2-tributylstannyl)vinyl amino acids, may be used for a-branch extension via cross-coupling.(link) Alternatively, acid-catalyzed hydrolysis effects protodestannylation concomitant with ester and amide cleavage, and provides a mechanism for late stage radiolabel introduction.

scheme6

     Returning to the bioorganic side, the (2*Z-fluoro)vinyl a-branch appears to be one of the more interesting new triggers in this work. A number of potential &suicide* mechanism(s) for this functionality can be envisioned. In order to carry out a first test of the viability of this strategy for PLP enzyme inactivation, we have chosen lysine DC as a model AADC. We have been able to obtain this enzyme from the bacterium Hafnia alvei, in house.

  • David B. Berkowitz,* Kannan R. Karukurichi, Roberto de la Salud-Bea, Gourhari Maiti, Jill M. McFadden and Michelle L. Morris ※Stereoselective Synthesis of Quaternary, alpha-Vinyl Amino Acids  and their alpha-(2*Z-Fluoro)vinyl Congeners 每 Promising Candidates for PLP Enzyme Inactivation.§ ACS Symposium-In-Print 2009, 1009, 288-303. pdf
  • David B. Berkowitz*, Kannan R. Karukurichi, Roberto de la Salud-Bea, David L. Nelson, and Christopher D. McCune, ※Use of fluorinated functionality in enzyme inhibitor development: Mechanistic and analytical advantages,§ J. Fluorine Chem. 2008, 129, 731每742. pdf

  • Kannan R. Karukurichi, Roberto de la Salud-Bea, Wan Jin Jahng, David B. Berkowitz* ※Examination of the New alpha-(2*Z-Fluoro)vinyl Trigger with Lysine Decarboxylase: The Absolute Stereochemistry Determines the Reaction Course,§ J. Am. Chem. Soc. 2007, 129, 258-259. pdf

  • David B. Berkowitz,* Bradley D. Charette, Kannan R. Karukurichi and Jill M. McFadden ※ alpha-Vinylic Amino Acids: Natural Occurrence, Asymmetric Synthesis and Biochemical Mechanisms .§ Tetrahedron: Asymmetry 2006, 17, 869-882. pdf

  • David B. Berkowitz,* Bin Wu and Huijie Li ※A Formal [3,3]-Sigmatropic Rearrangement Route to Quaternary alpha-Vinyl Amino Acids: Use of Allylic N-PMP Trifluoroacetimidates.§ Org. Lett. 2006, 8, 971-974. pdf

  • Weijun Shen, Molly K. McGath, Ruby Evande and David B. Berkowitz,* A Continuous Spectrophotometric Assay for Human Cystathionine Beta-Synthase. Analytical Biochemistry 2005, 342, 103-110. pdf

  • David B. Berkowitz, *Roberto de la Salud-Bea and Wan-Jin Jahng, Synthesis of Quaternary Amino Acids Bearing a (2'Z)-Fluorovinyl alpha-Branch: Potential PLP Enzyme Inactivators. Org. Lett. 2004 6, 1821-1824. pdf
  • David B. Berkowitz,* Esmort Chisowa and Jill M. McFadden, Stereocontrolled Synthesis of Quaternary beta, gamma-Unsaturated Amino Acids: Chain Extension of D- & L- alpha-(2-Tributylstannyl)Vinyl Amino Acids, Tetrahedron Symposia-in-Print 2001, 57, 6329-6343. pdf
  • David B. Berkowitz,* Jill M. McFadden, Esmort Chisowa and Craig L. Semerad Organoselenium-Based Entry into Versatile, alpha-(2-Tributylstannyl)vinyl Amino Acids in Scalemic Form: A New Route to Vinyl Stannanes, J. Am. Chem. Soc. 2000, 122, 11031-11032. pdf
  • David B. Berkowitz,* Jill M. McFadden and Marianne K. Sloss Engineering Acyclic Stereocontrol in the Alkylation of Vinylglycine-Derived Dianions: Asymmetric Synthesis of Higher alpha-Vinyl Amino Acids, J. Org. Chem. 2000, 65, 2907-2918. pdf
  • David B. Berkowitz,* Jill McFadden, Marianne K. Smith and Michelle L. Pedersen Synthesis of alpha-Vinyl Amino Acids. Methods in Molecular Medicine, Peptidomimetics Protocols (Wieslaw M. Kazmierski, volume Ed.; John M. Walker, series Ed.; Humana Press, Totowa, NJ), Vol. 23, Chapter 27, 467-488, 1999. pdf
  • David B. Berkowitz,* Wan-Jin Jahng and Michelle L. Pedersen (㊣)-alpha-Vinyllysine and (㊣)-alpha-Vinylarginine are Time-Dependent Inhibitors of Their Cognate Decarboxylases. Bioorganic and Medicinal Chemistry Letters 1996, 6, 2151-2156. pdf
  • David B. Berkowitz,* Michelle L. Pedersen and Wan-Jin Jahng Synthesis of Higher alpha-Chlorovinyl and alpha-Bromovinyl Amino Acids: The Amino Protecting Group Determines the Reaction Course, Tetrahedron Letters 1996,37, 4309-4312. pdf
  • David B. Berkowitz* and Marianne K. Smith, A Convenient Synthesis of L-Vinylglycine from L-Homoserine Lactone. Synthesis, 1996, 39-41. pdf
  • David B. Berkowitz* and Michelle L. Pedersen Free alpha-Oxiranyl Amino Acids. J. Org. Chem. 1995, 60, 5368-5369. pdf
  • David B. Berkowitz* and Marianne K. Smith Enantiomerically Enriched alpha-Methyl Amino Acids. Alkylation of an Acyclic, Chiral Alanine Dianion with a High Diastereofacial Bias. J. Org. Chem. 1995, 60, 1233-1238. pdf
  • David B. Berkowitz,* James A. Pumphrey and Quanrong Shen Enantiomerically Enriched alpha-Vinyl Amino Acids via Lipase-Mediated Reverse Transesterification, Tetrahedron Letters 1994, 35, 8743-8746. pdf
  • David B. Berkowitz* and Michelle L. Pedersen Simultaneous Amino and Carboxyl Group Protection for alpha-Branched Amino Acids. J. Org. Chem. 1994, 59, 5476-5478. pdf
  • Michelle L. Pedersen and David B. Berkowitz* Formal alpha-Vinylation of Amino Acids. Use of a New Benzeneselenolate Equivalent. J. Org. Chem.1993, 58, 6966-6975. pdf
  • Michelle L. Pedersen and David B. Berkowitz* A Reagent for the Efficient Cleavage of N-Benzoylhomoserine Lactones: Access to alpha-(2-phenylseleno)ethyl Amino Acids. Tetrahedron Letters 1992, 33, 7315-7318. pdf

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Chemoenzymatic and Ring E-Modular Entry into the (-)-Podophyllotoxin & Etoposide Drug Families

       In a total synthetic venture, a chemoenzymatic and ring-E modular route to the (-)-podophyllotoxin skeleton has been developed as a bioorganic tool to permit systematic SAR (Structure-Activity Relationship) studies on ring E. Podophyllotoxin is a tubulin-binding lignan isolated from the apple mandrake plant in North America (Podophyllum peltatum) and India (Podophyllum emodi). It has been used in Native American folk medicine for multifarious ailments for generations. It displays antimitotic activity, presumably through the inhibition of tubulin polymerization. (-)-Podophyllotoxin itself is used clinically for the treatment of venereal warts.

       A number of semi-synthetic derivatives of the natural product are used in cancer chemotherapy. The most widely used include Etoposide, Teniposide and Etopophos. Etoposide, in particular, is one of the most common single agents used to treat small cell ung cancer, testicular cancer and Kaposi*s sarcoma. There is a vast clinical literature on this compound. Etoposide is believed to act on a different target than its core aglycon; namely the drug is known to inhibit the religation step along the topoisomerase II reaction coordinate. This leads to the build-up of a covalent enzyme-DNA intermediate which is appears to provide a signal for apoptosis.

       Given the inherent difficulties in obtaining high resolution X-ray diffraction data for tubulin, and the sheer size of topoisomerase II (eukaryotic enzyme = dimer of 1429 AA subunits), information about interaction of each drug with its putative target is lacking. Furthermore, there is a significant body of literature that focuses on an alternative potential mode of action of these drugs. Namely, this involves oxidative demethylation of ring E to produce, catechol, semiquinone and o-quinone structures. This ring E oxidation chemistry can be demonstrated, in vitro, with cytochrome P-450. Its relevance to the anti-proliferative activity of either compound remains an open question. Our synthesis was therefore designed as a bioorganic tool to better define the &pharmacophores* of these two drug families, with a particular focus on establishing SAR in ring E.

       Though a number of total syntheses of (-)-podophyllotoxin had been reported prior to our work, philosophically and practically, our approach differs from these in several fundamental ways: (1) Our retrosynthetic analysis takes the natural product back to an intermediate with a plane of symmetry. No previous strategy had sought to exploit C s symmetry. (2) Ours is the first catalytic asymmetric synthesis. (3) Absolute stereochemistry is introduced by means of an enzyme-catalyzed transformation upon an advanced meso intermediate. Though enzymes are now quite often employed as chiral reagents in synthesis, their application is largely in the domain of small molecule building blocks, and rarely on substrates of high carbon count, multiple steps into a total synthesis. (4) Ring E is introduced late in the synthesis, via a diastereoselective conjugate addition (Scheme 7), allowing for a comprehensive SAR study of this sector of the natural product.

  • David B. Berkowitz ; Sylvain Broussy; Argiris Efstratiadis;  Apostolos Klinakis; Matthias Szabolcs.  (Columbia University In the City of   New York, USA; University of Nebraska).    ※Analogues of (-)-Picropodophyllin,  Synthesis and Uses Thereof.§ PCT Int. Appl.  2009, 114pp. CODEN: PIXXD2 WO 2009100349  A1  20090813
  • Wilstermann, A. M.; Bender, R. P.; Godfrey, M.; Choi, S.; Anklin, C.; Berkowitz, D. B.; Osheroff, N.; Graves, D. E. ※Topoisomerase II-Drug Interaction Domains: Identification of Substituents on Etoposide that Interact with the Enzyme.§ Biochemistry 2007, 46, 8217-8225. pdf

  • David B. Berkowitz,* Sungjo Choi, Debnath Bhuniya and Richard K. Shoemaker Novel 'Reverse Kahne-Type Glycosylation': Synthesis of O-, N- and C-Linked Epipodophyllotoxin Conjugates. Org. Lett. 2000, 2, 1149-1152. pdf

  • David B. Berkowitz,* Sungjo Choi and Jun-Ho Maeng Enzyme-Assisted Asymmetric Total Synthesis of (-)-Podophyllotoxin and (-)-Picropodophyllin.    J. Org. Chem. 2000, 65, 847-860. pdf

  • David B. Berkowitz,* Ryan Hartung and Sungjo Choi Enzymatic Hydrolysis of Advanced Synthetic Intermediates: On the Choice of the Organic Cosolvent. Tetrahedron: Asymmetry, 1999, 10, 4513-4520. pdf

  • David B. Berkowitz,* Jun-Ho Maeng, A.H. Dantzig, R.L. Shepard & B.H. Norman Chemoenzymatic and Ring E-Modular Approach to the (-)-Podophyllotoxin Skeleton. Synthesis of 3',4',5'-Tridemethoxy-(-)-podophyllotoxin, J. Am.Chem. Soc.(Communication) 1996, 118, 9426-9427. pdf

  • David B. Berkowitz* and Jun-Ho Maeng Enantioselective Entry into Benzoxabicyclo[2.2.1]heptyl Systems by Enzymatic Desymmetrization: Toward  Chiral Building Blocks for Lignan Synthesis, Tetrahedron: Asymmetry 1996,7,1577-1580. pdf

  • Quanrong Shen, Darby G. Sloss, David B. Berkowitz*  Displacement of Primary Sugar Triflates with C-Nucleophiles: D-Glucopyranose and D-Ribofuranose Functionalization and Chain Extension, Synthetic Communications 1994, 24, 1519-1530.pdf

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 Surrogates for Biological Phosphates: Synthesis and Biochemical Study

     Digestive phosphatases, much like digestive proteases, are ubiquitous. Thus, there is great interest in the medicinal and bioorganic chemistry communities in developing surrogate functionalities for the biological phosphate ester and amide bonds. Ideally, these surrogates would be resistant to hydrolases, but retain the favorable structural and functional properties of the native functionality. Phosphonates have been used as phosphate mimics for half a century. More recently, spurred on by the work of G. M. Blackburn (Sheffield), there has been heightened interest in a-fluorinated phosphonates, as potentially better mimics of phosphates, at least in terms of polarity and acidity (i.e. lower 2 nd pKa).

    One of our most utilized contributions is the divergent synthetic approach to the whole set of phosphonates, differing in the degree of alpha-fluorination (i.e bridging CH2, CHF and CF2) from a common precursor (Scheme 8). The chemistry involves the displacement of primary triflates, and turns out to be quite practical, particularly for carbohydrate systems. (link) The anions derived from both a-mono- and difluorinated methyl phosphonate esters, are unstable, with alpha-elimination known to occur in the difluoro case above about 每40 ∼C. This led to our application of the especially reactive triflate coupling partner, and to the installation of an anion-stabilizing sulfonyl group in our approach to the mono-fluoro targets. (link)

      Among the more important targets that we have been able to access are included the difluorinated phosphonate analogues of the phosphorylated amino acids, L-serine, L-threonine and L-allothreonine (Scheme 9). (link) This is particularly significant given the importance of amino acid phosphorylation/dephosphorylation as a signal transduction mechanism. These surrogates can be incorporated into peptides and provide a hydrolytically stable analogue switching signal that is fixed in either the ※ON§ or the ※OFF§ position, depending upon the function of phosphorylation. As can be imagined, this is a potentially powerful bioorganic tool. A most interesting demonstration of this has been reported by Appella. His group found that while it was impossible to raise antibodies to a pSer-containing peptide, based on the N-terminal sequence of p53, simple replacement of the pSer residue at the 6-position with our pSer mimic solved the problem. The initial antigen presumably suffered phosphate ester cleavage, at the hands of phosphatases in the rabbit. The surrogate functionality was both phosphatase-resistant, and resembled the native structure sufficiently that the antibodies raised against its peptide specifically recognize Ser-6-phosphorylated p53. This allowed Appella to study the degree of phosphorylation of this important tumor suppressor protein, in response to DNA damage (E. Appella and coworkers J. Biol. Chem.2000, 275, 23199-23203- pubMed link).

      Our own bioorganic studies in this area have been in several systems, all involving sugar phosphate mimics. Thus, a complete &fluorinated phosphonate scan* of the glucose 6-hosphate dehydrogenase (G6PDH) active site revealed that the a-monofluorinated phosphonate was the best phosphate mimic here, provided that it had the &correct* (S)-stereochemistry (Scheme 10). (link) This result is a reminder that the somewhat neglected monofluoro compounds warrant greater study. The paper also serves notice to the community that kinetic constants derived from assays of diastereomeric mixtures of ( a-monofluoro)phosphonates may not be particularly meaningful. In the G6PDH case, an order of magnitude difference in K m was seen between the two stereoisomers.

      Another system under current study in our lab include the enzyme glucosamine 6-phosphate synthase (GlmS), the enzyme that performs an &aminative tautomerization* of fructose 6-phosphate (F6P), employing glutamine as the ammonia source. This work is collaborative with Bernard Badet (CNRS-Gif-sur-Yvette, France).

      Recently, in collaboration with Richard G. MacDonald at the University of Nebraska Medical Center, we have begun to examine how mono- and bivalent analogues of mannose 6-phosphate bind to the important receptor known as the M6P/IGF2R (Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor). Self-cross metathesis is utilized as a method to rapidly assemble bivalent M6P-presenting ligands as &measuring sticks* (Scheme 10) to test for bivalent ligand-receptor interactions. (link)

  • Kaushik Panigrahi, MariJean Eggen, Jun-Ho Maeng, Quanrong Shen, David B. Berkowitz* ※The a,a-Difluorinated Phosphonate L-pSer-Analogue: An Accessible Chemical Tool for Studying Kinase-Dependent Signal Transduction, in Vitro and In Living Cells.§ Chemistry and Biology  2009, 16(9), 928-936 pdf
  • Fei, Xiang; Connelly, Christopher M.; MacDonald, Richard G.; Berkowitz, David B.. "A set of phosphatase-inert "molecular rulers" to probe for bivalent mannose 6-phosphate ligand-receptor interactions." Bioorganic & Medicinal Chemistry Letters 2008, 18(10), 3085-3089. pdf

  • Bradley D. Charette, Richard G. MacDonald, Stefan Wetzel, David B. Berkowitz* and Herbert Waldmann* ※PSSC (Protein Structure Similarity Clustering): Dynamic Treatment of PDB Structures Facilitates Clustering, Angew. Chem. Int. Ed. 2006, 45, 7766-7770. pdf

  • David B. Berkowitz,* Gourhari Maiti, Bradley D. Charette, Christine D. Dreis and Richard G. MacDonald* Mono- and Bivalent Ligands Bearing Mannose 6-Phosphate (M6P) Surrogates: Targeting the M6P/Insulin-like Growth Factor II Receptor. Org. Lett. 2004, 6, 4921-4924.pdf

  • David B. Berkowitz* and Mohua Bose (alpha-Monofluoroalkyl)phosphonates: A Class of Isoacidic and &Tunable* Mimics of Biological Phosphates, Journal of Fluorine Chemistry 2001, 112, 13-33 (invited).pdf

  • David B. Berkowitz,* Mohua Bose and Nathan G. Asher, A Convergent Triflate Displacement Approach to (alpha-Monofluoroalkyl)phosphonates, Org. Lett. 2001, 3, 2009-2012.pdf

  • David B. Berkowitz,* Mohua Bose, Travis J. Pfannenstiel and Tzanko Doukov alpha-Fluorinated Phosphonates as Substrate Mimics for Glucose 6-Phosphate Dehydrogenase: The CHF Stereochemistry Matters, J. Org. Chem. 2000, 65, 4498-4508.pdf

  • David B. Berkowitz,* Debnath Bhuniya and Gorka Peris Facile Installation of the Phosphonate and (alpha, alpha-Difluoromethyl)phosphonate Functionalities Equipped with Benzyl Protection. Tetrahedron Letters, 1999, 40, 1869-1872.pdf

  • David B. Berkowitz,* MariJean Eggen, Quanrong Shen and Richard K. Shoemaker Ready Access to Fluorinated Phosphonate Mimics of Secondary Phosphates. Synthesis of the (alpha, alpha-Difluoroalkyl)phosphonate Analogues of L-Phosphoserine, L-Phosphoallothreonine and L-Phosphothreonine, J. Org. Chem. 1996, 61, 4666-4675.pdf

  • David B. Berkowitz* and Darby G. Sloss Diallyl Lithiodifluoromethylphosphonate: A New Reagent for the Introduction of the (Difluoromethylene)phosphonate Functionality. J. Org. Chem. 1995, 60, 7047-7050.pdf

  • David B. Berkowitz,* Quanrong Shen and Jun-Ho Maeng Synthesis of the (alpha, alpha-Difluoroalkyl)phosphonate Analogue of Phosphoserine. Tetrahedron Letters 1994, 35, 6445-6448.pdf

  • David B. Berkowitz*, MariJean Eggen, Quanrong Shen and Darby G. Sloss Synthesis of (alpha, alpha -Difluoroalkyl)phosphonates by Displacement of Primary Triflates. J. Org. Chem. 1993, 58, 6174-6176.pdf

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Publications and Patents

David B. Berkowitz ; Sylvain Broussy; Argiris Efstratiadis;  Apostolos Klinakis; Matthias Szabolcs.  (Columbia University In the City of   New York, USA; University of Nebraska).    ※Analogues of (-)-Picropodophyllin,  Synthesis and Uses Thereof.§ PCT Int. Appl.  2009, 114pp. CODEN: PIXXD2 WO 2009100349  A1  20090813

Kaushik Panigrahi, MariJean Eggen, Jun-Ho Maeng, Quanrong Shen, David B. Berkowitz* ※The a,a-Difluorinated Phosphonate L-pSer-Analogue: An Accessible Chemical Tool for Studying Kinase-Dependent Signal Transduction, in Vitro and In Living Cells.§ Chemistry and Biology  2009, 16(9), 928-936 pdf

David B. Berkowitz,* Kannan R. Karukurichi, Roberto de la Salud-Bea, Gourhari Maiti, Jill M. McFadden and Michelle L. Morris ※Stereoselective Synthesis of Quaternary, alpha-Vinyl Amino Acids  and their alpha-(2*Z-Fluoro)vinyl Congeners 每 Promising Candidates for PLP Enzyme Inactivation.§ ACS Symposium-In-Print 2009, 1009, 288-303. pdf

Broussy, Sylvain, Cheloha, Ross W., and Berkowitz, David B.. "Enantioselective, Ketoreductase-Based Entry into Pharmaceutical Building Blocks: Ethanol as Tunable Nicotinamide Reductant." Organic Letters 2009, 11(2), 305-308. pdf

David B. Berkowitz*, Kannan R. Karukurichi, Roberto de la Salud-Bea, David L. Nelson, and Christopher D. McCune, ※Use of fluorinated functionality in enzyme inhibitor development: Mechanistic and analytical advantages,§
J. Fluorine Chem. 2008, 129, 731每742.pdf

Fei, Xiang; Connelly, Christopher M.; MacDonald, Richard G.; Berkowitz, David B.. "A set of phosphatase-inert "molecular rulers" to probe for bivalent mannose 6-phosphate ligand-receptor interactions." Bioorganic & Medicinal Chemistry Letters 2008, 18(10), 3085-3089. pdf

Sangeeta Dey, Douglas R. Powell, Chunhua Hu, and David B. Berkowitz,* "Cassette"-ISES (In Situ Enzymatic Screening) Identifies Complementary Chiral Scaffolds for Hydrolytic Kinetic Resolution Across a Range of Epoxides. Angew. Chem. Int. Ed. 2007, 46, 7010-7014. pdf

Wilstermann, A. M.; Bender, R. P.; Godfrey, M.; Choi, S.; Anklin, C.; Berkowitz, D. B.; Osheroff, N.; Graves, D. E. ※Topoisomerase II-Drug Interaction Domains: Identification of Substituents on Etoposide that Interact with the Enzyme.§ Biochemistry 2007, 46, 8217-8225. pdf

Kannan R. Karukurichi, Roberto de la Salud-Bea, Wan Jin Jahng, David B. Berkowitz* ※Examination of the New a-(2*Z-Fluoro)vinyl Trigger with Lysine Decarboxylase: The Absolute Stereochemistry Determines the Reaction Course ,§ J. Am. Chem. Soc. 2007, 129, 258-259. pdf

Bradley D. Charette, Richard G. MacDonald, Stefan Wetzel, David B. Berkowitz* and Herbert Waldmann* (co-corresponding authors) ※PSSC (Protein Structure Similarity Clustering): Dynamic Treatment of PDB Structures Facilitates Clustering, Angew. Chem. Int. Ed.2006, 45, 7766-7770. pdf

David B. Berkowitz,* Bradley D. Charette, Kannan R. Karukurichi and Jill M. McFadden ※ a-Vinylic Amino Acids: Natural Occurrence, Asymmetric Synthesis and Biochemical Mechanisms .§ Tetrahedron: Asymmetry 2006, 17, 869-882. pdf

David B. Berkowitz, Sangeeta Dey, Kannan R. Karukurichi and Weijun Shen ※A Method for Measuring the Stereoselectivity and Relative Rate for Organic Chemical Reactions.§ U.S. Patent Application, Serial No. 11/434,247, filed May 16, 2006.

David B. Berkowitz,* Bin Wu and Huijie Li ※A Formal [3,3]-Sigmatropic Rearrangement Route to Quaternary a-Vinyl Amino Acids: Use of Allylic N-PMP Trifluoroacetimidates.§ Org. Lett. 2006, 8, 971-974. pdf

Sangeeta Dey, Kannan R. Karukurichi,Weijun Shen, and David B. Berkowitz,* Double-Cuvette ISES: In Situ Estimation of Enantioselectivity and Relative Rate for Catalyst Screening.J. Am. Chem. Soc.2005, 127, 8610-8611.pdf

Weijun Shen, Molly K. McGath, Ruby Evande and David B. Berkowitz,* A Continuous Spectrophotometric Assay for Human Cystathionine Beta-Synthase.Analytical Biochemistry 2005, 342,1 103-110.pdf

David B. Berkowitz,* Gourhari Maiti, Bradley D. Charette, Christine D. Dreis and Richard G. MacDonald* Mono- and Bivalent Ligands Bearing Mannose 6-Phosphate (M6P) Surrogates: Targeting the M6P/Insulin-like Growth Factor II Receptor. Org. Lett. 2004, 6, 4921-4924.pdf

David B. Berkowitz* and Gourhari Maiti Following an ISES Lead: The First Examples of Asymmetric Ni(0)-Mediated Allylic Amination. Org. Lett. 2004, 6, 2661-2664.pdf

David B. Berkowitz, *Weijun Shen and Gourhari Maiti In Situ Enzymatic Screening (ISES) of P,N-Ligands for Ni(0)-Mediated Asymmetric, Intramolecular Allylic Amination. Tetrahedron: Asymmetry200415, 2845-2851. pdf

David B. Berkowitz, *Roberto de la Salud-Bea and Wan-Jin Jahng, Synthesis of Quaternary Amino Acids Bearing a (2'Z)-Fluorovinyl alpha-Branch: Potential PLP Enzyme Inactivators. Org. Lett. 2004, 6, 1821-1824.pdf

M.J. Huang,* S. Regunathan, M. Botta, K. Lee, E. McClendon, G. B. Yi, M. L. Pedersen, D. B. Berkowitz, G. Wang, M. Travagli and J. E. Piletz Structure-Activity Analysis of Guanidine Group in Agmatine for Brain Agmatinase, Annals of the New York Academy of Sciences 2003 , 1009, 52-64.

David B. Berkowitz, * Mohua Bose and Sungjo Choi ※Biphasic Process For In Situ Enzymatic Screening To Optimize Variables In Organic Reactions.§ U.S. Pat. Appl. Publ. (2003), 47 pp. CODEN: USXXCO US 2003148257 A1 20030807 CAN 139:164340 AN 2003:609937

David B. Berkowitz, * Mohua Bose and Sungjo Choi In Situ Enzymatic Screening (ISES): A Tool for Catalyst Discovery and Reaction Development. Angewandte Chemie International Edition, English 2002 , 41, 1603-1607. pdf

David B. Berkowitz * and Mohua Bose ※(alpha-monofluoroalkyl)phosphonates: A Class of Isoacidic and &Tunable* Mimics of Biological Phosphates,§ Journal of Fluorine Chemistry 2001 , 112, 13-33 (invited).pdf

David B. Berkowitz, * Esmort Chisowa and Jill M. McFadden, Stereocontrolled Synthesis of Quaternary beta, gama -Unsaturated Amino Acids: Chain Extension of D- & L- alpha -(2-Tributylstannyl)Vinyl Amino Acids, Tetrahedron Symposia-in-Print 2001 , 57, 6329-6343. pdf

David B. Berkowitz, * Mohua Bose and Nathan G. Asher, A Convergent Triflate Displacement Approach to ( alpha -Monofluoroalkyl)phosphonates, Org. Lett. 2001 , 3, 2009-2012. pdf

David B. Berkowitz, * Jill M. McFadden, Esmort Chisowa and Craig L. Semerad Organoselenium-Based Entry into Versatile, alpha -(2-Tributylstannyl)vinyl Amino Acids in Scalemic Form: A New Route to Vinyl Stannanes, J. Am. Chem.Soc. 2000, 122, 11031-11032.pdf

David B. Berkowitz, * Mohua Bose, Travis J. Pfannenstiel and Tzanko Doukov alpha-Fluorinated Phosphonates as Substrate Mimics for Glucose 6-Phosphate Dehydrogenase: The CHF Stereochemistry Matters, J. Org. Chem. 2000, 65, 4498-4508. pdf

David B. Berkowitz Ring E-Modified Analogues of (-)-Podophyllotoxin and a Method for Their Synthesis, U.S. Patent No. 6,051,721 April 18, 2000.

David B. Berkowitz, * Sungjo Choi, Debnath Bhuniya and Richard K. Shoemaker Novel 'Reverse Kahne-Type Glycosylation': Synthesis of O-, N- and C-Linked Epipodophyllotoxin Conjugates. Org. Lett. 2000, 2, 1149-1152.pdf

David B. Berkowitz, * Jill M. McFadden and Marianne K. Sloss Engineering Acyclic Stereocontrol in the Alkylation of Vinylglycine-Derived Dianions: Asymmetric Synthesis of Higher a -Vinyl Amino Acids, J. Org. Chem. 2000, 65, 2907-2918. pdf

David B. Berkowitz, * Sungjo Choi and Jun-Ho Maeng Enzyme-Assisted Asymmetric Total Synthesis of (-)-Podophyllotoxin and (-)-Picropodophyllin. J. Org. Chem. 2000, 65, 847-860.pdf

David B. Berkowitz and Michelle L. Pedersen alpha -Oxiranyl Amino Acids, U.S. Patent No. 6,008,386 December 28, 1999.

David B. Berkowitz, * Ryan Hartung and Sungjo Choi Enzymatic Hydrolysis of Advanced Synthetic Intermediates: On the Choice of the Organic Cosolvent. Tetrahedron: Asymmetry, 1999, 10, 4513-4520. pdf

David B. Berkowitz, * Debnath Bhuniya and Gorka Peris Facile Installation of the Phosphonate and ( alpha, alpha-Difluoromethyl)phosphonate Functionalities Equipped with Benzyl Protection.Tetrahedron Letters, 1999, 40, 1869-1872.pdf

David B. Berkowitz, * Jill McFadden, Marianne K. Smith and Michelle L. Pedersen Synthesis of alpha-Vinyl Amino Acids. Methods in Molecular Medicine, PeptidomimeticsProtocols (Wieslaw M. Kazmierski, volume Ed.; John M. Walker, series Ed.; Humana Press, Totowa, NJ), Vol. 23, Chapter 27, 467-488, 1999.pdf

David B. Berkowitz & Michelle L. Pedersen A Method for the Synthesis of alpha-Oxiranyl Amino Acids, U.S. Patent No. 5,705,660; January 6, 1998

David B. Berkowitz, * Wan-Jin Jahng and Michelle L. Pedersen (㊣)- a-Vinyllysine and (㊣)- alpha-Vinylarginine are Time-Dependent Inhibitors of Their Cognate Decarboxylases. Bioorganic and Medicinal Chemistry Letters 1996, 6, 2151-2156.pdf

David B. Berkowitz, * Jun-Ho Maeng, A.H. Dantzig, R.L. Shepard & B.H. Norman Chemoenzymatic and Ring E-Modular Approach to the (-)-Podophyllotoxin Skeleton. Synthesis of 3',4',5'-Tridemethoxy-(-)-podophyllotoxin, J. Am. Chem. Soc. (Communication) 1996, 118, 9426-9427.pdf

David B. Berkowitz, * MariJean Eggen, Quanrong Shen and Richard K. Shoemaker Ready Access to Fluorinated Phosphonate Mimics of Secondary Phosphates. Synthesis of the ( alpha, alpha-Difluoroalkyl)phosphonate Analogues of L-Phosphoserine, L-Phosphoallothreonine and L-Phosphothreonine, J. Org. Chem. 1996, 61, 4666-4675.pdf

David B. Berkowitz, * Michelle L. Pedersen and Wan-Jin Jahng Synthesis of Higher alpha-Chlorovinyl and alpha-Bromovinyl Amino Acids: The Amino Protecting Group Determines the Reaction Course, Tetrahedron Letters1996, 37, 4309-4312.pdf

David B. Berkowitz * and Jun-Ho Maeng Enantioselective Entry into Benzoxabicyclo[2.2.1]heptyl Systems by Enzymatic Desymmetrization: Toward Chiral Building Blocks for Lignan Synthesis, Tetrahedron: Asymmetry1996, 7, 1577-1580.

David B. Berkowitz * and Marianne K. Smith, A Convenient Synthesis of L-Vinylglycine from L-Homoserine Lactone. Synthesis , 1996, 39-41. pdf

David B. Berkowitz * and Darby G. Sloss  Diallyl Lithiodifluoromethylphosphonate: A New Reagent for the Introduction of the (Difluoromethylene)phosphonate Functionality. J. Org. Chem. 1995 , 60 , 7047-7050.pdf

David B. Berkowitz * and Michelle L. Pedersen Free alpha -Oxiranyl Amino Acids. J. Org. Chem. 1995 , 60 , 5368-5369. pdf

David B. Berkowitz * and Marianne K. Smith Enantiomerically Enriched alpha -Methyl Amino Acids. Alkylation of an Acyclic, Chiral Alanine Dianion with a High Diastereofacial Bias. J. Org. Chem. 1995, 60 , 1233-1238.pdf

David B. Berkowitz, * James A. Pumphrey and Quanrong Shen Enantiomerically Enriched a-Vinyl Amino Acids via Lipase-Mediated Reverse Transesterification, Tetrahedron Letters 1994, 35, 8743-8746.pdf

David B. Berkowitz, * Quanrong Shen and Jun-Ho Maeng Synthesis of the (alpha, alpha-Difluoroalkyl)phosphonate Analogue of Phosphoserine.  Tetrahedron Letters 1994, 35, 6445-6448.pdf

David B. Berkowitz * and Michelle L. Pedersen Simultaneous Amino and Carboxyl Group Protection for alpha-Branched Amino Acids. J. Org. Chem.1994, 59, 5476-5478.pdf

Quanrong Shen, Darby G. Sloss, David B. Berkowitz * Displacement of Primary Sugar Triflates with C-Nucleophiles: D-Glucopyranose and D-Ribofuranose Functionalization and Chain Extension, Synthetic Communications 1994, 24 , 1519-1530.

Michelle L. Pedersen and David B. Berkowitz * Formal alpha-Vinylation of Amino Acids. Use of a New Benzeneselenolate Equivalent.J. Org. Chem. 1993, 58, 6966-6975. pdf

David B. Berkowitz * , MariJean Eggen, Quanrong Shen and Darby G. Sloss Synthesis of (alpha , alpha -Difluoroalkyl)phosphonates by Displacement of Primary Triflates. J. Org. Chem. 1993, 58 , 6174-6176. pdf

Michelle L. Pedersen and David B. Berkowitz* A Reagent for the Efficient Cleavage of N-Benzoylhomoserine Lactones: Access to alpha-(2-phenylseleno)ethyl Amino Acids. Tetrahedron Letters 1992, 33, 7315-7318. pdf

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