Glycoconjugates for Biological Applications

DING Fei，LU Wangting

（Institute for Interdisciplinary Research，Jianghan University，Wuhan 430056，Hubei，China）

0 Introduction

Glycans bound with proteins or lipids via covalent bonds were defined as glycoconjugates，containing glycopro⁃teins，proteoglycans and glycolipids.Almost a century ago，the German scientist Otto Warburg observed that cancer⁃ous tissues consume large amounts of glucose compared to non-transformed tissue and have high rates of aerobic gly⁃colysis［1-2］，which is known as the Warburg effect.This is recognized as one of the landmark of cancer［3］.Glycolytic enzymes，as well as the insulin-independent glucose transporter GLUT-1，are widely overexpressed in human can⁃cers［4-5］and high expression levels of these proteins in tumor biopsy samples correlate with poor cancer prognosis，making them promising therapeutic targets［6-9］.

1 Conjugates for Drug Targeting Delivery

Delivering a cancer drug to its proposed site selectively has obvious therapeutic advantages，including reduced toxicity to the normal cells and smaller dose to be used.This can be achieved by exploiting an existing（endogenous）interaction or activity at the desired site or creating a new one.Typically，the former involves the exploitation of a binding interaction of either a ligand at that site for an introduced receptor（targeting receptor）or of a receptor at that site for an introduced ligand（targeting ligand）.Since the feasibility of using carbonhydrate ligands to target protein receptors at desired sites，termed"glycotargeting"was first demonstrated in 1971［10］，the potential of using carbonhydrates to create a truly targeted（or actively-targeted）drug delivery system has been made clear.However，after research for almost half of a century，to date，there is very few therapeutic system on the market，and there still exist many challenges，for example，avoiding to be filtrated into kidney and rapidly clearance.Therefore，the use of macromolecular constructs that allowing longer circulation time and giving access to additional chemical functionality or more precise delivery，is an attractive alternative option.

1.1 Folate-drug conjugates

Anticancer agents conjugated to molecules allow for preferential delivery to cancer cells.The often reported drug conjugation for improved cancer selectivity and uptake is folate conjugation.Cancer cells prefer tode novo synthesize nucleotide during the salvage pathway that is often favored by normal tissues［11］.This preference of cancer cells means that they must take up large amounts of folate.For this reason，about 2/5 of human cancers overexpressed folate receptor α（FR-α）［12-13］.

Kataoka，et al.［14］prepared a multifunctional polymeric micelle that was implemented with intracellular pH-dependent drug releasing functionality and folate-mediated cancer cell targeting property simultaneously（as shown in Fig.1）.By precision synthesis and preparation of the pH-sensitive micelles with different folate contents，the ligand-installed micelles for active drug targeting can be more effective than free drug in terms of antitumor activity，safety，pharmacokinetics and bioavailability.

Fig.1 Design of polymeric micelles and the rationale for active drug delivery.Chemical structures of folate-poly（ethylene glycol）-poly（aspartate-hydrazone-adriamycin）and methoxy-poly（ethylene glycol）-poly（aspartatehydrazone-adriamycin）block copolymers（（a）and（b））.As shown in panel（c）,these polymers can self-assemble into the polymeric micelles that can effectively achieve systemic drug delivery and tumor-specific accumulation（（1）and（2））.The micelles are then uptaken by the cancer cells via fluid endocytosis（3）or receptor mediated endocytosis（3'）depending on surface design for accelerating endocytosis（e.g.folate conjugation）.Anticancer drugs,adriamycin（ADR）,that are conjugated through a pH-sensitive hydrazone bond can be released from the micelles in the acidic intracellular compartments such as endosomes or lysosomes where pH ranges from 5 to 6（4）

1.2 Sugar（or glucose）-conjugated drugs

In 2001，Mikuni，et al.［15］reported the glycoconjugates of docetaxel，including conjugates with glucose，galactose，mannose，and xylose.These compounds were shown to have a 3 to 18 fold improvement in activity compared to the aglycone against B16 murine melanoma cells.Following this research，in 2008，a publication highlighted the in vivo potency of galactose-conjugated docetaxel（Fig.2（a））in a syngeneic P388 murine leukemia tumor model compared to the aglycone［16］，but did not elucidate the mechanism of the entry mode or potential products cleavage by the conjugate.To determine whether this glycoconjugate was able to phenocopy paclitaxel's method of inducing cell death，NPC-TW01 human nasopharyngeal carcinoma cells were treated with the vehicle（not loaded drug），drug alone（paclitaxel），or 2-D-glucose-conjugated paclitaxel（Fig.2（b））for 24 h and stained for tubulin distribution and chromosome morphology.While vehicle-treated cells showed diffuse tubulin distribution（Fig.2（c））and diffuse chromosomal distribution（Fig.2（f）），the cells treated with both paclitaxel and glucoseconjugated paclitaxel showed tubulin accumulation around cell nuclei and nuclear chromatin condensation after 24 h，indicating cell apoptosis（Fig.2（d）-（e）and（g）-（h））.However，in time course studies，cells treated with paclitaxel showed a more pronounced and rapid chromatin condensation than cells treated with glucoseconjugated paclitaxel［17］.

Fig.2 （a）1-α-D-galactose conjugated to position 12 of docetaxel via a short linker；（b）1-methylglucose conjugated to position 2'of paclitaxel via a short linker；fluorescent microscopy images of NPC-TW01 human nasopharyngeal carcinoma cells treated with paclitaxel and 2-D-glucose-conjugated paclitaxel；（（c）-（e））Tubulin distribution is visualized above；and（（f）-（h））chromosome morphology below

Vecchio，et al.［18］reported the synthesis of a glucose conjugate，8-hydroxyquinolines that was a class of metal-binding compounds.The 8-hydroxyquinolones have shown to be promising in early stage of clinical trials as anticancer agents due to their scavenging of copper（II），a necessary co-factor in tumor growth［19］.1-O-glucose conjugates of both clioquinol（GluCQ）（Fig.3（a））and another 8-hydroxyquinolone，8-quinolnyl（GluOHQ）（Fig.3（b））were synthesized to improve targeting of these compounds to tumor cells.

Fig.3 （a）Clioquinol conjugated to β-D-glucose via a C1 ether linkage；（b）8-quinolnyl conjugated to β-D-glucose via a C1 ether linkage

2 Conjugates for Cancer Imaging/Sensing

Cancer-associated markers（proteins，gene or carbonhydrate）are present in the blood，and are important for early diagnosis of cancer.Yoon，et al.［20］developed a sensitive biosensing system using lectins for breast cancer diagnosis based on the analysis of tumor-associated O-linked glycoprotein such as cancer antigen 15-3（CA15-3）（Fig.4（a））.Based on the difference between the glycan moieties in normal and cancer cells，CA15-3 was biospecifically detected usingSambucus nigraagglutinin（SNA）and peanut agglutinin（PNA）lectins.These lectins were conjugated with fluoro-microbeads and used as a detection molecule for an antibody-lectin sandwich assay.A fluoro-microbead guiding chip（FMGC）containing multiple sensing and fluidic channels was designed to measure CA15-3 proteins using the lectin-based assay.A capture antibody against CA15-3 that binds to the peptide backbone was immobilized on the gold-patterned sensing surface.On the modified FMGC，antibody-lectin sandwich assay for target CA15-3 was conducted.The developed PNA-and SNA-based assays，which exhibited a detection limit of 1.2 and 0.4 U/mL，worked well over the clinically range from 1.25 to 25 U/mL CA15-3（Fig.4（b）-（c））.The developed biosensor showed good sensitivity，fast response and reproducibility with a small volume of sample.This lectin-based sandwich assay provides a promising tool for the quantitative diagnosis of CA15-3 and glycated markers in clinical applications.

Fig.4 （a）The molecular structure of the CA15-3 antigen.Mucin（MUC）backbone and the differently expressed O-linked glycan chains found in normal and cancerous cells are schematically shown；（b）Design of a fluoromicrobead guiding chip（FMGC）and a schematic diagram of the antibody-lectin sandwich assay on the FMGC；（c）Quantitative analysis of fluorescence images obtained from FMGC by counting the number of beads on the patterned area using Image J software.Bead quantities in the area were automatically calculated using the"analyze particles"function

Wong，et al.［21］developed a fluorescent labeling technique based on the Cu（I）-catalyzed［3+2］cycloaddi⁃tion，or click chemistry，which allows rapid，versatile，and specific covalent labeling of cellular glycans bearing azide groups（Fig.5（a））.Using this click-activated fluorescent probe，we demonstrate incorporation of an azidocontaining fucose analog into glycoproteins via the fucose salvage pathway.Distinct fluorescent signals were observed by flow cytometry when cells treated with 6-azidofucose were labeled with the click-activated fluorogenic probe or bi⁃otinylated alkyne.The intracellular localization of fucosylated glycoconjugates was visualized by using fluorescence microscopy（Fig.5（b）-（c））.This technique will allow dynamic imaging of cellular fucosylation and facilitate stud⁃ies of fucosylated glycoproteins and glycolipids.

Zhang，et al.［22］reported on a coumarin based fluorogenic probe，which can be used as a bioorthogonal-label⁃ing tool for glycoproteins.They designed and fabricated an alkyne-functionalized fluorogenic glycoproteomic probe（probe 1）for imaging sialoglycoconjugates in living cells under no-wash conditions with good signal above back⁃ground（Fig.6）.Probe 1 was able to image glycoproteins efficiently in living cells without the need for the washing step.Moreover，the trifluoroethyl ester protected terminal carboxyl group at the 4 position of probe 1 could be used for further modification before bioorthogonal labeling.

Fig.5 （a）General strategy for glycan labeling；（b）Treatment with tunicamycin suppressed cell surface fluorescence（cells cultured with natural fucose，azidofucose,or azidofucose in the presence of tunicamycin）；（c）Labeling with biotinylated alkyne 13 was performed with ultra avidin-fluorescein,then analyzed by flow cytometry（cells treated with fucose or azidofucose）

Fig.6 General strategy for fluorogenic labeling of sialoglycoconjugates in living cells.Covalent modification of the target glycan with probe 1 results in production of fluorescently labeled glycoproteins bearing azide modified mannose（Ac4ManNAz）

3 Prospect

Carbonhydrates have been used successfully as non-proteolysable scaffolds in the design of peptidomimetics.The unusually high levels of secondary structure that exist in the mono-carbonhydrate structures allows to design new conjugates in which carbonhydrates provide not only the surface ligands but also an extremely well defined and predictable backbone.Since the application of glycoconjugates in therapeutic strategies becomes more widespread，certain features must be addressed.Carbonhydrate science is no longer a question of chemistry or biology，it is both.We hope more cooperation between making glycoconjugates and applying them will be underway.

［1］WARBURG O.On the origin of cancer cells［J］.Science，1956，123（3191）：309-314.

［2］WARBURG O，WIND F，NEGELEIN E.The metabolism of tumors in the body［J］.General Physiology，1927，8（6）：519-530.

［3］HANAHAN D，WEINBERG R A.Hallmarks of cancer：the next generation［J］.Cell，2011，144（5）：646-674.

［4］ALTENBERG B，GREULICH K O.Genes of glycolysis are ubiquitously overexpressed in 24 cancer classes［J］.Genomics，2004，84（6）：1014-1020.

［5］MEDINA R A，OWEN G I.Glucose transporters：expression，regulation and cancer［J］.Biological Research，2002，35（1）：9-26.

［6］OHBA S，FUJII H，ITO S，et al.Overexpression of GLUT-1 in the invasion front is associated with depth of oral squamous cell carcinoma and prognosis［J］.Oral Pathology and Medicine，2010，39（1）：74-78.

［7］VARKI A.Biological roles of oligosaccharides：all of the theories are correct［J］.Glycobiology，1993，3（2）：97-130.

［8］KOUKOURAKIS M I，GIATROMANOLAKI A，SIVRIDIS E，et al.Prognostic and predictive role of lactate dehydrogenase 5 ex⁃pression in colorectal cancer patients treated with PTK787/ZK 222584（vatalanib）antiangiogenic therapy［J］.Cancer Research and Clinical Oncology，2011，17（14）：4892-4900.

［9］GONG L，CUI Z Q，CHEN P C，et al.Reduced survival of patients with hepatocellular carcinoma expressing hexokinase II［J］.Medical Oncology，2012，29（2）：909-914.

［10］ROGERS J C，KORNFELD S.Hepatic uptake of proteins coupled to fetuin glycopeptide［J］.Biochemical and Biophysical Re⁃search Communications，1971，45（3）：622-629.

［11］VISENTIN M，ZHAO R，GOLDMAN I D.The antifolates［J］.Hematol Oncol Clin North Am，2012，26（3）：629-648.

［12］PARKER N，TURK M J，WESTRICK E，et al.Folate receptor expression in carcinomas and normal tissues determined by a quantitative radio ligand binding assay［J］.Analytical Biochemistry，2005，338（2）：284-293.

［13］LOW P S，KULARATNE S A.Folate-targeted therapeutic and imaging agents for cancer［J］.Current Opinion in Chemical Biolo⁃gy，2009，13（3）：256-262.

［14］BAE Y，NISHIYAMA N，KATAOKA K，et al.In vivo antitumor activity of the folate-conjugated pH-sensitive polymeric micelle selectively releasing adriamycin in the intracellular acidic compartments［J］.Bioconjugated Chemistry，2007，18（4）：1131-1139.

［15］MANDAI T，OKUMOTO H，MIKUNI K，et al.Synthesis and biological evaluation of water soluble taxoids bearing sugar moieties［J］.Heterocycles，2001，54（2）：561-566.

［16］MIKUNI K，NAKANISHI K，HARA K，et al.In vivo antitumor activity of novel water-soluble taxoids［J］.Biological and Phar⁃maceutical Bulletin，2008，31（6）：1155-1158.

［17］LIN Y S，TUNGPRADIT R，SINCHAIKUL S，et al.Targeting the delivery of glycan-based paclitaxel prodrugs to cancer cells via glucose transporters［J］.Medical Chemistry，2008，51（23）：7428-7441.

［18］OLIVERI V，GIUFFRIDA M L，VECCHIO G，et al.Gluconjugates of 8-hydroxyquinolines as potential anticancer prodrugs［J］.Dalton Transactions，2012，41（15）：4530-4535.

［19］SCHIMMER A D，JITKOVA Y，GRONDA M，et al.A phase I study of the metal ionophore clioquinol in patients with advanced hematologic malignancies［J］.Clinical Lymphoma Myeloma and Leukemia，2012，12（5）：330-336.

［20］PARK Y M，KIM S J，YOON C H，et al.Lectin-based optical sensing for quantitative analysis of cancer antigen CA15-3 as a breast cancer marker［J］.Sensors and Actuators B，2013，186（9）：571-579.

［21］SAWA M，HSU T L，WONG C H，et al.Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo［J］.Proc Natl Acad Sci USA，2006，33（103）：12371-12376.

［22］RONG L，LIU L H，ZHANG X Z，et al.A coumarin derivative as a fluorogenic glycoproteomic probe for biological imaging［J］.Chemical Communication，2014，50（6）：667-669.