Bio


Professor Carolyn Bertozzi's research interests span the disciplines of chemistry and biology with an emphasis on studies of cell surface sugars important to human health and disease. Her research group profiles changes in cell surface glycosylation associated with cancer, inflammation and bacterial infection, and uses this information to develop new diagnostic and therapeutic approaches, most recently in the area of immuno-oncology.

Dr. Bertozzi completed her undergraduate degree in Chemistry at Harvard University and her Ph.D. at UC Berkeley, focusing on the chemical synthesis of oligosaccharide analogs. During postdoctoral work at UC San Francisco, she studied the activity of endothelial oligosaccharides in promoting cell adhesion at sites of inflammation. She joined the UC Berkeley faculty in 1996. A Howard Hughes Medical Institute Investigator since 2000, she came to Stanford University in June 2015, among the first faculty to join the interdisciplinary institute ChEM-H (Chemistry, Engineering & Medicine for Human Health). Named a MacArthur Fellow in 1999, Dr. Bertozzi has received many awards for her dedication to chemistry, and to training a new generation of scientists fluent in both chemistry and biology. She has been elected to the Institute of Medicine, National Academy of Sciences, and American Academy of Arts and Sciences; and received the Lemelson-MIT Prize, the Heinrich Wieland Prize, and the ACS Award in Pure Chemistry, among many others. Her efforts in undergraduate education have earned the UC Berkeley Distinguished Teaching Award and the Donald Sterling Noyce Prize for Excellence in Undergraduate Teaching.

Today, the Bertozzi Group at Stanford studies the glycobiology underlying diseases such as cancer, inflammatory disorders such as arthritis, and infectious diseases such as tuberculosis. The work has advanced understanding of cell surface oligosaccharides involved in cell recognition and inter-cellular communication.

Dr. Bertozzi's lab also develops new methods to perform controlled chemical reactions within living systems. The group has developed new tools for studying glycans in living systems, and more recently nanotechnologies for probing biological systems. Such "bioorthoganol" chemistries enable manipulation of biomolecules in their living environment.

Several of the technologies developed in the Bertozzi lab have been adapted for commercial use. Actively engaged with several biotechnology start-ups, Dr. Bertozzi founded Redwood Bioscience of Emeryville, California, and has served on the research advisory board of GlaxoSmithKline.

Academic Appointments


Administrative Appointments


  • Investigator, Howard Hughes Medical Institute (2000 - Present)

Honors & Awards


  • Arthur C. Cope Award, American Chemical Society (2017)
  • National Academy of Sciences Award in the Chemical Sciences, National Academy of Sciences (2016)
  • Ernest Orlando Lawrence Award, U.S. Department of Energy (2015)
  • Heinrich Wieland Prize, Heinrich Wieland Prize (2012)
  • Lemelson-MIT Prize, Massachusetts Institute of Technology (2010)
  • Ernst Schering Prize, Ernst Schering Research Foundation (2007)
  • Distinguished Teaching Award, UC Berkeley College of Chemistry (2001)
  • Award in Pure Chemistry, American Chemical Society (2001)
  • MacArthur Foundation “Genius” Award, MacArthur Foundation (1999)
  • Arthur C. Cope Scholar Award, American Chemical Society (1999)
  • Honorary Degree, Freie University Berlin (2014)
  • Honorary Doctorate Degree, Duke University (2014)
  • Hans Bloemendal Award, Radboud Univ. Nijmegen (2013)
  • Honorary Doctorate Degree, Brown University (2012)
  • Tetrahedron Young Investigator Award, Executive Board of Editors and the Publisher of Tetrahedron Publications (2011)
  • Albert Hofmann Medal, U. Zurich (2009)
  • Harrison Howe Award, Rochester Section, American Chemical Society (2009)
  • W. H. Nichols Award, New York Section, American Chemical Society (2009)
  • Li Ka Shing Women in Science Award, Li Ka Shing Foundation Women in Science Program (2008)
  • Roy L. Whistler International Award in Carbohydrate Chemistry, International Carbohydrate Organization (2008)
  • Willard Gibbs Medal, Chicago Section, American Chemical Society (2008)
  • T.Z. and Irmgard Chu Distinguished Professorship in Chemistry, UC Berkeley (2005-14)
  • Havinga Medal, U. Leiden (2005)
  • Agnes Fay Morgan Research Award, Iota Sigma Pi (2004)
  • Fellow, American Association for the Advancement of Science (2002)
  • Irving Sigal Young Investigator Award, Protein Society (2002)
  • Donald Sterling Noyce Prize for Excellence in Undergraduate Teaching, UC Berkeley College of Chemistry (2001)
  • Department of Chemistry Teaching Award, UC Berkeley (2000)
  • Merck Academic Development Program Award, Merck (2000)
  • Presidential Early Career Award in Science and Engineering (PECASE), The U.S. White House (2000)
  • Camille Dreyfus Teacher-Scholar Award, Camille and Henry Dreyfus Foundation (1999)
  • Beckman Young Investigator Award, Arnold and Mabel Beckman Foundation (1998)
  • Glaxo Wellcome Scholar, Glaxo Wellcome (1998)
  • Prytanean Faculty Award, Prytanean Women's Honor Society, UC Berkeley (1998)
  • Research Innovation Award, Research Corporation (1998)
  • Young Investigator Award, Office of Naval Research (1998)
  • Horace S. Isbell Award in Carbohydrate Chemistry, American Chemical Society (1997)
  • New Investigator Award in Pharmacology, Burroughs Wellcome (1997)
  • Sloan Research Fellow, Alfred P. Sloan Foundation (1997)
  • Pew Scholars Award in the Biomedical Sciences, Pew Charitable Trusts (1996)
  • Young Investigator Award, Exxon Education Fund (1996)
  • Dreyfus New Faculty Award, Camille and Henry Dreyfus Foundation (1995)

Boards, Advisory Committees, Professional Organizations


  • Member, National Academy of Inventors (2013 - Present)
  • Member, Institute of Medicine (2011 - Present)
  • Member, German Academy of Sciences Leopoldina (2008 - Present)
  • Member, National Academy of Sciences (2005 - Present)
  • Member, American Academy of Arts and Sciences (2003 - Present)
  • Chair, Scientific Advisory Board, Redwood Bioscience
  • Board Member, Board of Scientific Counslors, Broad Institutue
  • Board Member, Catalent Biologics Board
  • Board Member, Research Advisory Baord, Glaxo Smithkline

Professional Education


  • Postdoc, UC San Francisco, Immunology
  • PhD, UC Berkeley, Chemistry (1993)
  • AB, Harvard University, Chemistry (1988)

2017-18 Courses


All Publications


  • Precision glycocalyx editing as a strategy for cancer immunotherapy. Proceedings of the National Academy of Sciences of the United States of America Xiao, H., Woods, E. C., Vukojicic, P., Bertozzi, C. R. 2016; 113 (37): 10304-10309

    Abstract

    Cell surface sialosides constitute a central axis of immune modulation that is exploited by tumors to evade both innate and adaptive immune destruction. Therapeutic strategies that target tumor-associated sialosides may therefore potentiate antitumor immunity. Here, we report the development of antibody-sialidase conjugates that enhance tumor cell susceptibility to antibody-dependent cell-mediated cytotoxicity (ADCC) by selective desialylation of the tumor cell glycocalyx. We chemically fused a recombinant sialidase to the human epidermal growth factor receptor 2 (HER2)-specific antibody trastuzumab through a C-terminal aldehyde tag. The antibody-sialidase conjugate desialylated tumor cells in a HER2-dependent manner, reduced binding by natural killer (NK) cell inhibitory sialic acid-binding Ig-like lectin (Siglec) receptors, and enhanced binding to the NK-activating receptor natural killer group 2D (NKG2D). Sialidase conjugation to trastuzumab enhanced ADCC against tumor cells expressing moderate levels of HER2, suggesting a therapeutic strategy for cancer patients with lower HER2 levels or inherent trastuzumab resistance. Precision glycocalyx editing with antibody-enzyme conjugates is therefore a promising avenue for cancer immune therapy.

    View details for DOI 10.1073/pnas.1608069113

    View details for PubMedID 27551071

  • Chemically tunable mucin chimeras assembled on living cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kramer, J. R., Onoa, B., Bustamante, C., Bertozzi, C. R. 2015; 112 (41): 12574-12579

    View details for DOI 10.1073/pnas.1516127112

    View details for Web of Science ID 000363130900023

    View details for PubMedID 26420872

  • CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Shieh, P., Dien, V. T., Beahm, B. J., Castellano, J. M., Wyss-Coray, T., Bertozzi, C. R. 2015; 137 (22): 7145-7151

    Abstract

    Fluorescent bioorthogonal smart probes across the visible spectrum will enable sensitive visualization of metabolically labeled molecules in biological systems. Here we present a unified design, based on the principle of photoinduced electron transfer, to access a panel of highly fluorogenic azide probes that are activated by conversion to the corresponding triazoles via click chemistry. Termed the CalFluors, these probes possess emission maxima that range from green to far red wavelengths, and enable sensitive biomolecule detection under no-wash conditions. We used the CalFluor probes to image various alkyne-labeled biomolecules (glycans, DNA, RNA, and proteins) in cells, developing zebrafish, and mouse brain tissue slices.

    View details for DOI 10.1021/jacs.5b02383

    View details for Web of Science ID 000356322300038

    View details for PubMedID 25902190

  • Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis NATURE METHODS Woo, C. M., Iavarone, A. T., Spiciarich, D. R., Palaniappan, K. K., Bertozzi, C. R. 2015; 12 (6): 561-?

    Abstract

    Protein glycosylation is a heterogeneous post-translational modification (PTM) that plays an essential role in biological regulation. However, the diversity found in glycoproteins has undermined efforts to describe the intact glycoproteome via mass spectrometry (MS). We present IsoTaG, a mass-independent chemical glycoproteomics platform for characterization of intact, metabolically labeled glycopeptides at the whole-proteome scale. In IsoTaG, metabolic labeling of the glycoproteome is combined with (i) chemical enrichment and isotopic recoding of glycopeptides to select peptides for targeted glycoproteomics using directed MS and (ii) mass-independent assignment of intact glycopeptides. We structurally assigned 32 N-glycopeptides and over 500 intact and fully elaborated O-glycopeptides from 250 proteins across three human cancer cell lines and also discovered unexpected peptide sequence polymorphisms (pSPs). The IsoTaG platform is broadly applicable to the discovery of PTM sites that are amenable to chemical labeling, as well as previously unknown protein isoforms including pSPs.

    View details for DOI 10.1038/nmeth.3366

    View details for Web of Science ID 000355248100027

    View details for PubMedID 25894945

  • The cancer glycocalyx mechanically primes integrin-mediated growth and survival NATURE Paszek, M. J., DuFort, C. C., Rossier, O., Bainer, R., Mouw, J. K., Godula, K., Hudak, J. E., Lakins, J. N., Wijekoon, A. C., Cassereau, L., Rubashkin, M. G., Magbanua, M. J., Thorn, K. S., Davidson, M. W., Rugo, H. S., Park, J. W., Hammer, D. A., Giannone, G., Bertozzi, C. R., Weaver, V. M. 2014; 511 (7509): 319-?

    Abstract

    Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.

    View details for DOI 10.1038/nature13535

    View details for Web of Science ID 000338992200029

    View details for PubMedID 25030168

  • Imaging bacterial peptidoglycan with near-infrared fluorogenic azide probes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shieh, P., Siegrist, M. S., Cullen, A. J., Bertozzi, C. R. 2014; 111 (15): 5456-5461

    Abstract

    Fluorescent probes designed for activation by bioorthogonal chemistry have enabled the visualization of biomolecules in living systems. Such activatable probes with near-infrared (NIR) emission would be ideal for in vivo imaging but have proven difficult to engineer. We present the development of NIR fluorogenic azide probes based on the Si-rhodamine scaffold that undergo a fluorescence enhancement of up to 48-fold upon reaction with terminal or strained alkynes. We used the probes for mammalian cell surface imaging and, in conjunction with a new class of cyclooctyne D-amino acids, for visualization of bacterial peptidoglycan without the need to wash away unreacted probe.

    View details for DOI 10.1073/pnas.1322727111

    View details for Web of Science ID 000334288600021

    View details for PubMedID 24706769

  • Glycocalyx engineering reveals a Siglec-based mechanism for NK cell immunoevasion NATURE CHEMICAL BIOLOGY Hudak, J. E., Canham, S. M., Bertozzi, C. R. 2014; 10 (1): 69-U111

    Abstract

    The increase of cell surface sialic acid is a characteristic shared by many tumor types. A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here, we show that increasing sialylated glycans on cancer cells inhibits human natural killer (NK) cell activation through the recruitment of sialic acid-binding immunoglobulin-like lectin 7 (Siglec-7). Key to these findings was the use of glycopolymers end-functionalized with phospholipids, which enable the introduction of synthetically defined glycans onto cancer cell surfaces. Remodeling the sialylation status of cancer cells affected the susceptibility to NK cell cytotoxicity via Siglec-7 engagement in a variety of tumor types. These results support a model in which hypersialylation offers a selective advantage to tumor cells under pressure from NK immunosurveillance by increasing Siglec ligands. We also exploited this finding to protect allogeneic and xenogeneic primary cells from NK-mediated killing, suggesting the potential of Siglecs as therapeutic targets in cell transplant therapy.

    View details for DOI 10.1038/NCHEMBIO.1388

    View details for Web of Science ID 000328854900013

    View details for PubMedID 24292068

  • Osmosensory signaling in Mycobacterium tuberculosis mediated by a eukaryotic-like Ser/Thr protein kinase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hatzios, S. K., Baer, C. E., Rustad, T. R., Siegrist, M. S., Pang, J. M., Ortega, C., Alber, T., Grundner, C., Sherman, D. R., Bertozzi, C. R. 2013; 110 (52): E5069-E5077

    Abstract

    Bacteria are able to adapt to dramatically different microenvironments, but in many organisms, the signaling pathways, transcriptional programs, and downstream physiological changes involved in adaptation are not well-understood. Here, we discovered that osmotic stress stimulates a signaling network in Mycobacterium tuberculosis regulated by the eukaryotic-like receptor Ser/Thr protein kinase PknD. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress. Furthermore, Rv0516c disruption modified peptidoglycan thickness, enhanced antibiotic resistance, and activated genes in the regulon of the alternative σ-factor SigF. Phosphorylation of Rv0516c regulated the abundance of EspA, a virulence-associated substrate of the type VII ESX-1 secretion system. These findings identify an osmosensory pathway orchestrated by PknD, Rv0516c, and SigF that enables adaptation to osmotic stress through cell wall remodeling and virulence factor production. Given the widespread occurrence of eukaryotic-like Ser/Thr protein kinases in bacteria, these proteins may play a broad role in bacterial osmosensing.

    View details for DOI 10.1073/pnas.1321205110

    View details for Web of Science ID 000328858800008

    View details for PubMedID 24309377

  • Imaging the Glycosylation State of Cell Surface Glycoproteins by Two-Photon Fluorescence Lifetime Imaging Microscopy ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Belardi, B., de la Zerda, A., Spiciarich, D. R., Maund, S. L., Peehl, D. M., Bertozzi, C. R. 2013; 52 (52): 14045-14049

    View details for DOI 10.1002/anie.201307512

    View details for Web of Science ID 000328531100027

    View details for PubMedID 24259491

  • D-Amino Acid Chemical Reporters Reveal Peptidoglycan Dynamics of an Intracellular Pathogen ACS CHEMICAL BIOLOGY Siegrist, M. S., Whiteside, S., Jewett, J. C., Aditham, A., Cava, F., Bertozzi, C. R. 2013; 8 (3): 500-505

    Abstract

    Peptidoglycan (PG) is an essential component of the bacterial cell wall. Although experiments with organisms in vitro have yielded a wealth of information on PG synthesis and maturation, it is unclear how these studies translate to bacteria replicating within host cells. We report a chemical approach for probing PG in vivo via metabolic labeling and bioorthogonal chemistry. A wide variety of bacterial species incorporated azide and alkyne-functionalized d-alanine into their cell walls, which we visualized by covalent reaction with click chemistry probes. The d-alanine analogues were specifically incorporated into nascent PG of the intracellular pathogen Listeria monocytogenes both in vitro and during macrophage infection. Metabolic incorporation of d-alanine derivatives and click chemistry detection constitute a facile, modular platform that facilitates unprecedented spatial and temporal resolution of PG dynamics in vivo.

    View details for DOI 10.1021/cb3004995

    View details for Web of Science ID 000316375500003

    View details for PubMedID 23240806

  • A Pictet-Spengler ligation for protein chemical modification PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Agarwal, P., van der Weijden, J., Sletten, E. M., Rabuka, D., Bertozzi, C. R. 2013; 110 (1): 46-51

    Abstract

    Aldehyde- and ketone-functionalized proteins are appealing substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with α-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C=N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits the utility of such conjugates in biological systems. Here we introduce a Pictet-Spengler ligation that is based on the classic Pictet-Spengler reaction of aldehydes and tryptamine nucleophiles. The ligation exploits the bioorthogonal reaction of aldehydes and alkoxyamines to form an intermediate oxyiminium ion; this intermediate undergoes intramolecular C-C bond formation with an indole nucleophile to form an oxacarboline product that is hydrolytically stable. We used the reaction for site-specific chemical modification of glyoxyl- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin. In conjunction with techniques for site-specific introduction of aldehydes into proteins, the Pictet-Spengler ligation offers a means to generate stable bioconjugates for medical and materials applications.

    View details for DOI 10.1073/pnas.1213186110

    View details for Web of Science ID 000313630300024

    View details for PubMedID 23237853

  • Reactivity of Biarylazacyclooctynones in Copper-Free Click Chemistry JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Gordon, C. G., Mackey, J. L., Jewett, J. C., Sletten, E. M., Houk, K. N., Bertozzi, C. R. 2012; 134 (22): 9199-9208

    Abstract

    The 1,3-dipolar cycloaddition of cyclooctynes with azides, also called "copper-free click chemistry", is a bioorthogonal reaction with widespread applications in biological discovery. The kinetics of this reaction are of paramount importance for studies of dynamic processes, particularly in living subjects. Here we performed a systematic analysis of the effects of strain and electronics on the reactivity of cyclooctynes with azides through both experimental measurements and computational studies using a density functional theory (DFT) distortion/interaction transition state model. In particular, we focused on biarylazacyclooctynone (BARAC) because it reacts with azides faster than any other reported cyclooctyne and its modular synthesis facilitated rapid access to analogues. We found that substituents on BARAC's aryl rings can alter the calculated transition state interaction energy of the cycloaddition through electronic effects or the calculated distortion energy through steric effects. Experimental data confirmed that electronic perturbation of BARAC's aryl rings has a modest effect on reaction rate, whereas steric hindrance in the transition state can significantly retard the reaction. Drawing on these results, we analyzed the relationship between alkyne bond angles, which we determined using X-ray crystallography, and reactivity, quantified by experimental second-order rate constants, for a range of cyclooctynes. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity. Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARAC's central lactam and compound reactivity. Collectively, these results indicate that the distortion/interaction model combined with bond angle analysis will enable predictions of cyclooctyne reactivity and the rational design of new reagents for copper-free click chemistry.

    View details for DOI 10.1021/ja3000936

    View details for Web of Science ID 000304837800041

    View details for PubMedID 22553995

  • Mapping Yeast N-Glycosites with Isotopically Recoded Glycans MOLECULAR & CELLULAR PROTEOMICS Breidenbach, M. A., Palaniappan, K. K., Pitcher, A. A., Bertozzi, C. R. 2012; 11 (6)

    Abstract

    Asparagine-linked glycosylation is a common post-translational modification of proteins; in addition to participating in key macromolecular interactions, N-glycans contribute to protein folding, trafficking, and stability. Despite their importance, few N-glycosites have been experimentally mapped in the Saccharomyces cerevisiae proteome. Factors including glycan heterogeneity, low abundance, and low occupancy can complicate site mapping. Here, we report a novel mass spectrometry-based strategy for detection of N-glycosites in the yeast proteome. Our method imparts N-glycopeptide mass envelopes with a pattern that is computationally distinguishable from background ions. Isotopic recoding is achieved via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides bearing the recoded envelopes are specifically targeted for fragmentation, facilitating high confidence site mapping. This strategy requires no chemical modification of the N-glycans or stringent sample enrichment. Further, enzymatically simplified N-glycans are preserved on peptides. Using this approach, we identify 133 N-glycosites spanning 58 proteins, nearly doubling the number of experimentally observed N-glycosites in the yeast proteome.

    View details for DOI 10.1074/mcp.M111.015339

    View details for Web of Science ID 000306408500017

    View details for PubMedID 22261724

  • Elucidation and Chemical Modulation of Sulfolipid-1 Biosynthesis in Mycobacterium tuberculosis JOURNAL OF BIOLOGICAL CHEMISTRY Seeliger, J. C., Holsclaw, C. M., Schelle, M. W., Botyanszki, Z., Gilmore, S. A., Tully, S. E., Niederweis, M., Cravatt, B. F., Leary, J. A., Bertozzi, C. R. 2012; 287 (11): 7990-8000

    Abstract

    Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.

    View details for DOI 10.1074/jbc.M111.315473

    View details for Web of Science ID 000301349400015

    View details for PubMedID 22194604

  • Synthesis of Heterobifunctional Protein Fusions Using Copper-Free Click Chemistry and the Aldehyde Tag ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Hudak, J. E., Barfield, R. M., de Hart, G. W., Grob, P., Nogales, E., Bertozzi, C. R., Rabuka, D. 2012; 51 (17): 4161-4165

    View details for DOI 10.1002/anie.201108130

    View details for Web of Science ID 000303001000032

    View details for PubMedID 22407566

  • A Bioorthogonal Quadricyclane Ligation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sletten, E. M., Bertozzi, C. R. 2011; 133 (44): 17570-17573

    Abstract

    New additions to the bioorthogonal chemistry compendium can advance biological research by enabling multiplexed analysis of biomolecules in complex systems. Here we introduce the quadricyclane ligation, a new bioorthogonal reaction between the highly strained hydrocarbon quadricyclane and Ni bis(dithiolene) reagents. This reaction has a second-order rate constant of 0.25 M(-1) s(-1), on par with fast bioorthogonal reactions of azides, and proceeds readily in aqueous environments. Ni bis(dithiolene) probes selectively labeled quadricyclane-modified bovine serum albumin, even in the presence of cell lysate. We have demonstrated that the quadricyclane ligation is compatible with, and orthogonal to, strain-promoted azide-alkyne cycloaddition and oxime ligation chemistries by performing all three reactions in one pot on differentially functionalized protein substrates. The quadricyclane ligation joins a small but growing list of tools for the selective covalent modification of biomolecules.

    View details for DOI 10.1021/ja2072934

    View details for Web of Science ID 000296312200014

    View details for PubMedID 21962173

  • From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions ACCOUNTS OF CHEMICAL RESEARCH Sletten, E. M., Bertozzi, C. R. 2011; 44 (9): 666-676

    Abstract

    Bioorthogonal reactions are chemical reactions that neither interact with nor interfere with a biological system. The participating functional groups must be inert to biological moieties, must selectively reactive with each other under biocompatible conditions, and, for in vivo applications, must be nontoxic to cells and organisms. Additionally, it is helpful if one reactive group is small and therefore minimally perturbing of a biomolecule into which it has been introduced either chemically or biosynthetically. Examples from the past decade suggest that a promising strategy for bioorthogonal reaction development begins with an analysis of functional group and reactivity space outside those defined by Nature. Issues such as stability of reactants and products (particularly in water), kinetics, and unwanted side reactivity with biofunctionalities must be addressed, ideally guided by detailed mechanistic studies. Finally, the reaction must be tested in a variety of environments, escalating from aqueous media to biomolecule solutions to cultured cells and, for the most optimized transformations, to live organisms. Work in our laboratory led to the development of two bioorthogonal transformations that exploit the azide as a small, abiotic, and bioinert reaction partner: the Staudinger ligation and strain-promoted azide-alkyne cycloaddition. The Staudinger ligation is based on the classic Staudinger reduction of azides with triarylphosphines first reported in 1919. In the ligation reaction, the intermediate aza-ylide undergoes intramolecular reaction with an ester, forming an amide bond faster than aza-ylide hydrolysis would otherwise occur in water. The Staudinger ligation is highly selective and reliably forms its product in environs as demanding as live mice. However, the Staudinger ligation has some liabilities, such as the propensity of phosphine reagents to undergo air oxidation and the relatively slow kinetics of the reaction. The Staudinger ligation takes advantage of the electrophilicity of the azide; however, the azide can also participate in cycloaddition reactions. In 1961, Wittig and Krebs noted that the strained, cyclic alkyne cyclooctyne reacts violently when combined neat with phenyl azide, forming a triazole product by 1,3-dipolar cycloaddition. This observation stood in stark contrast to the slow kinetics associated with 1,3-dipolar cycloaddition of azides with unstrained, linear alkynes, the conventional Huisgen process. Notably, the reaction of azides with terminal alkynes can be accelerated dramatically by copper catalysis (this highly popular Cu-catalyzed azide-alkyne cycloaddition (CuAAC) is a quintessential "click" reaction). However, the copper catalysts are too cytotoxic for long-term exposure with live cells or organisms. Thus, for applications of bioorthogonal chemistry in living systems, we built upon Wittig and Krebs' observation with the design of cyclooctyne reagents that react rapidly and selectively with biomolecule-associated azides. This strain-promoted azide-alkyne cycloaddition is often referred to as "Cu-free click chemistry". Mechanistic and theoretical studies inspired the design of a series of cyclooctyne compounds bearing fluorine substituents, fused rings, and judiciously situated heteroatoms, with the goals of optimizing azide cycloaddition kinetics, stability, solubility, and pharmacokinetic properties. Cyclooctyne reagents have now been used for labeling azide-modified biomolecules on cultured cells and in live Caenorhabditis elegans, zebrafish, and mice. As this special issue testifies, the field of bioorthogonal chemistry is firmly established as a challenging frontier of reaction methodology and an important new instrument for biological discovery. The above reactions, as well as several newcomers with bioorthogonal attributes, have enabled the high-precision chemical modification of biomolecules in vitro, as well as real-time visualization of molecules and processes in cells and live organisms. The consequence is an impressive body of new knowledge and technology, amassed using a relatively small bioorthogonal reaction compendium. Expansion of this toolkit, an effort that is already well underway, is an important objective for chemists and biologists alike.

    View details for DOI 10.1021/ar200148z

    View details for Web of Science ID 000296075300003

    View details for PubMedID 21838330

  • Isotopic Signature Transfer and Mass Pattern Prediction (IsoStamp): An Enabling Technique for Chemically-Directed Proteomics ACS CHEMICAL BIOLOGY Palaniappan, K. K., Pitcher, A. A., Smart, B. P., Spiciarich, D. R., Iavarone, A. T., Bertozzi, C. R. 2011; 6 (8): 829-836

    Abstract

    Directed proteomics applies mass spectrometry analysis to a subset of information-rich proteins. Here we describe a method for targeting select proteins by chemical modification with a tag that imparts a distinct isotopic signature detectable in a full-scan mass spectrum. Termed isotopic signature transfer and mass pattern prediction (IsoStamp), the technique exploits the perturbing effects of a dibrominated chemical tag on a peptide's mass envelope, which can be detected with high sensitivity and fidelity using a computational method. Applying IsoStamp, we were able to detect femtomole quantities of a single tagged protein from total mammalian cell lysates at signal-to-noise ratios as low as 2.5:1. To identify a tagged-peptide's sequence, we performed an inclusion list-driven shotgun proteomics experiment where peptides bearing a recoded mass envelope were targeted for fragmentation, allowing for direct site mapping. Using this approach, femtomole quantities of several targeted peptides were identified in total mammalian cell lysate, while traditional data-dependent methods were unable to identify as many peptides. Additionally, the isotopic signature imparted by the dibromide tag was detectable on a 12-kDa protein, suggesting applications in identifying large peptide fragments, such as those containing multiple or large posttranslational modifications (e.g., glycosylation). IsoStamp has the potential to enhance any proteomics platform that employs chemical labeling for targeted protein identification, including isotope coded affinity tagging, isobaric tagging for relative and absolute quantitation, and chemical tagging strategies for posttranslational modification.

    View details for DOI 10.1021/cb100338x

    View details for Web of Science ID 000294081900008

    View details for PubMedID 21604797

  • Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Boyce, M., Carrico, I. S., Ganguli, A. S., Yu, S., Hangauer, M. J., Hubbard, S. C., Kohler, J. J., Bertozzi, C. R. 2011; 108 (8): 3141-3146

    Abstract

    Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked β-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.

    View details for DOI 10.1073/pnas.1010045108

    View details for Web of Science ID 000287580400017

    View details for PubMedID 21300897

  • In vivo imaging of membrane-associated glycans in developing zebrafish SCIENCE Laughlin, S. T., Baskin, J. M., Amacher, S. L., Bertozzi, C. R. 2008; 320 (5876): 664-667

    Abstract

    Glycans are attractive targets for molecular imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with azides. Subsequently, the embryos were reacted with fluorophore conjugates by means of copper-free click chemistry, enabling the visualization of glycans in vivo at subcellular resolution during development. At 60 hours after fertilization, we observed an increase in de novo glycan biosynthesis in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, we performed a spatiotemporal analysis of glycan expression and trafficking and identified patterns that would be undetectable with conventional molecular imaging approaches.

    View details for DOI 10.1126/science.1155106

    View details for Web of Science ID 000255454300046

    View details for PubMedID 18451302