glycorandomization
{{Short description|Technology enabling rapid molecule diversification}}
Glycorandomization, is a drug discovery and drug development technology platform to enable the rapid diversification of bioactive small molecules, drug leads and/or approved drugs through the attachment of sugars. Initially developed as a facile method to manipulate carbohydrate substitutions of naturally occurring glycosides to afford the corresponding differentially glycosylated natural product libraries,{{cite journal | last1 = Yang | first1 = J. | last2 = Hoffmeister | first2 = D. | last3 = Liu | first3 = L. | last4 = Thorson | first4 = J. S. | year = 2004| title = Natural product glycorandomization| journal = Bioorganic & Medicinal Chemistry| volume = 12 | issue = 7| pages = 1577–1584 | doi=10.1016/j.bmc.2003.12.046| pmid = 15112655 }}{{cite journal|last1=Langenhan|first1=JM|last2=Griffith|first2=BR|last3=Thorson|first3=JS|title=Neoglycorandomization and chemoenzymatic glycorandomization: Two complementary tools for natural product diversification|journal=Journal of Natural Products|date=Nov 2005|volume=68|issue=11|pages=1696–711|pmid=16309329|doi=10.1021/np0502084}}{{cite journal|last1=Griffith|first1=BR|last2=Langenhan|first2=JM|last3=Thorson|first3=JS|title='Sweetening' natural products via glycorandomization|journal=Current Opinion in Biotechnology|date=Dec 2005|volume=16|issue=6|pages=622–30|pmid=16226456|doi=10.1016/j.copbio.2005.10.002}} glycorandomization applications have expanded to include both small molecules (drug leads and approved drugs) and even macromolecules (proteins).{{cite journal|last1=Gantt|first1=RW|last2=Peltier-Pain|first2=P|last3=Thorson|first3=JS|title=Enzymatic methods for glyco(diversification/randomization) of drugs and small molecules|journal=Natural Product Reports|date=Oct 2011|volume=28|issue=11|pages=1811–53|pmid=21901218|doi=10.1039/c1np00045d}} Also referred to as 'glycodiversification',{{cite journal|last1=Thibodeaux|first1=CJ|last2=Melançon|first2=CE|last3=Liu|first3=HW|title=Unusual sugar biosynthesis and natural product glycodiversification|journal=Nature|date=Apr 26, 2007|volume=446|issue=7139|pages=1008–16|pmid=17460661|doi=10.1038/nature05814|bibcode=2007Natur.446.1008T|s2cid=4404027}} glycorandomization has led to the discovery of new glycoside analogs which display improvements in potency, selectivity and/or ADMET as compared to the parent molecule.
Classification
The traditional method for attaching sugars to natural products, drugs or drug leads is by chemical glycosylation. This classical approach typically requires multiple protection/deprotection steps in addition to the key anomeric activation/coupling reaction which, depending upon the glycosyl donor/acceptor pair, can lead to a mixture of anomers. Unlike classical chemical glycosylation, glycorandomization methods are divergent (i.e., diverge from a common starting material, see divergent synthesis) and are not dependent upon sugar/aglycon protection/deprotection or sugar anomeric activation. Two complementary strategies to achieve glycorandomization/diversification have been developed: an enzyme-based strategy referred to as 'chemoenzymatic glycorandomization' and a chemoselective method known as 'neoglycorandomization'. Both methods start with free reducing sugars and a target aglycon to afford a library of compounds which differ solely by the sugars appended to the target natural product, drug or drug lead.
= Chemoenzymatic glycorandomization =
Chemoenzymatic glycorandomization was inspired by the early pathway engineering work of Hutchinson and coworkers that suggested natural product glycosyltransferases were capable of utilizing non-native sugar nucleotide donors.{{cite journal|last1=Madduri|first1=K|last2=Kennedy|first2=J|last3=Rivola|first3=G|last4=Inventi-Solari|first4=A|last5=Filippini|first5=S|last6=Zanuso|first6=G|last7=Colombo|first7=AL|last8=Gewain|first8=KM|last9=Occi|first9=JL|last10=MacNeil|first10=DJ|last11=Hutchinson|first11=CR|title=Production of the antitumor drug epirubicin (4'-epidoxorubicin) and its precursor by a genetically engineered strain of Streptomyces peucetius|journal=Nature Biotechnology|date=Jan 1998|volume=16|issue=1|pages=69–74|pmid=9447597|doi=10.1038/nbt0198-69|s2cid=7304148}} The initial platform for chemoenzymatic glycorandomization was based upon a set of two highly permissive sugar activation enzymes (a sugar anomeric kinase and sugar-1-phosphate nucleotidyltransferase) to afford sugar nucleotide libraries as donors for these promiscuous glycosyltransferases where the permissivity of the corresponding sugar kinase{{cite journal|last1=Hoffmeister|first1=D|last2=Yang|first2=J|last3=Liu|first3=L|last4=Thorson|first4=JS|title=Creation of the first anomeric D/L-sugar kinase by means of directed evolution|journal=Proceedings of the National Academy of Sciences of the United States of America|date=Nov 11, 2003|volume=100|issue=23|pages=13184–9|pmid=14612558|doi=10.1073/pnas.2235011100|pmc=263743|bibcode=2003PNAS..10013184H|doi-access=free}} and nucleotidyltransferase{{cite journal|last1=Barton|first1=WA|last2=Lesniak|first2=J|last3=Biggins|first3=JB|last4=Jeffrey|first4=PD|last5=Jiang|first5=J|last6=Rajashankar|first6=KR|last7=Thorson|first7=JS|last8=Nikolov|first8=DB|title=Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization|journal=Nature Structural Biology|date=Jun 2001|volume=8|issue=6|pages=545–51|pmid=11373625|doi=10.1038/88618|s2cid=25049013}}{{cite journal|last1=Moretti|first1=R|last2=Chang|first2=A|last3=Peltier-Pain|first3=P|last4=Bingman|first4=CA|last5=Phillips GN|first5=Jr|last6=Thorson|first6=JS|title=Expanding the nucleotide and sugar 1-phosphate promiscuity of nucleotidyltransferase RmlA via directed evolution|journal=The Journal of Biological Chemistry|date=Apr 15, 2011|volume=286|issue=15|pages=13235–43|pmid=21317292|doi=10.1074/jbc.m110.206433|pmc=3075670|doi-access=free}} was expanded by enzyme engineering and directed evolution. The first application of this three enzyme (kinase, nucleotidyltransferase and glycosyltransferase) strategy enabled the product of a set of >30 differentially glycosylated vancomycins, some members of which were further diversified chemoselectively by virtue of the installation of sugars bearing chemoselective handles.{{cite journal|last1=Fu|first1=X|last2=Albermann|first2=C|last3=Jiang|first3=J|last4=Liao|first4=J|last5=Zhang|first5=C|last6=Thorson|first6=JS|title=Antibiotic optimization via in vitro glycorandomization|journal=Nature Biotechnology|date=Dec 2003|volume=21|issue=12|pages=1467–9|pmid=14608364|doi=10.1038/nbt909|s2cid=2469387}}{{cite journal|last1=Fu|first1=X|last2=Albermann|first2=C|last3=Zhang|first3=C|last4=Thorson|first4=JS|title=Diversifying vancomycin via chemoenzymatic strategies|journal=Organic Letters|date=Apr 14, 2005|volume=7|issue=8|pages=1513–5|pmid=15816740|doi=10.1021/ol0501626}}{{cite journal|last1=Peltier-Pain|first1=P|last2=Marchillo|first2=K|last3=Zhou|first3=M|last4=Andes|first4=DR|last5=Thorson|first5=JS|title=Natural product disaccharide engineering through tandem glycosyltransferase catalysis reversibility and neoglycosylation.|journal=Organic Letters|date=Oct 5, 2012|volume=14|issue=19|pages=5086–9|pmid=22984807|doi=10.1021/ol3023374|pmc=3489467}} This enzymatic platform has been further advanced through glycosyltransferase evolution{{cite journal|last1=Williams|first1=GJ|last2=Zhang|first2=C|last3=Thorson|first3=JS|title=Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution|journal=Nature Chemical Biology|date=Oct 2007|volume=3|issue=10|pages=657–62|pmid=17828251|doi=10.1038/nchembio.2007.28}} and capitalizing upon the discovery of the reversibility of glycosyltransferase-catalyzed reactions first discovered in the context of calicheamicin biosynthesis.{{cite journal|last1=Zhang|first1=C|last2=Griffith|first2=BR|last3=Fu|first3=Q|last4=Albermann|first4=C|last5=Fu|first5=X|last6=Lee|first6=IK|last7=Li|first7=L|last8=Thorson|first8=JS|title=Exploiting the reversibility of natural product glycosyltransferase-catalyzed reactions|journal=Science|date=Sep 1, 2006|volume=313|issue=5791|pages=1291–4|pmid=16946071|doi=10.1126/science.1130028|bibcode=2006Sci...313.1291Z|s2cid=38072017}}{{cite journal|last1=Gantt|first1=RW|last2=Peltier-Pain|first2=P|last3=Cournoyer|first3=WJ|last4=Thorson|first4=JS|title=Using simple donors to drive the equilibria of glycosyltransferase-catalyzed reactions|journal=Nature Chemical Biology|date=Aug 21, 2011|volume=7|issue=10|pages=685–91|pmid=21857660|doi=10.1038/nchembio.638|pmc=3177962}}
= Neoglycorandomization =
Neoglycorandomization is a chemoselective glycodiversification method inspired by the alkoxyamine-based ‘neoglycosylation’ reaction first described Peri and Dumy.{{cite journal | last1 = Peri | first1 = F. | last2 = Dumy | first2 = P. | last3 = Mutter | first3 = M. | year = 1998 | title = Chemo- and stereoselective glycosylation of hydroxylamino derivatives: A versatile approach to glycoconjugates | journal = Tetrahedron | volume = 54 | issue = 40| pages = 12269–12278 | doi=10.1016/s0040-4020(98)00763-7}} This reaction proceeds via an oxy-iminium intermediate to ultimately provide the more thermodynamically-favored closed ring neoglycoside. The neoglycosylation reaction is compatible with a wide range of saccharide and aglycon functionality where neoglycoside anomeric stereospecificity is a thermodynamically-driven. Importantly, structural and functional studies reveal neoglycosides to serve as good mimics of their O-glycosidic comparators. The first neoglycorandomization proof of concept focused upon digitoxin where the rapid generation and cancer cell line cytotoxicity screening of 78 digitoxigenin neoglycosides revealed unique analogs with improved anticancer activity and reduced potential for cardiotoxicity.{{cite journal|last1=Langenhan|first1=JM|last2=Peters|first2=NR|last3=Guzei|first3=IA|last4=Hoffmann|first4=FM|last5=Thorson|first5=JS|title=Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization|journal=Proceedings of the National Academy of Sciences of the United States of America|date=Aug 30, 2005|volume=102|issue=35|pages=12305–10|pmid=16105948|doi=10.1073/pnas.0503270102|pmc=1194917|bibcode=2005PNAS..10212305L|doi-access=free}} This platform has since been automated and used as an effective medicinal chemistry tool to modulate the properties of a range of natural products and pharmaceutical drugs.{{cite journal|last1=Goff|first1=RD|last2=Thorson|first2=JS|title=Neoglycosylation and neoglycorandomization: Enabling tools for the discovery of novel glycosylated bioactive probes and early stage leads|journal=MedChemComm|date=Aug 1, 2014|volume=5|issue=8|pages=1036–1047|pmid=25071927|doi=10.1039/c4md00117f|pmc=4111257}}
= Comparison =
Both chemoenzymatic glycorandomization and neoglycorandomization use free reducing sugars and unprotected aglycons and are thereby a notable advance over classical glycosylation methods. A notable advantage of the enzymatic approach is the use of the corresponding genes encoding for the permissive kinases, nucleotidyltransferases and/or glycosyltransferases for in vivo synthetic biology applications to afford in vivo glycorandomization.{{cite journal|last1=Williams|first1=GJ|last2=Yang|first2=J|last3=Zhang|first3=C|last4=Thorson|first4=JS|title=Recombinant E. coli prototype strains for in vivo glycorandomization|journal=ACS Chemical Biology|date=Jan 21, 2011|volume=6|issue=1|pages=95–100|pmid=20886903|doi=10.1021/cb100267k|pmc=3025069}} However, it is important to note the enzymatic platform is dependent upon the permissivity of the enzymes employed. In contrast, the main hurdle to chemoselective neoglycorandomization is installation of the alkoxylamine handle. Unlike the enzymatic approach, the anomeric stereoselectivity of the chemoselective method depends upon the reducing sugar used and can, in some cases, lead to anomeric mixtures.
Uses
Glycorandomization is used in the pharmaceutical industry and academic community to alter glycosylation patterns of sugar-containing natural products or to append sugars to drugs/drug leads. It provides a fast way to investigate the effect of subtle sugar modification on the pharmacological properties of the natural products analogues,{{cite journal|last1=Zhang|first1=J|last2=Hughes|first2=RR|last3=Saunders|first3=MA|last4=Elshahawi|first4=SI|last5=Ponomareva|first5=LV|last6=Zhang|first6=Y|last7=Winchester|first7=SR|last8=Scott|first8=SA|last9=Sunkara|first9=M|last10=Morris|first10=AJ|last11=Prendergast|first11=MA|last12=Shaaban|first12=KA|last13=Thorson|first13=JS|title=Identification of neuroprotective spoxazomicin and oxachelin glycosides via chemoenzymatic glycosyl-scanning.|journal=Journal of Natural Products|date=28 December 2016|pmid=28029796|doi=10.1021/acs.jnatprod.6b00949|pmc=5337260|volume=80|issue=1|pages=12–18}} thus, affording a new approach to drug discovery.