Comprehensive Natural Products II Vol.6 Carbohydrates, Nucleosides & Nucleic Acids

751 Pages·2010·19.32 MB·English

by  Mander L.

6.01 Overview and Introduction
RobertWoodwardandPengGeorgeWang,TheOhioStateUniversity,Columbus,OH,USA
ª2010ElsevierLtd.Allrightsreserved.
6.01.1 Introduction 1
6.01.2 Overview 1
6.01.1 Introduction
Given the prevalence of sugars in nature, it is no surprise that a thorough understanding of carbohydrate
chemistrycoupledwithglycobiologyisoftenessentialtoelucidatingthedetailsofcomplexbiologicalprocesses
suchaspolysaccharidebiosynthesis,theglycosylationofproteinsinbothprokaryoticandeukaryoticcells,and
glycoconjugate-induced immunological activity. This has enabled the extension of previously existing syn-
theticstrategies,aswellasthedevelopmentofnovelmethods,forthepreparationofcomplicatedcarbohydrate-
based biomolecules. For example, many excellent organometallic transformations havefoundapplicability in
glycoconjugate chemistry. Furthermore, the advance of physical organic chemistry has yielded a deeper
understanding of the mechanism of glycosylation reactions, consequently allowing several new glycosylation
approachestobedeveloped.Theseadvances,coupledwiththosemadeinbiologyandbiotechnology,havethus
further solidified thenecessity ofeffectively interweaving chemistry and biology. It is here, where chemistry
andbiologymeet,thatmanymorebreakthroughsacrossvariousdisciplineswillundoubtedlybemade.
6.01.2 Overview
The20additionalchaptersthatcomprisethisvolumeprovideadetailedlookatcarbohydratesandtheiruses
bothinalaboratorysettingandinnature,whilealsoexaminingavarietyofotherimportantbiomoleculesand
associatedprocesses.Thefirstthreechapters,contributedbyPengGeorgeWang,AshrafBrik,andZhongwu
Guo,considerhowcarbohydratescanbeutilizedforsyntheticpurposes.Bothenzymaticandchemicalmodesof
synthesisarediscussedwithregardtotheiruseforcomplexcarbohydrate,glycoprotein,andvaccineproduc-
tion. Applications of mass spectrometry to analysis of glycans are then explored in the next two chapters as
authorsKay-HooiKhooandJianjunLidiscusssomeofthemoreadvancedtechnologiesinthefield.
Chapters6.07–6.09offeralookattheburgeoningfieldofchemicalglycobiology.Abroadlookatthisareais
first presented by Jennifer Kohler in Chapter 6.07 with topics ranging from chemoenzymatic approaches to
understanding glycan structure and function to the use of photocross-linkers for the covalent trapping of
interactionsbetweenglycans.Thefinaltwochaptersofthissection,writtenbyAlanElbeinandHowardHang,
takeamorefocusedapproach,consideringhowcertainprocessescanbestudiedthroughtheuseofinhibitors
andprobes.
The next section of this volume introduces how carbohydrates are utilized in a variety of biosynthetic
pathways.Specifically,authorMiguelValvanofirstconsidersO-antigenbiosynthesis,aprocessthatisessential
intheproductionoflipopolysaccharide.Adiscussionofthegenerationofglycoproteinsinbothmammalianand
bacterial systems follows in the next two chapters with contributions from Inka Brockhousen and Mario
Feldman.Subsequently,thestructureandbiosynthesisofthemycobacterialcellwallandglycosaminoglycans
areexaminedbyDeanCrickandJianLiuinChapters6.13and6.14,respectively.Thereaderisthenoffereda
sectionbyJackPreiss,whichdetailstheprocessesofbacterialandmammalianglycogenbiosynthesis,whilealso
examining the production of starch in plants. Finally, Rajai Atalla’s contribution, Chapter 6.16, focuses upon
cellulosesandtheirvariousrolesinnature.
Intheclosingchaptersofthisvolume,avarietyofotherimportantbiomoleculesandrelatedprocessesare
highlighted. Specifically, Norman Lewis and Daneel Ferreira describe lignins and proanthocyanidins in
Chapters6.17 and 6.18, specifically looking atthe chemistry and biology of these highly aromatic molecules.
1
2 OverviewandIntroduction
ThefinalthreechaptersthendirectattentiontotheareasofDNAandRNAwithDarrellDavisfirstillustrating
the importance and utility of nucleoside analogs. Chapter 6.20, written by George Garcia, subsequently
examines how RNA is modified enzymatically and the corresponding consequences thereof before the
structuresandrolesofriboswitchesinregulatoryprocessesarepresentedbyTinaHenkininChapter6.21.
Asisquiteapparent,thetopicsthatappearwithinthisvolumevaryquiteextensively.However,whetheritis
adiscussionofthetransferofanoligosaccharylmoietytoaserineresidueinO-glycosylationortheregulation
oftranscriptionterminationthroughariboswitch,itishopedthatthereaderwillnotonlycomeawaywithan
appreciation for all of the biomolecules discussed, but more importantly recognize the diversity of processes
thatthesesmallmoleculesandassociatedpolymerscanperformand/orregulate.
BiographicalSketches
RobertWoodwardobtainedaB.A.inchemistry(2006)andaB.S.inbiology(2006)fromThe
Ohio State University. He is an NIH Chemistry–Biology Interface Program Predoctoral
Fellow currently pursuing a Ph.D. in organic chemistry at The Ohio State University. His
research focuses on the applications of synthesis for studying complex biological processes
suchaspolysaccharidebiosynthesisandoligosaccharidetransfer.
PengGeorgeWangobtainedaB.S.inchemistry(1984)fromNankaiUniversity,China,anda
Ph.D. in organic chemistry (1990) from the University of California, Berkeley. He then
conducted postdoctoral research at the Scripps Research Institute before becoming an
assistantprofessor in1994 attheUniversityof Miami inCoralGables.From 1997 to 2003,
he was a faculty member at Wayne State University. Since then, he has served in the
Departments of Biochemistry and Chemistry at The Ohio State University as Ohio
EminentScholarinMacromolecularStructureandFunction.
OverviewandIntroduction 3
ResearchintheWanglaboratoryispredominatelyfocusedonfourareasofglycoscience.
Glycochemistry:Workiscenteredonthegenerationofuncommonsugarlibrariesaswellas
the synthesis of key intermediates in carbohydrate-based biological processes, which are
essentialfortheirstudy.Glycobiology:Biochemicalcharacterizationofcarbohydrate-active
enzymes and investigations of the biological functions of carbohydrates in human diseases,
immunity, and general microbiology are performed. Glycotechnology: Biosynthetic path-
ways are engineered for the synthesis of glycopharmaceuticals, polysaccharides for vaccine
development, and biomedically important human glycoproteins. Glycoanalysis: Analysis of
carbohydrate composition, sequence, structure, and their interaction with proteins through
MS,NMR,QCM,andotheranalyticalmethodsisconducted.
6.02 Enzymatic Synthesis of Complex Carbohydrates
WeiZhaoandTiehaiLi,NankaiUniversity,Tianjin,China
RobertWoodward,ChengfengXia,andPengGeorgeWang,TheOhioStateUniversity,Columbus,
OH,USA
WanyiGuan,ShandongUniversity,Shandong,China
ª2010ElsevierLtd.Allrightsreserved.
6.02.1 Introduction 5
6.02.2 Glycosidases 8
6.02.2.1 ThreeMechanismsofGlycosidases 8
6.02.2.2 GlycosidasesinCarbohydrateSynthesis 8
6.02.2.3 Conclusion 14
6.02.3 Glycosynthases 15
6.02.3.1 MechanismsofGlycosynthases 15
6.02.3.2 GlycosynthasesinCarbohydrateSynthesis 17
6.02.3.3 Conclusion 22
6.02.4 Glycosyltransferases 23
6.02.4.1 SugarNucleotideBiosyntheticPathway 23
6.02.4.2 LeloirGlycosyltransferases 25
6.02.4.2.1 Basicprinciple 25
6.02.4.2.2 Enzyme-basedcomplexsaccharidesynthesis 25
6.02.4.2.3 Conclusion 46
6.02.5 Outlook 47
References 49
6.02.1 Introduction
Recently, oligosaccharides and glycoconjugates have emerged as a new and challenging research area at the
interface of biology and chemistry. Carbohydrates, which constitute one of the most abundant types of
biomolecules,playaverybroadsetofrolesinbiologicalscience,especiallyinphysiologicalandpathological
processes, molecular recognition, signal transduction, cell communication, cell differentiation, and develop-
mentalevents.1–6Infact,carbohydratecomplexeshavebeenwidelyusedaspotentialpharmaceuticalsforthe
prevention of infection, the neutralization of toxins, and the immunotherapy of cancer. Therefore, further
growthinresearchonthebiologicalfunctionsofthevariedglycanstructureswill,undoubtedly,becloselytied
totheavailabilityofbioactivecarbohydrates.7–9
Complexsaccharidesarehighlydiverse instructureandbiologicalfunctions.Theyareessentialformany
fieldsofresearch,forexample,biochemicalstudiesinglycobiology,aspotentialdrugsdirectedtoenzymesor
receptorsinvolvedintheirfunctionandmetabolism,andasadvancedmaterialsduetotheirbiocompatibility,
structure-forming capacity, and environmentally benign properties.10–15 The development of efficient syn-
theticmethodologiesfortheirpreparationhasthereforebeeninhighdemand.Methodsforbothchemicaland
enzymaticsyntheseshaveexperiencednotableadvancesinthelastfewyearswiththeaimofproducingeither
polysaccharides resembling the natural products or novel polysaccharide mimics for biomedical applications
andbiomaterialsdevelopment.
Althoughchemicaltoolshavebeenprovenindispensableforstudiesinglycobiology,16mostoligosaccharides
and glycoconjugates and their derivatives are very difficult and costly to generate by chemical synthetic
approaches,duetoseverelimitationsincomplexcarbohydratessyntheses.SimilartoproteinsandDNA,glycans
too consist of limited types of building blocks, monosaccharides. However, oligomers come in a far greater
5
6 EnzymaticSynthesisofComplexCarbohydrates
diversity of structures because of varying glycosidic linkage patterns, stereochemistry, and branching. In
addition,unlikeDNAandpeptidesyntheses,whicharecommonlyperformedonsolidphaseusingcommercial
instruments, comparable methodologies for oligosaccharides are still in their infancy.17,18 Given that all
oligosaccharidesyntheticprotocolsareplaguedwiththeoftentediousprotectionanddeprotectionsteps,one-
pot enzymatic systems with high regio- and stereoselectivities are clearly attractive alternatives.19–22 It is no
wonder that Hurtley et al.23 predicted as early as in 2001 in Science that what has rescued the chemistry and
biologyofcarbohydrates,theCinderellafromtheshadows,isnofairygodmotherbutaplethoraofnewsynthetic
andanalyticmethods.Overtheyears,enzymaticapproacheshavebeengainingpopularityforthesynthesisof
oligosaccharidesandglycoconjugates,anditisbecomingincreasinglyfeasibletoproducecomplexcarbohydrates
onalargescale,followingenzymaticbiosyntheticpathways.Thebiosynthesisofnaturallyoccurringoligo-and
polysaccharidesisacomplexprocessthatinvolvestheformationofglycosidicbondsbetweentheirconstituent
monosaccharideunitsandsidechainmodificationstoproducespecificfunctionalgroupderivatives.
Among the numerous enzymes associated with carbohydrate processing in cells, the enzymes used in
enzymaticsynthesisbelongtothreecategories:glycosidases,glycosynthases,andglycosyltransferases(Table1).
Glycosidasesareenzymesthathydrolyzeoligosaccharidesandpolysaccharidesinvivo.Underappropriatecondi-
tions,however,theactivatedintermediatescanbeinterceptedbyothersugarstoformnewglycosidicbonds.Theycan
formglycosidiclinkagesunderinvitroconditionsinwhichacarbohydratehydroxylmoietyactsasamoreefficient
nucleophilethanwateritself.Theyhavebeenoftremendousbenefitintheenzymaticsynthesisofoligosaccharides
duetotheiravailability,stability,organicsolventcompatibility,andlowcost.24–26Nevertheless,traditionalglycosi-
dase-catalyzedtransglycosylationsstillsufferfromlowyieldsandpoorunpredictableregioselectivities.
Glycosynthases, a class of glycosidase mutants, have been developed to enhance the enzymatic activity
toward the synthesis of oligosaccharides, through mutation of a single catalytic carboxylate nucleophile to a
neutralaminoacidresidue(AlaorSer).Conversionofoneofthefreecarboxylatestoamethylgroupprovides
anactivesitethatretainsthecorrectstericenvironmentfortheformationofareactiveglycosyldonorbutlacks
a key catalytic group for cleavage. The resulting enzymes have no hydrolytic activity, but increased activity
toward the synthesis of oligosaccharides, using glycosylfluorides as activated donors.27–30 The efficiency of
glycosidasesisemphasizedbythefactthatsomecanacceptunnaturalsubstrates,displayingmodificationsofthe
sugarmoietyand/oravarietyofaglyconegroups.
Glycosyltransferasesareenzymesthatcantransfersugarmoietytoadefinedacceptor,soastoconstructa
specific glycosidic linkage. This ‘one enzyme-one linkage’ concept makes glycosyltransferases useful and
importantintheconstructionofglycosidiclinkagesincomplexsaccharidesynthesis.31–33Glycosyltransferases
canbefurtherdividedintotwogroups:thetransferasesoftheLeloirpathwayandthoseofnon-Leloirpathways.
The Leloir transferases are responsible for the synthesis of most glycoconjugates in cells, especially in
mammaliansystems,34–36andarethefocusofthischapter.
Table1 Enzymaticformationofglycosidicbonds
DonorþAcceptorE(cid:2)nz!ymesProducts
Enzymes Glycosyldonor Advantage Disadvantage
Glycosidase Nitrophenylglycoside Easytoperform Lowyield
Lowcost Lowregioselectivity
Glycosynthase Glycosylfluoride Highyield Hardtoobtainenzyme
Difficulttopredictresults
Leloirglycosyl- Sugarnucleotide Highyield Highcost
transferase Highregio-and
stereoselectivities
Essentialforimportant
sequences
Non-Leloir Sugarphosphateor Highyield Notusefulforimportantsugar
glycosyltransferase glycoside Highregio-and sequences
stereoselectivities
EnzymaticSynthesisofComplexCarbohydrates 7
Scheme1 Enzymaticglycosylationandregenerationofsugarnucleotide.
Leloirglycosyltransferase-catalyzedsynthesesstartwiththeconversionofmonosaccharidesintoactivated
sugar nucleotides (donors), which then donate the sugars to various acceptors through the action of specific
glycosyltransferases(Scheme1).
Glycosyltransferases are able to construct highly regiospecific and stereoselective glycosidic linkages.
Although they are generally specific to substrates, minor modifications on donor and acceptor structurescan
be tolerated.37,38 Many efforts have been geared toward the genetic engineering and recombinant sources of
glycosyltransferases.39–43 Most glycosyltransferases can be expressed at high levels in mammalian systems.
However, this expression procedure is too tedious and expensive to be applied in practical transferase
production. Efforts have been made in expressing mammalian enzymes in insects, plants, yeast, and bacterial
cells,buthigh-levelexpressionremainsdifficult.Fortunately,glycosyltransferasesfrombacterialsourcescanbe
easilyclonedandexpressedinEscherichiacoli,inlargequantities.44Theyalsohaveabroaderrangeofsubstrates
whencomparedwithmammalianglycosyltransferases.Furthermore,somebacterialtransferaseswerefoundto
produce mammalian-like oligosaccharide structures, which makes these enzymes quite promising in the
synthesis of biologically important oligosaccharides.45–47 The recent expansion in genomic sequencing has
allowedformanyglycosyltransferasestobecharacterizedandexpressedinrecombinantform.
The second obstacle to enzymaticcarbohydrate synthesis is the sugar nucleotides. Although all thecommon
sugar nucleotides are now commercially available, these materials are prohibitively expensive. Since a sugar
nucleotide only serves as an intermediate in the enzyme-catalyzed glycosylation, the most efficient synthetic
approachistoregenerateitinsitu.Inaddition,thelowconcentrationofsugarnucleotideregeneratedcanavoidits
inhibitoryeffectontheglycosyltransferaseandincreasethesyntheticefficiency.Theideaofinsituregenerationof
sugarnucleotideswasfirstdemonstratedin1982byWongandWhitesidesintheirworkonuridine59-diphospho-
(cid:2)-D-galactopyranose (UDP-Gal) regeneration (Scheme 2).48 Since then, this revolutionary concept has been
adoptedinotherregenerationsystemsandfurtherdevelopedinglycoconjugatesyntheses.49–55
Scheme2 WhitesidesandWong’ssugarnucleotideregenerationcycle.
8 EnzymaticSynthesisofComplexCarbohydrates
6.02.2 Glycosidases
6.02.2.1 ThreeMechanismsofGlycosidases
Glycosidasesaredegradingenzymesthatcatalyzethehydrolysisofglycosidicbondsinvivo,56buttheirnormal
hydrolytic reaction can be reversed under appropriate conditions. Therefore, glycosidases have been exten-
sivelystudiedasbiocatalystsforoligo-andpolysaccharidebiosynthesis.Theyarestableenzymesandeasyto
produce,andalargenumberofenzymesfromdifferentorganismswithdifferentspecificitiesareavailable.In
addition, the glycosyl donors required are inexpensive compounds and easy to obtain in the gram scale.
Through X-ray crystallographic analysis and site-directed mutagenesis, three reaction mechanisms have
been found to exist for all glycosidases. The first is the inverting mechanism, which generally involves the
nucleophilic attack of water at the anomeric center concomitant with the acid-catalyzed departure of the
leavinggroup.Thisreactionoccursviaasingle-displacementmechanismwhereinonecarboxylicacidactsasa
general base and the other as ageneralacid (Scheme 3(a)).57 The second is the retaining mechanism, which
occursviaadouble-displacementmechanismwhereinonecarboxylicacidactsasageneralacid–baseandthe
otherasanucleophile.Allreactionsproceedintwoorderedsteps.Inthefirststep,thecarboxyloxygenatthe
anomericcenter,actingasnucleophile,attacksthesubstrate,resultingintheformationofatransientcovalent
glycosyl–enzymeintermediate.Theothercarboxylgroupconcomitantlyfacilitatesdepartureoftheaglycone
leavinggroupbyprovidingageneralacid.Inthesecondstep,theresidueactingasageneralacidinthefirststep
nowactsasageneralbase,promotingtheattackofwaterattheanomericcenter,cleavingtheintermediateto
yieldthehemiacetalproductwithretainedstereochemistry(Scheme3(b)).Whenusedforsyntheticapplica-
tions, glycosidases often displayonlymoderate regioselectivity andconversionyields. Nevertheless, theyare
generally considered to be useful because they are readily available and catalytically versatile.58–61 The
efficiency of glycosidases is emphasized by the fact that some can accept unnatural substrates, displaying
modifications of the sugar moiety and/or a variety of aglycone groups.62–64 The third mechanism is also a
retaining in stereochemistry, which involves assistance from the neighboring 2-acetamido group of the
substrate.So,thisreactionisoftennamedasasubstrate-assistedmechanism(Scheme3(c)).65
All of the three mentioned mechanisms involve oxocarbenium ion-like transition states and a pair of
carboxylicacidsattheactivesite;however,theydifferinseveralaspects.Theinvertingmechanismisaone-
stepreactionthatresultsintheformationofaproductwithinvertedstereochemistry attheanomericcenter.
Theothertwoalternativesareretaininginstereochemistryattheanomericcenteranddifferfromeachother
primarilyinthenatureoftheintermediate;inthesecondmechanism,thisspeciesisacovalentenzymeadduct,
whereasinthethirdcaseitisbelievedtobeabicyclicoxazolineoroxazoliniumion.66
6.02.2.2 GlycosidasesinCarbohydrateSynthesis
Carbohydrates play important structural and functional roles in numerous physiological processes, including
various disease states.9 Most biologically important glycoconjugates (oligosaccharides, glycoproteins, and gly-
colipids) and their derivatives are difficult to obtain in large quantities, whether from natural or synthetic
sources. The use of glycosidases for carbohydrate synthesis is currently being pursued by several research
groups.Theenzymaticactionofretainingglycosidasesisbasedontheformationofaglycosylesterofenzyme,
whichquicklyreactswithanucleophilepresentinthereactionmixturetoformtheproducts.Ifthenucleophile
isawatermolecule,thenthehydrolysisreactiontakesplace.Transglycosylationproductformationisobservedif
othernucleophilesarepresentinthereactionmixture.Theformationofself-transglycosylationproductsusually
occurswhentheglyconmoietyoftheenzymeglycosylesteristransferredtoanothermoleculeofthesubstrate
itself.Underappropriateconditions,however,theactivatedintermediatescanbeinterceptedbyothersugarsto
form new glycosidicbonds (Scheme4).56 Reverse hydrolysis (equilibrium-controlled synthesis) andtransgly-
cosylation (kinetically controlled process) are two mechanisms used in glycosidase-catalyzed synthesis of
complex saccharides. Equilibrium-controlled synthesis offers only modest yields of oligosaccharide products,
whilekineticallycontrolledsynthesis,whichrequiresaretainingglycosidase,providesbetteryields(10–40%).
Comparedwithglycotransferases,glycosidaseshavemanyadvantagesincarbohydratesynthesis.Duetothe
highspecificity,glycosyltransferasesareincapableofsynthesizinganaloguesofthenaturallyoccurringcomplex
EnzymaticSynthesisofComplexCarbohydrates 9
(a)
–O O
O O O O
H δ+ H
O
O O O O O R
R δ–R
O H δ–O H OH
– H O H OH
O
O O
O
(b)
O O O O
H δ+ H
O O O O
R δ– R
δ–
– –
O O OH O
H
O O O
O
H
O
O
O O O O
H δ+ H
O O
OH O
δ– H
– δ–
O O
O O
(c)
O O –O O –O O O O
HO H HOR HO HOR′′ HO H HO H
RH′OO O OR HHOO O HHOO O O R′ RH′OO O OR′
HN N HN HN
O + O + O O
Oxazolinium ion R″ = H O or other acceptor
2
Scheme3 Generalglycosidasemechanisms.(a)Aninverting(cid:3)-glycosidase.(b)Aretaining(cid:3)-glycosidaseproceeding
throughanintermediatewitha4C1conformation.(c)Substrate-assistedmechanism.
saccharides, which would be useful tools for the exploration of oligosaccharide functions. Alternatively,
carbohydrate synthesis by glycosidases is especially useful where a glycosyltransferase is not available or
difficult to obtain. Glycosidases also have the advantage of not requiring expensive sugar nucleotides as the
sugardonor.Theycangenerateglycosidicbondsusingrelativelysimpleglycosyldonorsandreadilyavailable
10 EnzymaticSynthesisofComplexCarbohydrates
O O –O O –O O HO O
HHOOHO HOO HOR HHOOHOHOO HOR +–RR′′OOHH HHOOHOHOO HOR′ HHOOHO HOO OR
O– O O O O O O– O
Glu Glu
Glu Glu
Scheme4 Catalyticmechanismsofretainingglycosidases.
robust enzymes. Therefore, they offer the opportunity to synthesize oligosaccharides inexpensively. In addi-
tion,glycosidasesareabundantandcanoftenbeuseddirectlywithoutpurification.Moreover,theyalsohave
many advantages in the enzymatic synthesis of oligosaccharides due to their availability, stability, organic
solventcompatibility,andlowcost.25,26,67
Traditionalglycosidase-catalyzedtransglycosylationreactionsstillsufferfromlowyieldsandpoorregios-
electivities.So,itisnotgenerallyeconomicalforlarge-scalecarbohydratesynthesis.Avarietyofstrategieshave
been employed in the glycoside syntheses, including either thermodynamic-controlled (increasing substrate
concentrations, decreasing product concentrations by absorption, elevating reaction temperatures, adding
water-miscible organic cosolvents) or kinetic-controlled (using activated glycosyl donors and exogenous
nucleophiles) protocols, and several large-scale glycosidic bond-forming reactions have been reported in the
pastyears.68–72
SomeofthenovelstrategiesaredepictedinScheme5.Glycosidase-catalyzedsynthesisofdisaccharides,for
example, can be coupled in situ with a glycosyltransferase reaction to improve the overall yield
(Scheme 5(a)).73 Another interesting way to improve the yield and facilitate product isolation of glycosi-
dase-catalyzed glycosidation reactions has been demonstrated in the galactosidase-catalyzed synthesis of
N-acetyllactosamine, one of the intermediates in the synthesis of SLex.22 The key was the use of 6-oxo
p-nitrophenyl galactose, prepared by enzymatic oxidation of the corresponding galactose derivative with
galactose oxidase,as the glycosyl donor. The 6-oxo derivatives are less proneto be hydrolyzed and result in
improvedyieldsofthe69-oxodisaccharide.Reductionofthealdehydewithsodiumboronhydrideaffordedthe
desiredproductandalsofacilitatedisolationduetotheformationofaboroncomplex(Scheme5(b)).Chitinase,
which normally works to hydrolyze the polymer chitin, has been used to synthesize artificial chitin in
quantitativeyield.74Thekeytothispolymerizationreactionistheuseofatransitionstateanaloguesubstrate
andperformingthereactionatahighpHwheretheenzymecanactivatethesubstratebutcannothydrolyzethe
products(Scheme5(c)).
(cid:2)-L-Arabinofuranosidaseofbacterialorigin(AbfD3),aversatileglycosidaseofcarbohydrate-actingenzymes,
hasbeensuccessfullyemployedforthesynthesisofnovelalkyl-glycosides75andfuranose-containingdisacchar-
ides.76,77 Evidences proved that AbfD3 can catalyze the self-condensation of both p-nitrophenyl (cid:2)-L-
arabinofuranoside and p-nitrophenyl (cid:3)-D-galactofuranoside. Similarly, mixed disaccharides could be obtained
whenthesep-nitrophenylfuranosidesserveasdonorsand(cid:3)-D-xylosidesserveasacceptors.Dependingonthe
donor/acceptorratio,thereactionsoccurredwithvariabledegreesofregioselectivitytowardboth(1!2)and
(1!3) linkages, which reflects the glycosidic linkage specificity of AbfD3 operating in hydrolytic mode.78
Nugier-Chauvinandcoworkers79haveinvestigatedthespecificityofan(cid:2)-L-arabinofuranosidaseusingC2-and
C5-modified (cid:2)-L-arabinofuranosides. They attempted to manipulate the regioselectivity of AbfD3-catalyzed
glycosylationreactionsbysynthesizingaseriesofunnaturaldonorsthatdisplaystructuralmodificationsattheir
C2 or C5 position. Three donors, p-nitrophenyl 5-deoxy-5-fluoro-(cid:2)-L-arabinofuranoside (1), p-nitrophenyl
5-deoxy-(cid:2)-L-arabinofuranoside (2), and p-nitrophenyl 2-deoxy-(cid:2)-L-arabinofuranoside (3), were synthesized
(Scheme6).Subsequently,theseglycosidesweretestedtodetermineifthesedeoxygenatedanaloguescouldbe
recognizedassubstratesbyAbfD3inenzyme-catalyzedreactions.Totestthesuitabilityofmodifiedarabino-
furanosyl donors for AbfD3-catalyzed synthesis, compounds 1–3 were incubated with AbfD3. The results
clearly demonstrated that, besides cleavage, the AbfD3 furanosidase is able to catalyze transglycosylation
reactionswithC5-modifiedp-nitrophenylarabinofuranosidesandconfirmedthatAbfD3possessestheability
EnzymaticSynthesisofComplexCarbohydrates 11
Scheme5 Novelstrategiesforglycosidase-catalyzedtransglycosylationreactions.(a)Glycosidase-catalyzedsynthesis
coupledinsitutoimproveoverallyields.(b)Utilizationofa6-oxop-nitrophenylgalactosedonorasameanstoimprovethe
yield.(c)SynthesisofartificialchitininquantitativeyieldatahighpH.
Scheme6 p-Nitrophenyl-activatedC2-andC5-modified(cid:2)-L-arabinofuranosides.
to synthesize both (cid:2)-(1!2)- and (cid:2)-(1!3)-linked regioisomeric disaccharides from the C5 deoxygenated
substrate 2 (Scheme 7). However, when the 2-deoxy analogue 3 was incubated with AbfD3 under the same
conditions, HPTLC analysis failed to reveal any reaction products. Moreover, the free sugar could not be