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Carbohydrate Chains: Enzymaticand Chemical SynthesisThomas J. Tolbert and Chi-Huey WongScripps Research Institute, La Jolla, California, USA

Carbohydrate chains play many important roles in biology,

covering the surfaces of cells, mediating cell–cell recognition

events, and forming major classes of biologically active

molecules. Though carbohydrate chains are very important

in biology, their study is frequently problematical because

in vivo biosynthesis of carbohydrate chains often produces

heterogeneous mixtures that are hard to purify in sufficient

quantities for biochemical and structural studies. The in vitroenzymatic and chemical synthesis of carbohydrate chains offers

another route to these interesting biomolecules that allows

sufficient quantities of homogeneous oligosaccharides to be

produced for biological studies.

Difficulties in the Synthesis

of Carbohydrate Chains

The synthesis of oligosaccharides can present several

technical difficulties that do not occur in the synthesisof other biopolymers. Unlike nucleic acids andproteins, the linkage between each subunit, i.e.,sugar, of a carbohydrate chain forms a chiral centerat the sugar’s anomeric carbon that has two possibleconfigurations, termed the a- or b -anomer (Figure 1B).Each time a sugar is joined to a carbohydrate chainthrough a glycosidic linkage, the stereochemistry of the bond that is formed must be controlled ordirected in some manner to insure that the correctanomer is produced. In addition, carbohydrate chainsare not just simple linear chains as DNA and proteinsare, but can also be branched chains with increasedcomplexity. Glycosidic linkages can be formedthrough each hydroxyl of a sugar, and when multiplehydroxyls on a single sugar form glycosidic linkages,branched oligosaccharide structures result. Dis-tinguishing between the different hydroxyl moietieson sugars is another difficulty of carbohydratechain synthesis. Each sugar of a carbohydratechain can have several hydroxyl moieties that arenearly equivalent in chemical reactivity, making them

difficult to differentiate from one another and addingseveral steps to most chemical syntheses. To overcomethese difficulties, several enzymatic and chemicalmethods have been developed.

Enzymatic Synthesisof Oligosaccharides

Enzymes have frequently been employed to synthesizecarbohydrate chains, because they offer a few technicaladvantages over traditional organic synthesis. Enzymesgenerally do not require protecting groups to selectthe correct chemical moiety on their substrates, and incarbohydrate chain synthesis this is a great advantage.The many nearly chemically equivalent hydroxylmoieties of sugars make selective chemical protectionof sugar hydroxyls a laborious task. The use of

enzymes in carbohydrate chain synthesis can eliminatethe need for both selective hydroxyl protection beforeforming the glycosidic linkage, and removal of theprotecting groups after forming the glycosidic linkage,greatly reducing the number of steps required forsynthesis of an oligosaccharide. Another advantage of the use of enzymes in carbohydrate chain synthesis isthe stereoselectivity of glycosidic bond-formingenzymes, which often produce a single anomer duringthe formation of a glycosidic bond. In contrast,chemical methods frequently produce mixtures of anomers during glycosidic bond formation, whichmust be purified from one another after formation of

the glycosidic bond. Though enzymatic synthesis of oligosaccharides has many advantages, it is oftenlimited by the availability of specific enzymes neededto form certain types of carbohydrate structures, butwith the increased number of enzymes being discov-ered by genomic research this should improve in thefuture. Two classes of enzymes that have been used toform carbohydrate chains will be discussed furtherbelow, glycosidases and glycotransferases.

Encyclopedia of Biological Chemistry, Volume 1. q 2004, Elsevier Inc. All Rights Reserved. 307



C ARBOHYDRA TE CHAIN S YNTHE SIS

UTILIZING GLYCOSIDASES

Glycosidases are enzymes that normally break glycosidicbonds during glycoprocessing or catabolism of oligo-

saccharides, but by placing glycosidases under certaincontrolled reaction conditions they can be utilized toform, rather than break, glycosidic bonds. Most often,glycosidases are used to form glycosidic bonds intransglycosylation reactions, where a glycosidic bondis broken in a glycosyl donor glycoside, and a newglycosidic bond is formed with a glycosyl acceptor(Figure 2A). Several approaches can be utilized to favorformation of the desired glycosidic bond, including theuse of activated glycosyl donors such as p-nitrophenylglycosides, elevated concentrations of glycosyl donorsand acceptors, organic cosolvents, and the use of mutated glycosidases with reduced hydrolysis activity.

The use of glycosidases to form glycosidic bondsgenerally results in low to medium yields rangingfrom 20% to 40%, although yields as high as 90%have been reported using mutated glycosidases. Glyco-sidase catalyzed synthesis generally has good stereo-selectivity, forming a single a- or b -anomer, butsometimes has low regioselectivity for the differenthydroxyls on the acceptor sugar resulting in multipleproducts being formed. Though glycosidase reactions

can suffer from low yield and low regioselectivity,there is a wide range of glycosidases available tocatalyze the formation of many different types of gly-cosidic linkages.

C ARB OHY DRA TE CHAIN S YNTHE SI S

UTILIZING GLYCOTRANSFERASES

Glycotransferases are enzymes that catalyze the transferof activated monosaccharide donors to carbohydratesduring the biosynthesis of oligosaccharides. They arevery useful in synthesizing oligosaccharides in vitrobecause they exhibit high regioselectivity and stereo-selectivity in the formation of glycosidic bonds. Glyco-transferases that utilize nucleotide sugars as activatedmonosaccharide donors have been used most oftenin the in vitro synthesis of carbohydrate chains(Figure 2B). A wide variety of oligosaccharide struc-tures can be produced using the available glycotrans-ferases and activated nucleotide sugars such asUDP-glucose (UDP-Glc), UDP-N-acetylglucosamine(UDP-GlcNAc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-glucuronicacid (UDP-GlcUA), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), and CMP-sialic acid (CMP-NeuAc).Generally, each glycotransferase is selective for a specific

OO

OH

NHAc

OOH

O

HOO

OH

HO

OH

O

OHHO

OH

OO

OHCH3

OH

H3C

HO

HO

OR

OHO

HO

HO

H2N

O

O

O

NH2

HONH2O

O

NH2

HO

H2N OH

HOH2N

HO

HOOH

AcHN O

O

O

OHHO

HO2C

OHO

OO

OH

NHAcHO

HO OH

ORO

OH

OOH

CO2HOHO

O

NHAc

OSO3 –

O

OSO3 –

– O3SHN

OHO

O

O

O

OSO3 –

HO O

O

– O3SHNHO

– O3SO

O

OH

HO2C

HOO

HO

OMeOH

OH

HOO

HOOH

OMe

OH

Blood group O antigen(covers surface of type O human blood cells)

Neomycin B(aminoglycoside antibiotic)

Sialyl Lewis X antigen

(involved in recruiting leukocytesto areas of inflammation)

B a -Anomer b -Anomer

O

HO

HO

OH

OO

HO

NHAc

OH

OO

HO

O

OH

OHO

OO

OHO

OH OH

OH

OO

HOHO C13H27

OH

NHCOC15H31

O

OHHO

OH

Heparin pentasaccharide(inhibitor of blood clotting)

Globo H(cancer antigen)A

FIGURE 1 Carbohydrate chains: (A) some biologically important oligosaccharides and (B) 1-O-methyl-glucopyranosides,

a- and b -anomers.

308 CARBOHYDRATE CHAINS: ENZYMATIC AND CHEMICAL SYNTHESIS



nucleotide-sugar donor, and also has specificity forcertain oligosaccharide structures in its substrates.Because of this it is often difficult to make unnatural

oligosaccharides using glycotransferases, but in somecases relaxed substrate and nucleotide-sugar specificityhas been utilized to make unnatural carbohydrate chains.Formation of oligosaccharides with glycotransferasesrequires the substrate oligosaccharide, manganese, thenucleotide-sugar donor, and the glycotransferase itself in an appropriate buffer. Glycotransferase-catalyzedsynthesis of carbohydrate chains usually results inhigh yields, approaching 100%, but the enzymaticreaction can suffer from product inhibition that hasto be overcome to achieve those high yields. The use of glycotransferases in oligosaccharide synthesis is limitedby the high cost of nucleotide sugars and the availability

of glycotransferases that form certain oligosaccharidestructures. Sugar-nucleotide recycling alleviates someof the former problem while genomic sequenc-ing and research has started to alleviate some of thelatter problem.

Product Inhibition in Glycotransferase Reactions

Though glycotransferases can be used to form oligo-saccharides in nearly 100% yield, they suffer fromproduct inhibition from the nucleoside diphosphatesand nucleoside monophosphates that are produced asthe activated nucleotide-sugar donors are transferredto the growing carbohydrate chain. This productinhibition can drastically slow the enzymatic reactionand also reduce the yield of product, and so it isdesirable to remove the nucleotide by-products of theglycotransferase reactions. Two approaches can beused to overcome product inhibition in glycotransfer-ase reactions use of phosphorylases and nucleotide-sugar recycling (Figure 2B).

Redu ction of Glyc otra nsfe rase Inhibiti on with

Phosphorylases Nucleoside diphosphates and mono-phosphates can be converted into nucleosides, which

do not inhibit glycotransferases, using phosphorylases.This is a very simple method, which requires only one ortwo additional enzymes to be added to the glycotrans-ferase reaction. Unfortunately this method requires astoichiometric amount of nucleotide-sugar donor forformation of the desired oligosaccharide, which can bequite costly on larger scales since nucleotide sugars aregenerally very expensive.

Reduction of Glycotransferase Inhibition with Nucleotide-

Sugar Recycling Another method to remove nucleotideby-products of glycotransferase reactions is to regener-ate the nucleotide-sugar donors from the nucle-otide by-products using an enzymatic recycling

reaction. This method of overcoming product inhibitionis somewhat more complicated than using phosphory-lases, requiring several additional enzymes for thenucleotide-sugar recycling reaction, but it allows acatalytic amount of nucleotide-sugar donor to be usedin the glycotransferase reaction. Since nucleotide-sugarsare generally expensive, this method is desirable forlarger-scale glycotransferase reactions.

Chemical Synthesis

of Oligosaccharides

Chemical synthesis of oligosaccharides is often themethod of choice for constructing carbohydrate chainsbecause chemical methods are flexible, can be used toproduce both natural and unnatural oligosaccharides,and are also not limited by the availability of specificenzymes. Formation of glycosidic bonds by chemicalmethods usually relies upon activation of a leavinggroup on the anomeric carbon of a glycosyl donor with a

OOR

O OOR

O

NO2

ONO2HO

OOR

O OOR

NDP

Glycosidase

Acceptor

Donor

NDP-sugar

Glycosyl-transferase

Glycotransferaseinhibition

N + 2Pi

Phosphorylases

Nucleotide-sugarrecycling

HO O HO O

HOHO

HO

A B

FIGURE 2 Enzymatic synthesis of oligosaccharides using: (A) glycosidases and (B) glycotransferases.

CARBOHYDRATE CHAINS: ENZYMATIC AND CHEMICAL SYNTHESIS 309



Lewis acid, which once activated will react with a freehydroxyl upon a glycosyl acceptor (Figure 3A). A widevariety of glycosyl leaving groups have been utilized forcarbohydrate chain synthesis some of which are shownin Figure 3B.

CONTROL OF STEREOCHEMISTRY IN CHEMICAL GLYCOSIDIC

BOND FORMATION

Control of the stereochemistry of the anomeric linkagein the products of glycosylation reactions can be verycomplex, and many factors including types of protect-ing groups on the sugars, solvent, temperature, andleaving groups can be used to influence the a / b ratio of the products formed in glycosylation reactions. Ingeneral, the anomeric effect, stabilization of axialorientation over equatorial orientation of electronwithdrawing groups attached to the C1 of pyranose

sugars, can be utilized to produce a-linked glucosidesand galactosides. Participation of protecting groups of the 2-OH, such as acetate, and the use of polarsolvents in glycosylation reactions can direct productstoward b -linked glucosides and galactosides throughdioxocarbenium or solvent intermediates (Figure 3A).Sometimes it is difficult to obtain the desired anomerusing chemical synthesis, as in the case of sialic acidglycosides, where the natural sialic acid linkage isexclusively the a-anomer, but the b -anomer is obtainedfrom most chemical glycosylation reactions. Control-ling the anomeric outcome of chemical glycosylationreactions can still be a difficult problem, and requiresseveral approaches for different types of glycosidiclinkages and sugars.

PROTECTION OF SUGAR H YDROXYL S IN

CHEMICAL C ARBOHYDRA TE S YNT HESIS

Chemical oligosaccharide synthesis relies heavily uponsugar protecting groups to both distinguish betweensugar hydroxyls with similar chemical reactivity andalso to control the stereochemical outcome, either a orb , of glycosylation reactions. Because of this, chemicaloligosaccharide synthesis typically requires a largeamount of protecting group manipulations. Normally,protecting groups must be placed on hydroxyls that arenot going to be used to form glycosidic linkages toprevent them from reacting in glycosylation reactions. Inaddition, the synthesis of carbohydrate chains longerthan two sugars requires sugar subunits that can act bothas glycosyl donors and acceptors, and this usuallyrequires selective protection and deprotection of thehydroxyls that are to form glycosidic linkages. Selectiveprotection of sugar subunits for glycosylation reactionsoften requires many chemical steps to produce eachselectively protected subunit for the synthesis of an

oligosaccharide, and this is one factor that cancontribute to the large number of steps required forchemical oligosaccharide syntheses. Some protectinggroups commonly used in chemical carbohydrate chainsynthesis include benzyl ethers, p-methoxybenzyl ethers,tert -butyl-diphenylsilyl ethers, tert -butyl-dimethylsilylethers, allyl ethers, acetate esters, benzoate esters,levulinoyl esters, and dimethyl acetals.

SOLUTION PHASE CHEMICAL

OLIGOSACCHARIDE S YNT HESIS

In conventional solution-phase oligosaccharide syn-thesis, a donor sugar is activated by a Lewis acid at theanomeric carbon, and then reacted with an acceptor

A

O

L

OSOPh

O

X

OSR

B

O

O

O O

R

H

O

R′

OO P

O

OR

OR

O

O POR

OR

O

O

OR′

O

O

CCl3

NH

OS OEt

S

O

O

O OR′

Trichloroacetimidates

(TMSOTf)

Glycosyl halides

X = Cl, Br, (AgOTf),or F (SnCl

2 /AgClO

4)

Thioglycosides

(NIS/TfOH or DMTST)

Glycosyl sulfoxides

(Tf2O)

Glycosyl phosphites

(TMSOTf)

Pentenyl glycosides(NIS/Et

3SiOTf)

Glycals

(1. DMDO, 2. ZnCl2)

Glycosyl phosphates(TMSOTf)

Lewisacid

– L – + and

+

Dioxocarbenium

(participation of C-2 substituent)

a b

Xanthates

(DMTST, Cu(OTf)2)

Activation Glycosidation

FIGURE 3 Chemical glycosylation reactions. (A) Mechanism of glycosylation. (B) Some commonly used glycosylation leaving groups andthe reagents used to activator them (in parentheses). Abbreviations: L, leaving group; R, variable group; TMSOTf, trimethylsilyl triflate; NIS,

N-iodosuccinimide; TfOH, triflic acid; Et3SiOTf, triethylsilyl triflate; Tf 2O, triflic anhydride; DMDO, 3,3-dimethyldioxirane; DMTST,dimethylthiosulfonium triflate.




OOP

O

OPOOP O

OPOP

HO

OPO

OP

OP

OP

OOP

O

OPHO

OPO

OP

OP

OP

HO

OOP

O

OPOP

OOP

O

OPOOP O

OPOP

HO

OPOR

OP

OP

OP

O

OPOP

PO

OP

OOP

O

OPOOP

O

OPOP

O

OOP

OR

OPOP

OP

OOP

O

OPHO

OPOR

OP

OP

OP

HO

OOP

OR

OP

OP

3

4

2

1

3

2

1

2

1

1

O

OP

PO

OP

3

2

1

2

1

1

O

OPOP

PO

OP

OOP

O

OPOOP

O

OPOP

O

OOP

OR

OPOP

OP3

4

2

1

4

O

OP

OP

PO

OP

STol

HO

OP

4

Most reactive 2nd Mo

+

(1) 1st glycosylation (purification)(2) 1st deprotection (purification)

(3) 2nd glycosylation (purification)

(4) 2nd deprotection (purification)

(5) 3rd glycosylation (purification)

(1) 1st glycosylation(2) 1st deprotection

(3) 2nd glycosylation

4) 2nd deprotection

(5) 3rd glycosylation

(6) Cleavage from resin (purification)

Solution-phase synthesis

3 glycosylation reactions2 deprotections5 purifications

Solid-phase synthesis

3 glycosylation reactions2 deprotections1 purification1 cleavage from resin

OptiMer's pro

1 one-pot rea1 purification

A B C

O

O

O

O

F‘IGURE 4 Chemical synthesis of oligosaccharides: (A) solution-phase synthesis, (B) solid-phase synthesis, and (C) OptiMer’s



sugar that contains a free hydroxyl. Once the glycosidicbond has been formed, the product disaccharide must bepurified away from other glycosylation reaction reagentsand side products, and then selectively deprotected tounmask a single hydroxyl so that it can then act as aglycosyl acceptor in the next glycosylation reaction(Figure 4A). Purification is often necessary after eachglycosylation and deprotection step in the preparation

of an oligosaccharide by solution phase synthesis. Whencombined with the number of steps necessary to produceselectively protected sugar subunits, the number of chemical steps and purifications necessary to constructeven small oligosaccharides can be very large.

A UTOMATED OLIGOSACCHARIDE

S YNTHE SIS

Because of the large amount of work necessary toconstruct even relatively small oligosaccharidesby solution-phase synthesis, many approaches

toward automating oligosaccharide synthesis havebeen developed in hopes of minimizing the number of chemical steps and purifications required to synthesizeoligosaccharides. Two approaches to automated oligo-saccharide synthesis will be discussed below: solid-phaseoligosaccharide synthesis and programmable one-potoligosaccharide synthesis.

Solid-Phase Chemical Oligosaccharide Synthesis

In solid-phase oligosaccharide synthesis, either theglycosyl donor or acceptor of a glycosylation reactionis attached to the solid phase. The advantage of this is

that large excesses of the other components of theglycosylation reaction can be used to increase the rate of the reaction and insure that it goes to completion, andthen rinsed away by simple filtration. This has thepotential of increasing the yield of glycosylation reac-tions and also eliminates many of the laboriouspurification steps that are necessary in solution-phaseoligosaccharide synthesis (Figure 4B). Unfortunatelythere are many technical difficulties that must beovercome to successfully apply solid-phase synthesis tothe production of oligosaccharides. Solid supports thatfunction well with many solvents must be selected, sincea wide variety of solvents are used to affect the outcomeof glycosylation reactions. Linkers must be developedthat are not sterically hindering for glycosylationreactions, stable to glycosylation conditions, and easilycleaved after the oligosaccharide has been synthesized.Strategies for protecting sugar monomers that allowhighly flexible and selective deprotection of sugarhydroxyl groups in the presence of many other protectedhydroxyls must be worked out. Since the reactionconditions of glycosidic bond formation can vary widely

depending on oligosaccharide structure and the types of glycosidic linkages to be formed, there has been greatdifficulty in developing broadly applicable generalmethods for solid-phase oligosaccharide synthesis.Nevertheless, there have been many notable examplesof successful solid-phase syntheses including the syn-thesis of a dodecasaccharide by both the Nicolaouand Seeberger groups.

Programmable One-PotOligosaccharide Synthesis

One-pot reaction approaches that involve conductingseveral sequential glycosylation reactions in one reactionflask have been developed to facilitate automatedoligosaccharide synthesis. The one-pot approach reliesupon using a reactivity profile of protected sugars todetermine what sequence of sugars to add to glycosyla-tion reactions to obtain the desired products. Protectedsugars are added to one-pot reactions in the order of most reactive to least reactive, thereby controlling the

order of glycosidic bond formation during the synthesisof carbohydrate chains (Figure 4C). Since it has beenshown that the types of protecting groups and type of anomeric activating groups used on sugars can greatlyalter the speed at which a sugar will react in aglycosylation reaction, a wide range of reactivities canbe obtained for a single type of sugar, allowing wideflexibility in oligosaccharide synthesis using thisapproach. The one-pot approach greatly reduces thenumber of steps necessary to construct oligosaccharidesand eliminates several tedious purifications by combin-ing multiple glycosylation reactions into a single one-potreaction. The one-pot approach also minimizes protect-ing group manipulations, eliminating protecting groupmanipulations after construction of the building blocks.A large number of p-methylphenyl thioglycosides havebeen synthesized and had their reactivities measured tofacilitate the use of this strategy. A computer program,OptiMer, has been developed that uses these p-methyl-phenyl thioglycosides as a set of building blocks tochoose from to build oligosaccharides. When acarbohydrate chain structure is entered as input, theOptiMer program will calculate the best set of reactantsfor the construction of that carbohydrate chain.Because the programmable one-pot strategy requires a

minimum of protecting group manipulations and has alarge library of building blocks, a wide range of oligosaccharide structures can be synthesized rapidlyusing this method.

SEE A LSO THE FOLLOWING A RTICLES

Enzyme Reaction Mechanisms: Stereochemistry †

Oligosaccharide Chains: Free, N -Linked, O-Linked




GLOSSARY

anomeric carbon The carbon of a cyclic sugar which forms a

hemiacetal or hemiketal. In the linear form of the sugar, the carbonthat will becomethe anomericcarbon when the sugar cyclizes is the

carbonyl carbon.

anomeric effect The stabilization of axial orientation over equatorialorientation of electron withdrawing groups attached to the C1 of pyranose sugars.

anomers The pair of diastereomers, termed the a- or b -anomer, that

results when a linear sugar forms a cyclic hemiacetal or hemiketal.glycosidic linkage The linkage formed between the anomeric carbon

of a sugar and an alcohol.

FURTHER R EADING

Koeller, K. M., and Wong, C.-H. (2000). Synthesis of complex

carbohydrates and glycoconjugates: Enzyme-based and program-mable one-pot strategies. Chem. Rev. 100, 4465–4493.

Nicolaou, K. C., Watanabe, N., Li, J., Pastor, J., and Winssinger, N.

(1998). Solid-phase synthesis of oligosaccharides: Construction of a

dodecasaccharide. Angew. Chem. Int. Ed. 37, 1559.Plante, O. J., Palmacci, E. R., and Seeberger, P. H. (2001). Automated

solid-phase synthesis of oligosaccharides. Science 291, 1523.

Sears, P., and Wong, C.-H. (2001). Toward automated synthesis of oligosaccharides and glycoproteins. Science 291, 2344–2350.

Seeberger, P. H., and Haase, W.-C. (2000). Solid-phase oligosaccharide

synthesis and combinatorial carbohydrate libraries. Chem. Rev.100, 4349–4393.

Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G., and Marth, J.

(eds.) (1999). Essentials of Glycobiology. Cold Spring HarborLaboratory Press, Cold Spring Harbor.

Wong, C.-H., Halcomb, R. L., Ichikawa, Y., and Kajimoto, T. (1995).

Enzymes in organic synthesis: Application to the problems of carbo-hydrate recognition: Part 1. Angew. Chem. Int. Ed. 34, 412–432.

Wong, C.-H., Halcomb, R. L., Ichikawa, Y., and Kajimoto, T. (1995).Enzymes in organic synthesis: Application to the problems of carbo-

hydrate recognition: Part 2. Angew. Chem. Int. Ed. 34, 521–546.Zhang, Z., Ollman, I. R., Ye, X.-S., Wischnot, R., Baasov, T., and

Wong, C.-H. (1999). Programmable one-pot digosaccharidesynthesis. J. Am. Chem. Soc. 121, 734.

BIOGRAPHY

Thomas J. Tolbert is an Assistant Professor at Indiana University.

His principal research interests lie in the use of chemical and enzymaticsynthesis to solve biological problems. He holds a Ph.D. in Biochemistryfrom MIT, and a B.S. degree in chemistry from Purdue University.

Chi-HueyWong is Professorand Ernest W. Hahn Chair in Chemistryat

theScrippsResearch Instituteand also a Member of theSkaggs Insitutefor ChemicalBiology. His principal research interests are in the areas of bioorganic and synthetic chemistry and biocatalysis, with particular

focus on development of new chemoenzymatic methods to tacklemajor problems in carbohydrate-mediated biological recognitions. Hereceived his B.S. and M.S. degrees from National Taiwan University,

and Ph.D. in Chemistry from Massachusetts Institute of Technology.He is a Member of the National Academy of Sciences.

CARBOHYDRATE CHAINS: ENZYMATIC AND CHEMICAL SYNTHESIS 313

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