Post-Translational Glycosylation: Problems and Opportunities

Chi-Huey Wong Academia Sinica, Taipei, Taiwan

chwong@gate.sinica.edu.tw

Carbohydrate Chemistry and Drug Discovery

Chi-Huey Wong

The Scripps Research Institute wong@scripps.edu

The Human Genome and Post-Translational Glycosylation

46 Chromosomes Three billion nucleotides Only 1.5% encode genes ~30,000 genes >50% proteins glycosylated

Nature, 2001, vol. 409, no. 6822.

Molecular Recognition Involves Cell-Surface Carbohydrates

Sugars on Red Blood Cells Define Blood Type

OH OH O HO AcHN O O O HO OH OH HO OH O OH OH O O HO HO O O OH HO OH OH OH O OH OH O O O HO O OH OH OH O O

Type-A Type

Type-B Type B

Type-C Type O

Red Blood Cell

Red Blood Cell

Red Blood Cell

* Type-A persons contain antibodies against Type-B sugars * Type-B persons contain antibodies against Type-A sugars

HO

OH HO HO2C O O

AcHN HO OH X X = H, F

O HO HO

OH O

OH O HO

OH O NH O H N O C13H27 N F B N F H3C

CH3

O O N nH O

NH

IC50 = 1.9X10-12 M

Ac

H N

ONa O m

n 270 n:m 1.5:100

NaO

Angew. Chem. Int Ed. 1998, 37, 1524

Electrophilic Fluorination-Nucleophilic Addition Cl

+

N O N F
NuH
+

2 X-

Nu F

Selectfluor (OR)n (OR)n

NuH = ROH, RNH2, RSH, (RO)2P(O)OH X = BF4-, TfO-

J. Am. Chem. Soc. 1997, 119, 11743. Angew. Chem. Int. Ed. 2005, 44, 192.

Activation of Thioglycosides using Selectfluor

Cl + N (BF4-)2 + N F

O RO

SR

O RO

F SR
+

O RO H

BF4-

F SR O

ROH BF3-Et2O RO

O SR

O RO F RO

OR

Carbohydrate-protein Linkages
OH O O N H R O HO OH O R

HO HO AcHN

HO AcHN O N H

HO HO

O OH O N H R O

O-linked

GlcNAcb- O-Ser(Thr) OH O HO OO PO O OH O

O GalNAca -O-Ser(Thr)

Xylb- O-Ser(Thr) HO HO HO O HN O OH O HN HN Mana - C-Trp O

HO HO AcHN

OH O N H R

HO HO AcHN

OH O

H N

N H O GlcNAcb- O-SerP

O Fuca -O-Ser(Thr)

GlcNAcb- N-Asn

H2N-protein-CONH

GPI Anchor

O O P OO 6Mana 1,2Mana 1,6Mana 1,4GlcNAca 1 O R RO O O

-

N-linked

C-linked

OH HO O

OH O R O

OP O O

Glycoprotein Synthesis

Sugar Arrays

Directed Evolution: Aldolases Sugar Arrays Glyco-proteomics Drug Discovery

Drug Discovery

Synthesis of Homogeneous Glycoproteins

J. Am. Chem. Soc. 1991, 113, 683 Nature 2001, 409, 232

Engineering Enzymes for the Synthesis of Glycopeptides and Glycoproteins

Double Linker Strategy

tBu FMOC-NH Deprotect and cleave OH FMOC-NH CO2R

OMe

FMOC-Peptide

O N H

OMe

OCH2CONH PAM Acid Labile TFA Rink Acid, non-aqueous base stabile

Relative Free Energy, kcal/mol

Aminolysis

Hydrolysis 6.1

4.3

E:P2 0.86 E+S (peptide) ES':P1 E:S 1.6 ES'+P1 acyl enzyme

E + P2 (acid)

Reaction Progress
Cys S CO2 HN O N H O R H CO2 HN N H H N Cys S O R R

Hydrolysis
Ser O O H R OCys S O H R O-

Aminolysis
Cys S H N R R O-

Enzymatic Glycoprotein Synthesis

OMe MeO H N O O Fmoc-SPPS PG Fmoc PEPTIDE Sugar Cleavage, TFA NH2 Fmoc PEPTIDE Sugar O O H PEPTIDE O O O 4 O O Rink-AM O Fmoc O H N or PS PG PEPTIDE O O O O 4 MeO O O (CH2)3 O O or N H O PEGA Fmoc-AA O O N (CH2)3 Si O CPG O H

PS

PG-Removal, TFA

Pd(0), Nucleophile H PEPTIDE OH

PEGA = polyethylene glycol PEGA = (2-acrylamidoprop-1-yl) CPG = controlled-pore glass PS = polystyrene PG = acid sensitive PG = protecting group PEPTIDE Sugar

Engineered Subtilisin FMOC removal (DMF, morpholine) PEPTIDE OH JACS 1997, 119, 1734. JACS 1997, 119, 2114. JACS 1998, 120, 1979.

Glycosyltransferase

GLYCOPROTEIN

Angew. Chem. Int. Ed. 2002, 41, 2171

Glycoproteins Synthesis via Traceless Staudinger Ligation

O N3 AA AA O CF3 Glycan Glycoprotein Peptide O + H2N Polypeptide

Subtilisin

N3

Polypeptide

Glycan O S PPh2

Peptide NH Polypeptide

O R S + N3 R' PPh2 R

O S PPh2 N R' R

O N Ph2P R' H2O R S O N H R'

Iminophosphorane

ChemBioChem 2006, 7, 429

Interleukin 2 (IL2)
AcHN HO HO O OH OH HO HO2C O OH OH HO O O O AcHN O HN2-Ala-Pro-Thr-Ser HO OH OH HO HO2C O OH O HO O O O AcHN O HN2-Ala-Pro-Thr-Ser ThrCO2 H OH OH CO2H HO

O AcHN HO OH

O AcHN HO OH Thr-CO2H

133 amino acid protein O-glycosylated on Thr 3 Growth factor stimulating proliferation of T-cells Cancer treatment (renal cell carcinoma and metastatic melanoma) Glycosylation Effects Increased stability Decreased clearance from blood Increased solubility Altered activity

Sialyl Lewis X -- Bound Conformation and Key Groups

Transfer NOE Study

Sialyl Lewis-X Mimetic

OH OH OH H3C O OH OH O O O O OH
O HO HO HO OH

OH OH O NHAc O OH OH HO2C HO2C N H O H N O O HN O OH O OR

OOC

R=H IC50 (P-selectin) = 1 M

AcHN

IC50 (P-selectin) = 3 mM

Enzymatic Synthesis with Cofactor Regeneration

Glycosyltransferase
O O O S O P O OOR-OH

Sulfotransferase

R-OSO3-

A O

Pi +

Phosphoryl- transfer CO2 enzymes

OPO32CO2-

PAPS

O OH O P OO

O OP O O-

A O

Aryl sulfotransferase

O OH O P OPAP O-

+
O2N OH OO S O O

O2N

X = Sugar Nucleotide

A = Adenine R = oligosaccharide or glycopeptide

J. Am. Chem. Soc. 1992, 114, 9283.

Angew. Chem. Int. Ed. Engl. 1999, 38, 2747.

Synthesis of Oligosaccharides with Glycosyltransferases: Sugar Nucleotide Regeneration

Sugar-1 E1 Sugar-2 -CMP OPO32Pi E4 PPi E3 UTP Sugar-2 -P E2 O CO2Pyr
HO OH

Sugar-2

Sugar-1

Sugar-1 E1

Sugar-2

Sugar-1

CMP ATP Pyr E3 PEP

Sugar-2

-UDP

UDP CO2PEP Pi

PPi E4 CTP Pyr CDP E3 PEP

E2 ADP

Sugar-2

E1: Glycosyltransferase E2: Pyruvate kinase E3: Sugar nucleotide pyrophosphorylase E4: Pyrophosphatase E5: Myokinase E6: Sugar nucleotide synthase
HO2C HO HO AcHN O

OH H3C O HO OH O O O O OH

NHAc OH O OH

HO HO

HO COOO O HO O O AcHN n

OH OH

Sialyl Lex

n ~ 1500 Hyaluronic Acid

J. Am. Chem. Soc. 1992, 114, 9283 J. Am. Chem. Soc. 1995, 117, 5869

Enzyme Inhibitor Discovery: Synthesis and screening in situ in Microtiterplates

(a) Fucosidase
H3C

(Angew. Chem. Int. 42, 4661(2003), Chem. & Biol. 11, 1301 (2004))
H N OH OH NH2 + H3C H N OH OH H N O R

OH

RCOOH

OH

HBTU, DIEA

(60 various acids)

H3C

H N OH OH

OH

H N O

NH

H3 C

H N OH OH

OH

H N O

Ki = 0.46 0.03 pM

Ki = 5.5 0.2 pM

(b) Fucosyltransferase

(J. Am. Chem. Soc. 125, 9588 (2003))

GDP O O N H R N N N n N H R + N3

GDP

60 alkynes
GDP O N N N 4 N H

5 azides
GDP O N N N N H

Ki = 62 nM

Ki = 0.4 M

Sulfotransferase Catalysis
ROH
Sulfotransferase His 104 HN N
H RO + O S O
+

ROSO3-

Largely broken ~ 3 O P OR O-

NH2 N
-

NH2 N
-

N N O O P O OO

N N

Partially formed ~ 3

H3N Lys 102

O O O S O P O O OO O-

N O OH

PAPS O P O-

O OH O P OO-

PAP

Dissociative (sulfotrioxide-like) Transition State in the Forward Reaction

nuc = +0.33 lev = -0.45

Proc. Natl. Acad. Sci. USA 2003, 100, 910

HO

Ex = 360 nm Em = 449 nm

O3SO

(For screening) HO NO2

-

O3SO

NO2 (For regeneration)

Combinatorial Synthesis of Purine Derivatives

OMe R1NH2 O CHO OMe H N OMe N HN O OMe N Cl N N R1 N Aryl N R0 HN HN O N H N H O F OH R2HN N N R1 N N R0 HN N R3 N R4 N R1 R5O N N R0 N N N R1 N N R0 HN N R1 N N R0 O NHR1 OMe Cl OMe N N Cl N N R0 "R0" Mitsunobu Cl N N Cl N N H

Cl N

Dichloro heterocycles

Ki = 96 nM -Arylsulfotransferase-IV

12
N H

Ki = 5 nM

40,000 Compounds

Chapman, E.; Ding, S.; Schultz, P.G.; Wong, C.-H. J. Am. Chem. Soc. 2002, 124, 14524.

Rapid Identification of Sulfotransferase Inhibitors: Synthesis and Screening In situ in Microtiter Plates

HN HN N N H N N N Cl R N

R-X, TBAF, DMF/H2O

N

HN N Cl

N N N

N Cl

Ki= 96 nM

Ki= 9 nM
O F Br O Br Br Br CF3 O Br Br I H2N O O Br Br O O O Br CF3 Br

Br

Cl

Br

Br

Sugar-Assisted Glycopeptide Ligation

Brik, A., Yang, Y.Y., Ficht, S., Wong, CH, J. Am. Chem. Soc. 2006, 128 5627.

Building Blocks

-GalNAc Thr

-GalNAc Ser

-GlcNAc Asn

-GluNAc Thr

-GluNAc Ser

-O-Xylose Ser

3-O-thioacetyl-Glu Ser

Bacterial Protein Biosynthesis

DNA aminoacyl-tRNA synthetase
aa

86 tRNA genes in E. coli 21 aa-tRNA synthetases About 50 processing and base modification genes

ATP

mRNA tRNA

Several deacylation enzymes

R NH3+ O

NH3+ O

nascent peptide chain

O

22 rRNA genes At least 85 ribosomal protein genes At least 12 elongation and initiation factors

H2N EF-Tu

ribosome

Directed Evolution
Operation of Darwinian evolution under Experimental laboratory conditions;

i.e., The creation of molecular diversity in a test tube through iterative amplification, mutation, and selection/screening to find the molecule of interest.

Evolution of aminoacyl-tRNA synthetase

109 Mutant synthetases Undesired synthetases Desired synthetases

Positive selection
Add glycosylamino acid

CAT

Incoporation of Tyrosine

Incoporation of Glycosylamino acid

Undesired synthetases

Desired synthetases

Negative selection
Without glycosylamino acid

Incoporation of Tyrosine

Incoporation of Glycosylamino acid

_ * Barnase * *

Mutant synthetases

Die

* = TAG stop codon acetyltransferase CAT = chloramphenicol

Directed Evolution of Aminoacyl tRNA Synthase for Incorporation of Glycosylated Amino Acid into Proteins in E. coli
HO
AcO AcO OAc O NH3+ O O O-

Evolved E. coli

HO

OH OH O
HO

OH

AcHN

AcHN

AcHN

NH3+
O

NH3+ O O

AUC UAG

Science, 2004, 303, 371 J. Am. Chem. Soc. 2004, 126, 15654

Stop Codon

Sites of Amino Acid Mutation

I-90
Asp158Cys,Glu107Pro, Ile159Tyr, Leu162Arg
OH O O H2N COOH

AH1
Tyr32Phe, Ala67Pro
HO OH O O COOH

C10F
Tyr32Ala, His70Pro Ala67Ser, Leu98Ile
HO OH O O COOH

HO HO AcHN

HO AcHN

HO AcHN

H2N

H2N

Suppressor-tRNA technology : A novel vector

for efficient tRNA and tRNA-synthetase expression

Advantages:
1. Compatible with most commercially available E-coli expression vectors. 2. Control tRNA and tRNA synthetase ratio by changing the origin instead of the promoter

(Insert tRNA) (Change origin)

Vector Expression yield pWH-p15A ~5mg/L

pWH-RSF <1mg/L

pWH-CDF 40~50mg/L

Chemoenzymatic synthesis of neoglycoproteins

HO HO OH O AcHN O

N N N

HO HO

OH O AcHN

R E. coli + evolved MjTyrRS

OH O H HO N HO AcHN O

HO HO

OH O H N AcHN O

N N N

H2N R = N3or CH3CO-

COOH

HO OH O HO AcHNO HO OH O HO AcHNO OH O

HO HO

HO OH O HO AcHNO

O
AcHN

HO OH O HO AcHNO

NH2
OH O

NH2

N N N

HO HO

O AcHN N

Assembly of Glycophage: Vaccine Development

Carbohydrate Antigens

O O O O O O N

O O O O O O O N N O O O O N N N N N O O O O O O O O O O O O O O O O

O O

O O O

Carbohydrate Antigens

O O

2-

O OH + H

O OPO3 OH
2-

FDP Aldolase

2-

OH OPO3 OH OH
2-

O3PO

O3PO

Ser271 OH O O P O HO
-

Lys229
o

His

His His Zn
2+

NH OH O H

8.3A
2-

OOH O H O Tyr
2-

O3PO

OPO3 OH

2-

H3N Lys107

OPO3 OH

Type I Aldolase
Sygusch, J. et al Proc. Natl. Acad. Sci. USA

Type II Aldolase
Dreyer and Schultz J. Mol. Biol. 1996, 259, 458 Hunter, W. N. et al Structure 1996, 4, 1303

1987, 84, 7866

Blom, N. et al Nature Struct. Biol.

1997, 4, 36

PO OH

Donor: PO
O PO OH OH FDP aldolase O OH PO OH

Donor:

Donor:
-

O2C OH OP
-

O2C

O OP
-

OH

O O2C

OH OP
-

O O2C

OH CO2-

O2C OH DAHP synthetase O OH OH

OH 2-Keto-3-deoxy-6-Pgluconate aldolase OP O
-

2-Keto-4-OH-glutarate aldolase O HO CH3 OH

-

OH O PO

OH OH

O2C OH OH KDO synthetase O OH OH OH

OH

OH O2C

Fuculose-1-P aldolase

O2C OH OH KDO aldolase

CO2-

4-Me-4-OH-2-ketoglutarate aldolase OH OH
-

OH

OH

O2C AcHN OH Sialic acid synthetase O

OP
-

O O2C

OH

OH

O O2C

OH OH

Rhamnulose-1-P aldolase O PO OH OH O HO OH OP

AcHN

OH 2-Keto-3-deoxy-Larabinoate aldolase O OP OH
-

Donor:
HO OH HO NH2 D-Thr aldolase O OH O

Sialic acid aldolase NH2

-

O O2C

OH O2C

OH OH

Tagatose-1,6-P2 aldolase O

Donor:
H O H OH 2-Deoxyribose 5-P aldolase OH OP

OH

NH2 Ser hydroxymethyl transferase

-

2-Keto-3-deoxy-6-Pgalactonate aldolase O O2C OH CO2-

2-Keto-3-deoxy-Dxylonate aldolase O O2C OH

HO NH2 L-Thr aldolase

OH 2-Keto-3-deoxy-Dglucarate aldolase

Hydroxybutyrate aldolase

Aldolases in Organic Synthesis

X X O H Y R2
-CO 2-

R1
-CH2OPO3
=

Y
-OH -CH3 -H, -CH3, or ClCH2

Aldolase
X O Y R2 HO X O R2 X Y HO R2 Y HO R2 HO H N X S HO Y
R1 = OH R1 = N3 H2/Pd-C R1 = SH Ac2 O Et3SiH-BF3

-H -CH3 -CH3, CH2 F R1 = CH(CN)P(O R2 ) R1 = NO2

R1

HO Y HO

CN

Y X NO2 HO R2

R2

Angew. Chem. Int. Ed. 2000, 112, 1406

Mechanism of Glycosyltransfer Reaction

O Mn HO Me O COOH OH OH + O R -O2C HO Me O Acceptor O OH O O P O OH + H O2C
2+

N O
-

NH N NH2

O O HO

OH

-1,3-Fucosyltransferase Reaction

-1,3-Fucosyltransferase Reaction

Mn2+ HO Me OH N H OH HO Me OH N H OH OH HO Me N H OH OH OH Me Me NH2 HO OH NH HO HO ON R O

Biochemistry 1997, 36, 823

Proteoglycan Degradation in Arthritis

Chem. & Biol. 2001, 8, 701.

2-Deoxyribose-5-Phosphate Aldolase
H + O CH3 H OH O OPO32O H OH H OH OPO32-

Lys-201 H Asp-102 O O HN H CH2 NH3+ H O H O OPO32OH Lys-167 N H OH

OH H2 O

OPO32-

Ser-238, 239 Gly-205 Hydrophilic site

Hydrophobic site

Thr-170 Lys-172

Lys-167

Science, 2001, 294, 369

Sequential Asymmetric Aldol Reactions Catalyzed by DERA

R H O OH R O O

O H R

O H

DERA
OH

Br2, BaCO3
OH

R = H, OMe, Cl, N3, CH2CO2O N3 H O H O H

a) DERA (Ser238Asp) b)Br2, BaCO3 35% for 2 steps

N3

OH

H N

OH F

Atorvastin (LipitorTM)
J. Am. Chem. Soc. 1994, 116, 8422

DERA Products for Total Synthesis of Epothilone A

OH O HO H DERA, 48% HO O H Inverted Enantioselectivity OH H O O O

OH O H OH DERA, 48% O H OH

O H I S N

Normal Enantioselectivity

OAc

O HO O O OH O

S N

Epothilone A
Angew. Chem. Int. Ed. 2002, 41, 1404

Directed Evolution of N-Acetylneuraminic Acid Aldolase

O HO HO HO NHAc O OH N-Ac-D-mannosamine CO2HO OH O HO D-Sialic acid CO2OH

D-Neu5Ac

aldolase (Wild type)

HO AcHN

13 U/mg

Directed evolution (Error-prone PCR, 2nd generation)

O NHAc O OH N-Ac-L-mannosamine OH OH OH CO2HO

L-Neu5Ac aldolase Tyr98His, Phe115Leu, Val251Ile

CO2O

HO

OH

OH

OH NHAc

L-Sialic acid

0.2 U/mg

Directed Evolution of Sialic Acid Aldolase

PNAS 2005, 102, 9112
O HO HO HO NHAc O OH CO2D-Neu5Ac aldolase HO OH O HO OH CO2-

HO AcHN

N-Ac-D-mannosamine

D-Sialic acid
Kcat = 10.5 S-1

Directed Evolution

OH HO O OH L-Arabinose OH

L-KDO aldolase Y98H, F115L, V251I, V265I, Y281C, N153Y, E60A, D150G HO O CO2-

HO

OH O CO2-

OH L-KDO

OH

Kcat = 10.4 S-1

Docking of sialic acid glycal with mutations in aldolase variants N2C5 (Left) and N5B2 (Right)

Hsu et al. (2005) Proc. Natl. Acad. Sci. USA 102, 9122-9126

D- and L-KDO cleavage specificity constant of sialic acid aldolase variants through the progress of directed evolution

Hsu, Che-Chang et al. (2005) Proc. Natl. Acad. Sci. USA 102, 9122-9126

Mirror-Image Selection of D-Peptides for Binding to Sugars

HO OH HO AcHN HO
D-Sialic Acid

CO2 O O

D-sugar

L-sugar O

CO2

HO

HO

OH

OH NHAc

L-Sialic Acid

HO OH HO O O HO CO2
D-KDO

OH OH OH O O D-peptide L-peptide CO2

OH

L-KDO

peptide phage library

selection for binding to L-sugar

chemical synthesis of D-peptide

natural L-peptide binders

Mirror-Image Sugar and Phage Display

ChemBioChem. 2001, 2, 741

Programmable One-Pot Synthesis

J. Am. Chem. Soc. 1999, 121, 734

One-Pot 9 monosaccharides

1.5 x 107 possible tetrasaccharides

Determination of Relative Reactivity Competition Analysis Using HPLC

O 1 SR L+ D1 Activator (L+) k1 1 Acceptor O 1 OAcceptor (Donor1 - Acceptor) O

O 1

SR L+

Activator (L+) k2 1

Acceptor O 1

O OAcceptor (Donor2- Acceptor)

D2

v=k[Dx][L+]

k1 ln[D1]1 - ln[D1]0 = k2 ln[D2]1 - ln[D2]0

J. Am. Chem. Soc. 1999, 121, 734

G = RT ln

k1 k2

Promoters in One-Pot Glycosylation

O I N O O TfOH (cat) O By-product SMe S -OTf DMTST O STol + O STol N+ N+ F O S O O STol + O O N

STol

Low reactivity, Unstable promoter

HO

Cl 2X O O O STol

High yield Fast

N O O O STol

High yield

Tf 2 O O N O Tf 2 O S

STol

High yield, Broad temperature range

N-(Phenylthio)-e-caprolactam:

A New Promoter for Glycosylation

O BnO

OTf S CH3 D1 + N

OTf S R1 OTf N R2 OBz H3 C S S L1 CH2Cl2 O BnO S S D2 OTf CH3

O BnO O BzO

OBz O BzO OMe

HO BzO

O BzO OMe A1 O BnO D3 OTf

P1

92 %

Org. Lett. 2004, 6, 839-841

OAc AcO OAc OAc S-Tol O CO2Me N3 AcO (0.692) AcO AcO AcO OAc O S-Tol (1.0) OBz BzO OBz O BzO O BzO BzO BzO BzO (5.7) OBz O S-Tol PhthN (5.7) S-Tol PhthN (1.7) AcO OAc CO2Me OAc O S-Tol N3 AcO (2.23) OAc O S-Tol AcO (2.7) ONBz O S-Tol PhthN (3.2) HO BzO HO OBz OBz CO2Me O S-Tol AcHN BzO (7.9) BnO BnO OBn O AcO OAc O AcO O AcO OAc O S-Tol AcO

NBzO HO

ONBz O S-Tol OClBn (12.6) OH O S-Tol BzO (13.1)

BzO

OH OH O

S-Tol CO2Me

HO BzO

OBn O S-Tol PhthN (84.5) Ph O ClAcO O O BnO O S-Tol PhthN AcO AcO (94.6) O O O S-Tol PhthN (102.0) LevO PMBO S-Tol PhthN (102.8) OTBS O TBSO (110) STol BzO BzO Ph O O BnO (211) OTBS O STol O S-Tol PhthN (185.4) OAc O S-Tol NHTroc (197) BnO S-Tol

LevO PMBO

(4.1) OTBDPS O BzO S-Tol BzO PhthN (5.6) OBz O S-Tol

BzO BzO

AcHN BzO (23.5) Ph O ClAcO N3 (24.1) BzO OBz O O O

OTBDPS O S-Tol BzO (340.1)

BnO BnO

OBn O N3 (1276) STol

Ph O BnO BnO (7180) HO STol PMBO OTBDPS O S-Tol O O OBn BnO OBn (8417) BnO BnO BnO Ph (1.7x10 ) O O HO BnO (2.0x10 ) BnO BnO OH (5.2 x 104) BnO OBn OBn O BnO (4.0 x 10 ) BnO OBn O OBn S-Tol
4 4 4

O O S-Tol

Ph

O O AcO

Ph

S-Tol Ph

O O HO

OBn O STol

Ph AcO OAc MeO2C O O O O AcHN AcO OAc O LevO

(391) Ph O O BnO

AcO AcO

OAc O S-Tol AcO (14.3)

HO BzO (28.9) Ph O O ClAcO

S-Tol

ClAcO

OBn

OH O STol

OBz O S-Tol BzO (1.0) OBz O S-Tol BzO

AcO

OH OH

S-Tol CO2Me

HO AcO S-Tol OClAc

OBn O

OBn O O OBn O S-Tol BzO (208.6)

(440)

(1308) BnO OBn OBn O O OTBDP O S S-Tol PMBO BzO Ph O HO BzO (1791) BzO HO OTBDPS O S-Tol NHTroc (2000) OBn O S-Tol BnO (2656) OTBDPS O S-Tol PMBO BzO (2797) LevO OTBDPS O S-Tol HO (3531) HO (1320) O O

OBn O S-Tol

BzO BzO

HO BzO

O AcHN AcO (16.5) BzO BzO HO (17.6) BnO OBz O

TBSO HO BzO

OTBS O (442) STol

O S-Tol

(31.4) HO BzO BzO OTBS O

S-Tol

(1.3) AcO AcO OAc O

TBSO BzO HO

Ph

O O BzO

OH O STol

OBn O STol

S-Tol

Ph (40) STol O O O S-Tol BzO (118.7) TBSO HO TBSO OBz O

(233) LevO HO

(460) LevO PMBO BzO Ph O (482.9) O O BnO LevO (577.1) HO AcO OBn O S-Tol AcO (731.4) PMBO S-Tol OH O S-Tol BnO BnO

OBz BnO O O S-Tol BzO BzO (7.9) OBz O S-Tol BzO (9.4)

OBn OBn S-Tol O Ac N CO2Bn Bn BnO (17.9)

BnO

OBn OBn S-Tol O CO2Bn AcHN BnO (48.4)

ClAcO

OTBDPS O S-Tol O

STol CH2OAc

O OBn BnO OBn (254.3) BnO OBn O OBn O HO STol OTBDPS O S-Tol BzO

AcHN

AcO AcO

HO

OH CO2Me OAc O S-Tol AcHN AcO (19.4) BzO OTBS O S-Tol BzO (19.2)

ONBz O HO S-Tol HO PhthN (57) OAc O S-Tol NHTroc (57.3) TBSO BzO TBSO OBz O STol (60) HO BzO BzO (67.1) OBz O S-Tol

(123) STol Ph O O HO (131) LevO HO OBz O

(7.2x104) BnO OBn OBn O BnO (7.8 x 104) BnO OBn OBn O CH2OAc STol STol CH2OTBDPS

HO NBzO

(268.6)

AcO AcO

OAc O S-Tol PhthN (10.4) OBz O S-Tol PhthN (11.4)

AcO AcO

BzO

OTBDPS O S-Tol BzO (142.9)

AcHN

Ph O BzO BzO (285.7) Ph O O BnO (315) Ph O HO ClAcO (320.0) O O S-Tol OBn O BzO STol HO O O S-Tol PMBO

LevO

AcO

OAc S-Tol OAc O CO2Me AcHN AcO (2.55)

Ph

HO AcO

O O HO

O S-Tol PhthN (20.0)

OTBDPS BnO O BnO O S-Tol BnO BzO (148.6)

OBn LevO O

OTBDPS O S-Tol O O OBn

BnO OBn (780.0) OBz O S-Tol NHTroc (850) OH O O O BnO OBn (1155) OBn

BnO OBn O BnO STol LevO (4000) BnO OBn O BnO STol OAc (4800) BnO BnO BnO OBn O

AcHN

BnO 4 (8.0 x 10 ) BnO OBn OBn O STol

AcO

OAc OAc S-Tol O Ac N CO2Me Ac AcO (3.45) OBz OBz S-Tol O CO2Me AcHN BzO (3.5)

Ph AcO OAc OAc CO2Me O S-Tol AcHN AcO (11.5) OAc O CO2Me AcHN AcO (22.0) OH O S-Tol PhthN (22.6) O O S-Tol

HO BzO

OBz O S-Tol NHTroc (162.9)

AcHN

CH2OCH2OBn BnO 5 (2.25 x 10 ) OBn OBn O BnO 5 (3.3 x 10 ) STol CH2OH

LevO PMBO

TBSO

AcO

OAc OAc CO2Me O Ac N S-Tol Ac AcO (12.2)

BzO BzO

Ph

S-Tol

BnO

O O BzO O S-Tol PhthN (72.4)

OBn O OLev BnO OH BnO O BnO O S-Tol BzO (182.9)

AcHN

(5000) STol

Optimer Programmable One-Pot Synthesis

Science, 2001, 291, 2344

Programmed One-Pot Synthesis of the Cancer Antigen Globo H

HO HO O HO OH OH O O OH HO O OH O AcHN HO O OH O HO O OH O O OH HO

In
OH O OH OR

Globo H

HO

Out

Deprotection
BnO OBn O OH

O OBn

STol OBn 1 OBz O NBzO O ONBz STol OClBn O

BnO BnO

OBn O O O

BzO O

OBz O

NBzO O

ONBz O

Mix 1 + 2 + 3 62%
OBn O BnO BnO O OBn O BnO OR

BnO BnO

BzO O

TrocHN

ClBnO

TrocHN 2

BnO

OBn

OBn

BnO

HO BnO

OBn O BnO BnO 3 O

OBn O BnO OR

Angew. Chem. Int. Ed. 2001, 40, 1274

Reactivity-Based One-Pot Synthesis of Lewis Y: a Colon Cancer Related Antigen

BnO OBn O BnO HO
4

OBn O

BnO

OBn O

OBn O

O HO

STol

HO LevO

O AcO

O(CH2)5CO2Me

NHTroc

NHTroc

(RRV = 1.2 * 10 )
O OBn BnO STol OBn BnO OBn O O BnO O BnO OBn O OBn BnO OBn O O OBn OBn O BnO O

(RRV = 0) 2h NIS/TfOH promoted glycosylation 4h

(RRV = 7.2 * 104)

OBn O LevO O AcO

OBn O O(CH2)5CO2Me

NHTroc

NHTroc

global deprotection

Lewis Y: Colon cancer related antigen

Reactivity-Based One-Pot Synthesis of Fucosyl GM1: a Specific Small Cell Lung Cancer
AcO BnO BnO HO OBn O AcO O OAc O STol AcO AcO OAc MeO2C O OAc HO O BnO OBn O O BnO OBn O OBn O N Bn Cbz NHTroc

(RRV = 1500) 2h

(RRV = 0) 4h NIS/TfOH promoted glycosylation

BnO OBn O AcO O OAc O O OBn O BnO O OAc OAc O BnO OBn O OBn O N Bn Cbz

O BnO OBn

STol OBn

(RRV = 7.2 * 104)

BnO O O OBn BnO

TrocHN O MeO2C OBn AcO AcO AcO

global deprotection

Fucosyl GM1: Small cell lung cancer antigen

One-Pot Synthesis of Heparin Sulfates

OTBDPS HO BnO O OBz O BnO N3 OTBDPS BnO BnO OAc O STol N3
NIS, TfOH or N-(phenylthio) caprolactam, Tf2O

OAc O STol

HO BzO

OBn O O BnO

OAc O N3

OMe

BnO BnO

OAc O OTBDPS N3 O BnO O OBz O BnO OAc O N3 O OTBDPS OBn O O BnO BzO
1. HF.Pyr, THF 2. TEMPO, NaOCl 3. MeI, KHCO3

OAc O N3

OMe

BnO BnO

OAc O

OMe O OBz O BnO OAc O O N3 O

OMe OBn O O BnO BzO OAc O N3

N3 O BnO

OMe

Globo H: A hexasaccharide epitope on cancer cells

OH OH O HO O H3C HO OH O OH O OH OH O O NHAc OH O HO OH OH O O HO OH O OR OH OH OH O

More than 85% of breast cancer cells have Globo H. Also on prostate, ovarian, and colon cancers. Globo H-based vaccine for breast and prostate cancers Inhibition of fucosyltransferase Globo H array for antibody binding assay Globo-H or precursor on cancer stem cells

FucT

HO

Globo H and truncated analogs on microarray

PNAS 2006
HO 1) H 2N S S 1 O HO CuI, MeOH O
5

O O O HO O
5

NH2

HN

2 3 3 6 6
O O
5

3 3 6 6 6 6

O N H

2
N N

3 3 2 3

N3 2) NHS

Key:

Gal

Glc

GalNAc

Fuc

Probing the specificity of Globo H antibodies against Globo H and truncated sequences by microarray
mAB VK-9

mAB MBr1

2 2

2
6 6

3
3 3 6 6

Glycoproteomic Probes: Click-Induced Fluoroscence via Azide-Alkyne Ligation

PNAS 2006, 103, 12371

Imaging Post-Translational Glycosylation: Probing the Fucosyltransferase

Sawa M, Hsu TL, Itoh T, Sugiyama M, Hanson S, Vogt PK, Wong CH Proc. Natl. Acad. Sci. 2006, 103, 12371

Profiling the Substrate Specificity of Fucosytransferases using Glycan Array

2 4

GDP-fucose(N3) FucTs
3

Gal GlcNAc Fuc

Glycans Fabricated Microarray

Probe

Cu(I)

FucT I & II FucT III - VII

Fuc2Gal4GlcNAc-R Gal4[Fuc3]GlcNAc-R

Results of Micro-plate Assay

GDP-fucose analogs FucTs

GDP-6-N3-Fucose
1

GDP-6-Alkyne-Fucose
1

0.8

0.8

Absorbance

0.6

0.6

0.4

0.4

0.2

0.2

II

III

IV

VI

VII

II

III

IV

VI

VII

Human FucTs

Human FucTs

The activity of the enzymes was varied. FucT Reaction: rt, overnight. Click Reaction: tr, 1h. The values were calculated by subtracting background value (GDP-fucose) from the original data

Analysis of fucosylated glycoproteins on the cellsurface by flow cytometry

Sawa M, Hsu TL, Itoh T, Sugiyama M, Hanson S, Vogt PK, Wong CH Proc. Natl. Acad. Sci. 2006, 103, 12371

Double Staining Study: Fucosylation at Golgi

Double

Probe

Golgi marker (WGA-lectin)

Merge

Wheat Germ Agglutinin (WGA) lectin (Molecular Probes): A Golgi marker binding to sialic acid and N-acetylglucosaminyl residues of glycoproteins

Biosynthesis of N-Linked Glycoproteins

Modification of the sugar domain of natural products

R1O O O R2O RO
3

HO

O STol O O STol

building blocks
STol from database

natural product 1. protection 2. remove of glycon

OH

aglycon 1. one-pot reaction (2. switch protecting and/or activating groups)

RO O O

aglycon

coupling

RO

HO O

one-pot reaction

n deprotection

protected
aglycon

natural product derivative

aglycon

Angew. Chem. Int. Ed. 2003, 42, 4657

Inhibition of Peptidoglycan Biosynthesis

HO OH OH HO HO O HN HO OH OH O OHO O O O O OH CONH2 O O O O O NHAc OH O CO2O Me OH NH2 O Me O OH O O O Cl O OH O N H O N H Me H N Me Me HO OH

O O

NHAc

moenomycin A (transglycosylase Inhibitor)

RO O O H N H O H N

Cl H N O H O N H O NH2 OH OH

+ B:
OH O RO O AcNH HO O NHAc O OH O O O

H HO O OH O d AcHN
+

OH NHAc O O O RO O AcHN O P O OOH O P O O C55H89

HO

O R N H

H N O

O O

D-Ala-D-Ala-L-Lys-D-Glu-L-Ala-NH

O O O P O O- O P O C55H89

Vancomycin: D-Ala-D-Ala Binding

(transpeptidase Inhibition)

Transglycosylase Reaction membrane

Effects of Sugars on Vancomycin Activity

Angew. Chem. Int. Ed. 2003, 42, 4657.
HO NH3Cl O HO OH O OH O O HO O NH

HO HO HO OH HO OH H N O NH2 O NH NH2+ O NH

O O HO

OH O OH O O Cl H N O OH OH

O O Cl O N H O OH H N O NH2 O NH NH2+

O O Cl O

O N H

Cl H N O OH OH

N H

N H O

O2C HO

O2C HO

Vancomycin 2 g/ml

Gal-Glc Vancomycin >40 g/ml

HO HO

OH O

AcHN O O O HO NHAc HO OH HO O NH

OH O OH O O Cl H N O OH OH

HO O O Cl O N H O OH H N O NH2 O NH NH2+

HO HO

O O HO NH2 HO O NH O2C HO O N H

OH O OH O Cl H N O OH OH

O O Cl O N H O OH H N O NH2 O NH NH2+

N H

O2C HO

GlcNAc-GlcNAc-Glc Vancomycin >40 g/ml

6-amino-Gal-Glc Vancomycin 5 g/ml

Chemo-enzymatic synthesis of glucosaminecontaining vancomycin

Chemistry An Asian Journal 2006
OH O HO O HN HO O HO OH OH N H O Cl H N O N H O O O Cl OH H N O NH2 O N H H N 1) one-pot enzymatic glycosylation 2) reductive amination HO O HN HO O HO OH OH N H O Cl H N O N H O RHN O O O O O OH OH Cl OH OH H N O NH2 O N H H N

vancomycin aglycon

MRSA 33591 Vancomycin R = C10H21 R = C12H25 R = C14H29 R = C16H33 R = chlorobiphenyl 1 0.5 1 2 2 0.5

VSE 29212 2 0.5 0.5 1 2 0.5

VRE 51575 1000 10 2 1 2 10

RHN O O HO O HN HO O HO N H O ClH N O OH OH

OH O O O N H O OH Cl OH H N O NH2 OH O N H H N

Antibiotic activity of monoglycosylated vancomycin analogs

VSE 29212 2 5 10 5 2 0.5 0.5 1 2 10 5 0.5 VRE 51575 1000 >50 >50 >50 50 5-10 2 1 2 50 50 10

MRSA 33591 Vancomycin Vancomycin aglycon R=H R = C6H13 R = C8H17 R = C10H21 R = C12H25 R = C14H29 R = C16H33 R = C18H37 R = benzyl R = chlorobiphenyl 0.5-1 1 2 2 0.5-1 0.5 1 2 2 5 2 0.5

Strategies for Creating Carbohydrate Diversity

Building Blocks
1 2 3 4

Glycosyltransferase

Sulfotransferase

2 SO3-

Representative glycan structures on the array

Blixt, Ola et al. (2004) Proc. Natl. Acad. Sci. USA 101, 17033-17038

Programmable One-pot Synthesis

Photocleavable DIOS-MS Sugar Array

DIOS-MS

Development of a Photocleavable DIOS-MS Sugar Array

Angew. Chem. Int. Ed. 2006, 45, 2753-2757

Limit of Detection for Man1 and Man4

HO HO OH OH O O O OSu HO HO HO HO O
4

HO HO

OH OH O

O
( )4

OSu

O O
4

O NH2

NH

NH

5000

3000

1000

500

300

100

50

30

10

Man1 and Man4 detected by Con A-biotin (10 g/mL) and streptavidin-Cy3 (10 g/ml) LOD: 10 M x 500 pL = 6 fmol/spot ~ 5 pg/spot 1 mg sample 2 108 spots Man1 & Man4 Conc. Rang

1918 Spanish Influenza

From 1918-1919, an influenza pandemic killed more than 40 million people The influenza virus had a profound virulence. a mortality rate at 2.5% compared to the previous influenza epidemics , which were less than 0.1% Caused by the H1N1 virus

1918 reconstructed H1
Science. 2004 303, 1866-70.

Carbohydrate Receptors for Influenza

CO2O HO

OH HO AcHN

OH O

CO2O O HO

HO

HO HO O

OH OH HO AcHN HO O

HO O OH O

Human Influenza A receptor (Neu5Aca2-6Galb)

Avian Influenza A receptor (Neu5Aca2-3Galb)

OH HO AcHN

OH O

CO2O HO O
-

HO

HO OH OH HO AcHN HO O CO2

HO HO O

O HO

O OH

Swine Influenza A receptors

(Neu5Ac-2,3-Gal, Neu5Ac-2,6-Gal)

Biol Pharm Bull. 2005, 3, 399-408.

Carbohydrate microarray analysis

A/Vietnam/1203/2004 Human H5

Fuc NeuAc Gal GlcNAc GalNAc

Array regions:
glycoproteins 2-3 ligands 2-6 ligands 2-8 ligands other sulfated or negatively charged molecules

A/Duck/Singapore/Q-F119-3/1997 Avian H5

Computational Modeling structure of the complex of 2G12 with Gp120

HIV is a retrovirus and enclosed by an envelope, which is required for fusion to the host-cell surface. This is an essential step in HIV replication cycle. The HIV-1 envelope is a basic lipid bilayer that contains the gp120 surface envelope and the gp41 transmembrane envelope glycoproteins.

Science 2003, 300, 2065

35

Interactions of the Fab 2G12 Dimer with Man9GlcNAc2

I. Wilson et al., Science, 2003, 300, 2065.

Oligomannose inhibition of 2G12 binding to gp120

100 80

% inhibition

60 40 20 0
1 2 3 4 5 6 7 8 9

Antibody 2G12 Ligand Binding

45000 Relative Fluorescence Units 40000 35000 30000 25000 20000 15000 10000 Man 9 5000 0 105 200
HO HO HO HO HO HO HO HO HO OH O O O O O HO HO O

HO OH O HO HO HO OH HO O O HO O HO HO HO O HO O O OH HO HO O HO O O

HO HO HO HO HO HO

OH O O O O O O HO OH O O NH2 5

HO HO HO
NH2 5

HO HO HO

OH O O

Man 8
OH O O NH2 5

Glass slide microarray 6-10 fmol / spot

Ligands 105 - 200

PNAS 2004, 101, 17033

Man4-2G12

Overlap with Man4

H2O-mediated H-bonding

Man5-2G12

Man8-2G12 Dimer

Man8-2G12 (cross-link)

Overlap of Man4, Man7, Man8, and Man9 with 2G12

Multivalent HIV-1 Vaccine

S

M uc -1

NH NH

-1 uc M
HN O 5 HN NH HN O O HN 5 NH O HN

O S 5 NH NH O 5 N H S N H O 3 N H S NH 3

Muc-1

Carrier

Muc-1

O HN

O O

H N 5 S

H N

O 3

H N S

NH

O HN 5 NH NH S O NH NH S 3

1 ucM

Mu c

-1

The Immune Response Through MHC and NKT-Cell Pathways

Mode of glycolipid presentation by CD1d to NKT cells

Vi TCR -GalCer
3 2

Th1 cytokine (IFN- etc.) Th2 cytokine (IL-4 etc.)

NKT cell APCs

1

CD1d 2-microglobulin

Human V24i NKT cells bind to glycolipids in the context of CD1d.

Proc Natl Acad Sci U S A. 2005, 102, 1351; Nature 2005, 434, 520-5

7000 6000
IFN- (pg/ml)

300 250
IL-4 (pg/ml)

5000 4000 3000 2000 1000 0

-GalCer
OH HO HO OH O O

200 150 100 50 0

9
O HN

8
C25H51 OH

none
OH OH O O HN

-GalCer
C25H51 OH OH C14H29

8
OH

7
OH O O HN

2
C25H51 OH

none

Na O3SO

+-

-Galcer
OH Na O3S O HO
+-

OH
O

C14H29

HO O

Na+-O3S O HO

OH

C12H25

a-3-sulfo-Galcer (9)
C12H24 C8H17
O OH OH O O HO HN HO O OH C10H21 C12H25

b-3-sulfo-Galcer (8)
O O OH OH O HN HO OH HO O OH C10H21 C12H25

OH HN O O

OH

C12H25

sulfatide (7)

Sphingomonas GSL (1)

Sphingomonas GSL (2)

Overview of CD1d-sulfatide structure

J. Exp.Med. 2005, 202, 1517

O OH
-

O3SO HO OH

OH O

HN O O

sulfatide
OH OH

HO HO

OH O O

HN

-GalCer analog

OH HO HO

OH O O

O HN OH OH

-GalCer

Crystal structure of a bacterial glycolipid in complex with mCD1d. PNAS 2006

N42 2 1

1
A 2M 3
O OH HO HO OH O O OH HN O C10H21 C12H25

N165

Design of NKT Cell Activators

A
2 1

N42

1
2M A F

2
3 N165

C
R79

D
R79

D80

1
D80

Y73 C12

C12

T156

Comparative binding analysis

mCD1d-suflatide

mCD1d-GalCer analog (PBS-25)

hCD1a-sulfatide

Human NKT activation by -galcer analogs

J. Am. Chem. Soc. 2006
Relative activity to -galcer (%)
1200 1000 800 600 400 200
-GalCer

0 R=

19

20
S

21

22
O O

23

24

25

26

27

28

31

34
O

39

40

41

42

OCH3

CF3

i-Bu

HO HO

OH O

O HN

R OH

HO O OH

= IFN- = IL-4

Design of NKT cell activators

PNAS 2005, 102, 1351
O OH HO HO OH O HN O OH

C26
OH C12H25 C19H39

OH HO HO

OH O O

O HN OH

C8

OH

D80 Y73 F70 W40

C12 F114

+
Glycolipid pulsed DC
Wild type mice NKT cell activation

Enhanced Bacterial clearance

Bacteria

V14i NKT-cell deficient CD1d -/- mice

Reduced bacterial clearance

(I)
C57 Bl/6 + S. capsulata
1500000 1250000 1000000 750000 500000 250000 0

Analogs of -GalCer as Novel Antibiotics: Model I

CFU / g liver

* *
*
C1 PBS C3 C9 C11 C14 C15

*
C16 C17

treatment (100ug/kg by IP)

Acknowledgement
Dr. Michael Best Dr. Ashraf Brik Marian Bryan Michael Burkart Dr. Aileen Chang Dr. Grace DeSantis Dr. Simon Ficht Sarah Hanson Dr. Sherry Hsu C. Y. Huang Dr. J.-C. Lee Junji Liu Dr. Lei Liu Dr. Masaaki Sawa Desiree Thayer Dr. Thomas Tolbert Yu-Ying Yang Dr. C.-Y. Wu Peter Schultz Glycoprotein synthesis In vivo Jim Paulson Ola Blixt Glycoarrays Ian A. Wilson Protein Structure

Financial Support NIH, Academia Sinica

Using Enzymatic Synthesis to Tackle the Problem in Glycobiology

Chi-Huey Wong The Scripps Research Institute Wong@Scripps.edu www.scripps.edu/chem/wong

Combinatorial Reactions in Microplates for Screening In Situ

+

Common Core

Microtiter Plate
Reaction

In Situ Assay

Lead compounds

Micro scale In water or nontoxic water compatable solvents

No protection No isolation High yield

Directed Evolution of 2-Keto-3-Deoxy-D-Phosphogluconate (KDPG) Aldolase

O H + O -O2C OH OPO3
2-

KDPG aldolase -O2C

OH OH
2OPO3

Phosphatase

HO HO

O CO 2 OH D-sugar

DNA Shuffling Error-Prone PCR 2-Keto-3-deoxy-L-galactonate aldolase T84A, I92F, T105I, V118A, E138V*, G141N O H + O OH OH -O2C O OH OH OH HO

No requirement for phosphate Retention of facial selectivity Inversion of enantioselectivity

OH O OH CO2 -

-O2C
Kcat = 2 sec-1 (No activity for the wild type)

L-sugar

Chem. & Biol. 2000, 7, 873

Directed evolution of L-rhamnulose-1-phosplate aldolase to alter the donor substrate specificity

O H2O3PO OH O

WT RhaD
H2O3PO OH

OH

L-rhamnulose-1P
OH OH

DHAP
O

WT RhaD
O H2O3PO OH OH OH OH OH OH

O H2O3PO OH

L-fructose-1P

DHAP

Directed evolution
O

O HO OH

Mutated RhaD
OH OH HO

OH

OH OH OH

L-fructose

DHA

Metabolic pathway of L-rhamnose in E. coli and strategy for the in vivo selection
L-rhamnose Selection host: E. coli BW25113 (DE3) rhaDAB

Rhamnose isomerase (RhaA) L-rhamnose

Rhamnulose kinase (RhaB) L-rhamnulose-1P Rhamnulose-1 P aldolase (RhaD) L-lactoaldehyde

L-rhamnulose

Mutated RhaD
L-lactoaldehyde

+ DHA

Glycolysis TCA cycle

+ DHAP

Co-expression of RhaA and randomly mutated RhaD Selection in the mimimal media with L-rhamnose as a sole carbon source

In vivo selection for the evolved RhaD

(1) Enrichment with MM-rhamnose liquid culture
1% seed 0.2% seed

(2) Selection on MM-rhamnose plate

WT
1% Rha 0.5% Rha 0.5% Rha

vector

Selected Library 1
5 4 OD600 3 2 1 0 0 50

Selected Library 2

SL1 SL2 Non-mutated

100 Time (h) 150

Mutant#10

Mutant#9

Mutation sites in the evolved RhaD

Pi DHA T158S

C145Y

Assembly of Glycoprotein: Ligation of Glycopeptide to an Expressed Protein Fragment

E.Coli. His6-ENLYFQ-Cys6-IL2(42 mg/L) TEV protease

6 H2N-CTKKTQLQLEHLLLDLAQMILNGINNYKNPKL TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS KNFHLRPRDLISNIEVIVLELKGSETTFMCEYADETATIVE FLNRWITFSQSIISTLT-COOHO OH HO OH O O O HO AcHN O HO
HO AcHN HO OH OH HO2C O HOOH HOOH O O O O OH AcNH O

H2NAPTSS-SR

Chemical or Enzymatic Ligation

Sialyltransferase

RNA Polymerase II Repeat Glycosylation-Induced Conformational Change from Random Coil to -Turn
Ser3
HO N

Pro7

NH H O H H H N H N O H O AcHN O HO OH H HO H O N O

Pro4

Ser6

Thr5--D-GlcNAc ~90o (Thr5:H-C-C-H)

OH

J. Am. Chem. Soc. 1998, 120, 11567.

Chemical and Enzymatic Approaches to Tackle the Problem of Glycoiology

Chi-Huey Wong The Scripps Research Institute wong@scripps.edu www.scripps.edu/chem/wong

Hexosaminidase Inhibitor for Osteoarthritis (Ki = 2.7 nM)

HO

H N NHAc OH

HO

NH2 N NHAc OH

HO

HO

100% 90% 80% 70% Hex. activity (%) 60% 50% 40% 30% 20% 10% 0% 1 10 100 1000 Log Conc. 10000 100000 1000000 C7 C0

ChemBioChem 2006, 7, 165-173

Cell-based assay

Ligation Rate
OH HO HO O HS O N NH H

OH

Ac-L-Y-R-A-AA2-SR HO HO 6 M Gn.HCl, 37oC, 2% thiophenol

O HS

O N NH H

H2N-AA1-N-P-G-Y-S Entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -AA1--Gly Ala Val Asp Gly Ala Val Asp Gly Gly Asp Asn His His -AA2Gly Gly Gly Gly Gly Ala Ala Ala Ala Val His His Gly Gly His

Ac-L-Y-R-A-AA2-AA1-N-P-G-Y-S Ligation Junction t1/2 (h) Obsd Mass Calcd(Da) -AA2-AA1Gly-Asp ~24 1373.5 ? 0.2 1373.6 Gly-Gly ~6 1430.6 ? 0.2 1430.6 Gly-Ala ~12 1444.5 ? 0.2 1444.6 Gly-Val ~20 1472.5 ? 0.2 1472.7 Gly-Asp ~6 1488.5 ? 0.2 1488.7 Ala-Gly ~20 1444.5 ? 0.2 1444.6 Ala-Ala ~36 1458.5 ? 0.2 1458.7 Ala-Val --1486.2 ? 0.2 1486.3 Ala-Asp ~18 1504.2 ? 0.2 1503.6 Val-Gly --1472.4 ? 0.2 1472.2 His-Gly ~6 1511.1 ? 0.2 1511.6 His-Asp ~6 1569.2 ? 0.2 1469.7 Gly-Asn ~18 1489.1 ? 0.2 1488.9 Gly-His ~3 1511.1 ? 0.2 1511.6 His-His ~4 1591.3 ? 0.2 1591.7

Synthesis of [N-(thioacetyl)--Glucosamine]-Peptide

Brik, A., Yang, Y.Y., Ficht, S., Wong, CH, J. Am. Chem. Soc. 2006, 128 5627.

D-Fructose-6-phosphate aldolase (MipB)

O HO OH O O OPO3H2 OH HO OH

F6P aldolase (MipB/TalC)

OPO3H2 OH OH

DHA

D-G3P

D-F6P

Class I aldolase (Schiff base) Sequence and structural homology with transaldolase (28% homology with TA, no homology with FDP Aldolase)

Dihydroxyacetone is the natural substrate

M. Schurmann et al., J. Biol. Chem. (2001) S. Thorell et al., J. Mol. Biol. (2002)

Synthesis of D-deoxynojirimycin and its derivatives

OH N3 O + O HO X

MipB
rt, 12h
N3

OH O X OH OH X=H, OH, CH3

MipB gave 1:1 mixture of diastereomers

Pseudomonas LPL-3 Lipase
OAc N3 OEt OEt O O

HCl
N3 OH O

HO

X N3 OH

OH

O X OH

Pd/C, H2
HO

H N

X OH

MipB

OH

HCl

HO N3 OH O

X N3

OH

O X

Pd/C, H2
HO

H N

X OH

Lipase-catalyzed resolution

MipB

OH

OH

OH

Reactivity-Based One-Pot Synthesis of Poly N-acetyllactosamine Octasaccharides

BnO HO LevO AcO AcO AcO OAc O O BnO OBn O STol OBn O O AcO OBn O STol BnO HO LevO OBn O O AcO OBn O BnO O LevO OBn O O AcO OBn O OR

NHTroc

NHTroc

NHTroc

(RRV = 256) 2h

(RRV = 0) 4h NIS/TfOH promoted glycosylation

NHTroc

(RRV = 1.3 * 104)

AcO AcO AcO OAc O O BnO OBn O BnO O LevO OBn O O AcO OBn O BnO O LevO OBn O O AcO OBn O BnO O LevO OBn O O AcO OBn O OR

NHTroc

NHTroc

NHTroc

NHTroc

R= -(CH2)5CO2Me

global deprotection

Poly N-acetyllactosamine Octasaccharide

Differential expression of cell surface carbohydrate markers to identify cancer cells and/or their staging

Ex.: mature cancer cells vs. cancer stem cells cancer cells vs. normal cells

Enzymatic Synthesis and Glycobiology

Chi-Huey Wong The Scripps Research Institute wong@scripps.edu

Use of borate as a phosphate ester mimic

O HO OH + H O RhaD R sodium borate OH pH 7.6 O HO OH OH R = OH (L-fructose), 92% H (L-rhamnulose), 53% OH R

O HO O HO O HO OH + HOVO32OH + HOAsO32OH + ATP

Kinase
-2

O O3PO O
-2

O HO OH

O HO OAsO32RCHO

OH
HOAsO32-

O3AsO O

OH
OH O R OH OH OH OAsO32-

-2

O3VO

OH

OH O R

Using arsenate or vanadate is limited by toxicity and modest yields.

Drueckhammer, D.G., et. al. J. Org. Chem. 1989.

Apparent irreversible formation of L-fructose

Activities of RhaD with Nonphospharylated Sugars in the Presence of 200 mM Borate
substrate L-rhamnulose 1-phosphate L-fructose 1-phosphate L-hamnulose + borate L-fructose + borate DL-lactaldehyde + DHAP DL-lactaldehyde +DHA + borate DL-glyceraldehyde +DHAP DL-glyceraldehyde +DHA + borate Vmax (mol/min/mg) 2.2 0.71 0.035 Not detected 33 1.0 23 0.48
OH O HO OH + H OH O OH RhaD X sodium borate pH 7.6 HO HO O O O B O O OH O OH OH L-fructose workup O HO OH OH OH OH

Km (mM) 0.96 3.2

L-iminocyclitol synthesis using RhaD/borate

DHA + aldehyde O H OH N3 1. RhaD, borate 2. Pd/C, H2, MeOH HO HO OH O H N3 HO HO O H N3 O H N3 NHAc HO HO OH HO HO H N OH H N OH H N OH iminocyclitol H N OH

L-deoxymannojirimycin

OH

NHAc

Kinetics parameters of wild type and evolved sialic acid aldolase

Enzyme wild type Substrate
D-Sialic acid L-Sialic acid D-KDO L-KDO D-Sialic acid L-Sialic acid D-KDO L-KDO D-Sialic acid L-Sialic acid D-KDO L-KDO D-Sialic acid L-Sialic acid D-KDO L-KDO

Km (mM) 2.60 4.43 24.5 13.3 2.07 N.D.a 12.3 14.1 3.90 369.4 14.9 9.5 2.6 N.D.a 116.1 5.0

kcat (min1) 0.81 0.01 0.18 0.15 0.81 N.D.a 0.29 0.16 0.75 0.27 0.39 0.26 0.28 N.D.a 0.38 0.45

kcat/Km (mM-1min-1) 0.311 0.002 0.007 0.012 (100 %) ( 1.0 %) ( 2.3 %) ( 3.7 %)

epNanA.1.1

0.389 (125 %) N.D.a 0.024 ( 7.6 %) 0.011 ( 3.7 %) 0.195 0.001 0.026 0.028 (61.6 %) ( 0.2 %) ( 8.4 %) ( 8.9 %)

epNanA.2.5

epNanA.3.B7

0.109 (35.1 %) N.D.a 0.003 ( 1.1 %) 0.089 (28.5 %)

no detectable activity was observed

Hsu et al. (2005) Proc. Natl. Acad. Sci. USA 102, 9122-9126

Relative rates of substrates for sialic acid aldolase variants

Acceptor substrate N-acetyl-D-mannosamine N-acetyl-L-mannosamine D-mannosamine D-mannose L-mannose D-arabinose L-arabinose D-gulose L-gulose D-talose D-glucose D-xylose 3-bromo-3-deoxy-D-mannose N-acetyl-D-glucoseamine 3-bromo-3-deoxy-D-glucose Wild type 100 0.2 80 80 30 20 10 10 70 60 40 45 5 N.D. N.D. N2C5 100 0.5 90 110 15 35 40 N.D. 35 50 40 40 5 N.D. N.D. N3A4 100 1 165 95 70 70 65 110 15 105 20 55 10 10 5 N4E6 100 2 190 95 80 40 40 90 15 80 25 25 10 10 N.D. N5B2 100 2 170 95 75 80 85 70 15 70 20 25 10 10 N.D.

Hsu et al. (2005) Proc. Natl. Acad. Sci. USA 102, 9122-9126

Sialic acid aldolase variants for the use of preparative synthesis

Hsu et al. (2005) Proc. Natl. Acad. Sci. USA 102, 9122-9126

Overcoming the glycine limitation in Staudinger ligation

Change the substituents on phosphorus
O S P X CH3CN/H2O N3 O X NH2 50 oC Ac H N O N H NH2 O R N O S P Ar Ar

Ac

H N

X Cl H CH3 OMe

yield 43% 50% 55% 61%

R'

Change the ring size in the rearrangement

Ac

H N

O S P + N3 O yield = 35% NH2 CH3CN/H2O 50 C

o

Ac

H N

O N H NH2 O R' R

S N P Ar Ar

Sugar-Assisted Glycopeptide Ligation

OH

O Ac-Leu-Tyr-Arg-Ala-Gly

O S NH2
HO HO O HS

O NH O

Ligation Condition: 6M Gn.HCl, pH 8.5 2% thiophenol, 37 C

H2N

N H

H N O

O NH2

0h

OH HO HO O HS Ac-leu-Tyr-Arg-Ala-Gly-Ser-Phe-NH2 O NH O

8h

Ashraf Brik, Yu-Ying Yang, Simon Ficht, and Chi-Huey Wong, JACS, 2006, 128, 5626

Ligation Study with Glycopeptides Containing -GlcNAc Asn /-GalNAc Ser

OH OH HO HO O HS H2N O N NH H O HO HO O HS Ac-L-Y-R-A-G-G-N-F-NH2 O N NH H O

Ac-L-Y-R-A-G-SR
O N H Phe O

2% Thiophenol 6 M Gn.HCl, 37oC, pH 8.5 70%

HO HO O HS

OH O NH O

HO HO O

OH O NH O

Ac-L-Y-R-A-G-SR 2% Thiophenol 6 M Gn.HCl, 37oC, pH 8.5

HS Ac-L-Y-R-A-G-G-S-F-NH2

O H2N N H Phe O

70%

Fluorescence-Based Screening

Umbelliferone Excitation: 335 nm Emission: 460 nm

Fluorescent Substrate Cleavage by DERA Mutants

L Substrate Cleavage by DERA Enzymes (1 mg/mL)
8000 7000 Relative Fluorescence Units 6000 5000 4000 3000 2000 1000 0 5 10 15 20 25 Time (minutes) No Enzyme Wild Type A203G A237H A203P A203G A237H A203G A237H S238F

Enzyme Wild Type

Substrate D-coumarinyl L-coumarinyl

Vrel 100 2 100 17

A203,A237H,S238F

D-coumarinyl L-coumarinyl

Probing cancer patient serum for antibodies against Globo H and truncated sequences by microarray

Synthesis of 2-deoxy-L-ribose

Enzyme Kinetics

Mutant Wild Type A203G A203G, A237H A203G, A237H, S238F

Km 40 77.302 6.3 78

D Kcat 0.17 0.206 0.0165 0.105

Kcat/Km 0.00425 0.002665 0.002619 0.001346

Km 431 137 160 456

L Kcat 0.0069 0.0041 0.0034 0.0074

Kcat/Km 1.6E-05 2.99E-05 2.13E-05 1.62E-05

L/D % 0.376689 1.123018 0.811364 1.205514

Photocleavable Glyco-Array and Characterization with DIOS-MS

NH2 NO2 OH O O O O N O NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2

1. Et3N; 2. DSC/Et3N + porous Si 2 3. N3-minor/Et3N

OH O OH HO HO O HO O OH HO OH HO HO O HO O OH HO O O O O AcHN

O OH O O HO HO O AcHN O O (CH2)5NH

O O

NO2

O OH HO OH OH O

O NH

DIOS-MS

OH

porous Si 3

DIOS-MS: Desorption/Ionization On Silicon Mass Spectrometry Angew Chem. Int. Ed. 2006

Antibody 2G12 Ligand Binding

45000 Relative Fluorescence Units 40000 35000 30000 25000 20000 15000 10000 Man 9 5000 0 105 200
HO HO HO HO HO HO HO HO HO OH O O O O O HO HO O

HO OH O HO HO HO OH HO O O HO O HO HO HO O HO O O OH HO HO O HO O O

HO HO HO HO HO HO

OH O O O O O O HO OH O O NH2 5

HO HO HO
NH2 5

HO HO HO

OH O O

Man 8
OH O O NH2 5

Glass slide microarray 6-10 fmol / spot

Ligands 105 - 200

PNAS 2004, 101, 17033

Directed evolution of D-sialic acid aldolase to L-KDO aldolase

Hsu, Che-Chang et al. (2005) Proc. Natl. Acad. Sci. USA 102, 9122-9126

Three-step synthesis of sialic acid via modified Petasis coupling

OH O HO OH L-arabinose OH + MeO EtOH/H2O OH OH NR OMe OH OH overall yield = 55% de > 99% TFA/Ac2O OBu H O 2 B OBu stable B OH unstable OH OH OH R H O HO B OH H NR OH OH NHR NH2 Step 1 OH OH NHAc

OH OH

OH OH

NHAc + N O

CO2Et

dioxane, 30 oC 90% Step 2

OH OH

NHAc CO2Et

OH OH OH HO AcHN HO OH O CO2H

OH OH O de% = 82% Step 3 NaOH

N R

H2O

R OH N

COOH R

-elimination

R O N

COOH R

OH L-Sialic acid 3 steps, overall yield ~ 30%

CFG: Define Paradigms by which Glycan Binding Proteins Mediate Cell Communication
Glycan Ligand Glycan Cell Receptor Surface Glycan Targeting / Endocytosis
Cell Adhesion

Glycan Binding Protein One Cell Modulate Receptor Signaling

Pathogen

Antibodies

Pathogen (HIV-DCSIGN) Glycoprotein

TRANS

CIS

CD22 (Siglec-2)

Selectins

Enzymatic Synthesis with Cofactor Regeneration

Glycosyltransferase
O O O S O P O OOR-OH

Sulfotransferase

R-OSO3-

A O

Pi +

Phosphoryltransfer CO2enzymes

OPO32CO2
-

PAPS

O OH O P OO

O OP O O-

A O

Aryl sulfotransferase

O OH O P OPAP O-

+
O2N OH O O S O O
-

O2N

X = Sugar Nucleotide

A = Adenine R = oligosaccharide or glycopeptide

J. Am. Chem. Soc. 1992, 114, 9283.

Angew. Chem. Int. Ed. Engl. 1999, 38, 2747.

L-iminocyclitol synthesis using RhaD/borate

DHA + aldehyde O H OH N3 1. RhaD, borate 2. Pd/C, H2, MeOH HO HO OH 1 O H N3 HO HO O H N3 O H N3 NHAc HO HO OH HO HO H N OH H N OH H N HO OH iminocyclitol H N OH

L-deoxymannojirimycin

OH HO 2:1 ratio H N OH

OH

3a

3b

NHAc

Synthesis of Deoxynojirimycin with D-fructose-6-P aldolase

2-O PO 3

O OH

O HO

D-fructose-6-P aldolase (FSA)

OH
2-

OH O OH OH OH

O3PO D-Fructose-6-P

D-Glyceraldehyde-3-P

DHA

N3 OH

O HO OH

FSA
N3

OH O OH OH OH

Pd/C, H2 HO

H N

OH OH

OH 1-Deoxy nojirimycin (DNJ) H N HO OH 1-Deoxy mannojirimycin (manno-DNJ)

N3 OH

O HO OH

FSA
N3

OH O OH OH OH

Pd/C, H2

OH OH

Sugar-Triggered Fluoroscence via Azide-Alkyne Ligation

Intensity

(nm)

ex 357 nm

Convergent Glycopeptide Synthesis via Staudinger Ligation and Enzymatic Coupling

O N3 AA AA O CF3

H2N

Expressed peptide

Glycan
O

Subtilisincatalyzed Condensation Traceless Staudinger Ligation

Glycopeptide
S

N3

Polypeptide

Glycoprotein

PPh2

(solid phase synthesis) Subtilisincatalyzed Condensation

Staudinger Ligation

Fmoc-Ser-Gly-Ala-Gly-Phe-Gly-Phe-Gln-Glu-Ala-Tyr-Arg-Arg-Phe-Tyr-Gly-Pro-Val-OH Gal(OAc)4

2-Deoxyribose-5-phosphate Aldolase Stereochemical course

A
DH R DERA O D2O DD HD O S DERA O D2O HD O

1.2

1.1

1.0

0.9

0.8

1.2

1.1

1.0

0.9

0.8

1.2

1.1

1.0

0.9

0.8

1.2

1.1

1.0

0.9

0.8

B
re B: A:H N Lys167 H HS CH3 HR A: HN Lys167 H CH3 O

B:H

OPO3 = OH H si

OH OPO3 CH3
=

Science. 2001, 294, 369

Acknowledgement
Dr. Michael Best Dr. Ashraf Brik Marian Bryan Michael Burkart Dr. F. Burkhart Dr. Aileen Chang Dr. Fabio Fazio Dr. Simon Ficht Dr. Masakazu Fujio Sarah Hanson C. Y. Huang Dr. J.-C. Lee Dr. Lei Liu Fu-Sen Liang Ian Ollman Dr. Masaaki Sawa Dr. S Sucheck Desiree Thayer Dr. Thomas Tolbert Doug Wu Yu-Ying Yang Zhiyuan Zhang Peter Schultz Glycoprotein synthesis In vivo Jim Paulson Ola Blixt Glycoarrays Ian A. Wilson Protein Structure Mitch Kronenberg Moriya Tsuji CD1-d/NKT

Financial Support NIH, NSF, Skaggs, Optimer

Inhibitors of Fucosyl Transferase V and VI

HO HO OH OH OGDP H3CO OH OGDP

OH N

FucT V FucT VI

Ki = 13.1 M Ki = 11.1 M HO

8.25 M 6.18 M O N

OH O F OGDP

HN

N OGDP

FucT V FucT VI

Ki = 4.0 M Ki = 10 M

0.27 M 4.6 x 10-2 M

Angew. Chem. Int. Ed. 2002, 41, 3041

Enzyme Inhibitor Discovery: Synthesis and screening in situ in Microtiterplates

Fucosidase

H3C

H N OH OH

OH

NH2 +

RCOOH

H3C

H N OH OH

OH

H N O

HBTU, DIEA

(60 various acids)

H3 C

H N OH OH

OH

H N O

NH

H3 C

H N OH OH

OH

H N O

Ki = 0.46 0.03 pM

Ki = 5.5 0.2 pM

(Angew. Chem. Int. 42, 4661(2003), Chem. & Biol. 11, 1301 (2004))

Hemagglutinin (HA) of influenza A virus forms a trimer complex. The green ribbon shows the HA1 of a monomer.
173-193 host surface

140-163

60-120

275-306

virus surface

The segment colored in blue was used to raise antibodies against HA. (Protein: Struct. Funct. Gen. 40, 572, 2000). The one colored in red is proposed by Dr. Trees Chung. The segments colored in pink are suggested for further study as well.

Programmable one-pot synthesis of Globo H

B nO BnO O B n BzO O HO O OBz O STol N H T ro c (R R V = 6 4 4 ) O Me O S Tol OBn HO Ph O O B n OO BnO Ph O O R 1 OO R 1O OR1 O R 1O R 1O O OR1 O O (C H 2 ) 5 N H R 4 OBn O BnO O BnO OBn O BnO O (C H 2 ) 5 N H C b z

O Bn BnO (R R V = 7 2 0 0 0 )

R 1O R 1O Me R 1O

O R 1 R 2O O O O OR1 O

OR2 O

O NHR3

OR1

OR1

R 1 = B n , R 2 = O B z , R 3 = T ro c , R 4 = C b z R1 = R2 = R4 = H, R3 = Ac

Protein Synthesis on the Prokaryotic Ribosome

Ribosome structure: H.F. Noller et al., Science 1999, 285, 2095 V. Ramakrishnan et al., Nature 1999, 400, 833 T.A. Steitz et al., Nature 1999, 400, 841

Enzymatic Modification of Aminoglycosides Resistance Development

R HO HO HO OH H2N NH2 O OH O (R = NH2) O H2N H2N O O OH HO OH O OH NH2 H2N HO R1 O R
2

H2N O HO O O NH2 NH2 OH

NH2 Neomycin B OH Paromomycin

Acetylation Adenylation Phosphorylation

OH Kanamycin A NH2 Kanamycin B NH2 Tobramycin

Chem. & Biol. 1999, 6, 99

Pharmacophores from Aminoglycosides

NH2 O H2N H2N HO O HO O HO O OH NH2 NH2 N3 O N3 O BnO O BnO O OBn NH2 O H2N O HO O NH2 NH2 N3 N3

HO

1) TfN3, ZnCl2, (CuSO4), 2.5 h

BnO

2) BnBr, NaH, DMF 88%

N3 BnO

CuCl2 CH3CN 52%

BnO

N3 O N3 BnOO HO N3 N3

HO

J. Am. Chem. Soc. 2002, 124, 10773

Semisynthetic Tobramycin
NH2 HO O H2N HO HO HN HO 2 OH O O O NH2 NH2

Tobramycin

NH2 HO O H2N HO H2N HO O OH O O NH2 NH2

Organism (Tobramycin Sensitive) P. aeruginosa (ATCC 27853) P. aeruginosa (ATCC 35151) P. aeruginosa (PAE_NUH18) P. aeruginosa (PAE_NUH19) P. aeruginosa (PAE_NUH20) P. aeruginosa (PAE_NUH21) P. aeruginosa (PAE_NUH22) P. aeruginosa (PAE_NUH23) P. aeruginosa (PAE_NUH26) P. aeruginosa (PAO-1) S. aeruginosa (MSSA) E. coli (ATCC 25922) E. faecalis (ATCC 29212)

Tobramycin 1 ND 0.5 0.5 2 1 0.25 >32 2 0.25 0.5 4 4

Analog 0.78 1 1 <0.5 <0.5 <0.5 1.56 1 <0.5 0.5 1 2 5

HO

Analog

Synthesis of Biotinylated 16S RNA and Analogs for Binding Studies

Semisynthetic Azithromycin
N HO HO O O O O OMe OH OH O HO O NMe2

Staph aureus

MIC (mg/mL) A B 1.1 2.3 4.0 2.8 <0.12 1.0 >64 >64 >64 32

Azithromycin (A)

O OH HO HO O O O O OMe O HO O

NMe2

MSSA MRSA MLS-S MLS-C St. pneumo

O OBn O H2N OBn OBn

Analog (B)

Common Untranslated Region of Hepatitis RNA (antiviral)

HCV
G A U C U G C G A A A A G A C G C A C C C A U G G C G U U A G U A U G A G U G

Oncogenic RNA (Tyr Kinase)

A. BCR2-ABL2 (BCR exon 2 - ABL exon 2) 5'-GGCUGACCAUCAAUAAGGAAG-AAGCCCUUCAGCGGCCAGUA B. PAX3-FKHR 5'-GGAUUUAAGCAGAGUUCAA-AAGCCCUUCAGCGGCCAGUAG
C A U A C A C G U A G U A G G A C

GBV-B
G A G G U G G G A A A A C G U C C C A A C C A C C U UA G U A U G U A G

HoCV
G A U C G G U A A A A G C C G G C C A U G C C A U A G U A G G A C

BVDV
G A U C G G A G A A A A C C A U G C C

BDV
A A U U G U G C C

C U U

5'

3'

5'

3'

5'

3' 3'

G C C G G A

G G A C U

A G U A

C A A G C C G G

Monoamine Oxidase (Depression, Parkinson's Disease)

Human MAO A 5'-ATAAGGAATGAGCATGTTGATTACGTAGATG Human MAO B 5'-CTTAGGAACCAAAAGGTTAAATATGTGGACC

5'

3'

5'

3' 3' A

5'

CUA U UCGC C G GA C A C AG GG CUCG T C D C G AGC U G G GG A G C G A G U A UA GC CG A U A U C G G

A C C A G C G C G U G C C G U A A U

5'

C C A A U A U G C G C G U

PSGL-1 Tyrosine Sulfotransferases (Antiinflammatory)

A B 5'-GCCAACCCACCTAACTACGGAAAACCTGATCCC... 5'-GCAAACCCCCCCAACTATGGCAACCCTGACCCC...

Bacterial Transglycosylase mRNA

E. coli S. aureus H. pylori 5'-GAA GAC AGC CGC TTC TAC GAG CAT 5'-GAA GAC AAT CGT TTC TAC GAA CAT 5'-GAA GAC ACC CTC TTT TTT GAA CAT

Human

Transfer RNA (antibiotics)

Interleukin 1
Loop 1 Loop 2 5'-GATATGGAGCAACAAGTGGTGTTCTCCATGTCCTTT 5'-CGCCAGTGAAATGATGGCTTATTACAGTGGCAA

E. coli tRNA GGC

Ala

HO HO H2N O HO O HO O OH NH2 H2N O OH O OH

NH2 O

NH2
NH2 NH2 H2N

O NH2 O O NHCH3 OH NH2 OH O HO

Kd vs RNA (M) Compd 1 3 4 32 16S-AS

2.1

EcT G
19 2.8 12 11 1.8 0.62 4.9 5.3 14 1

HCV2 b
2.7 2.9 4.3 12 3 0.42 1.8 9.4

HCV3 d
2.2 2.9

HIVFSS
0.64 1.6 2.7 4.7 1.2 3.4

HIVPAS
14 2 15 19 1.1

hBcrAbl
1.3 2 10 3.2 3.5

hTSulT
65

3
NH2 HO H2N O NH2 O HO R O NH2

H3 C

0.2
1.3 5.9 1.7 4.3 3 1.8 3.8 6.6 2.6 4.6 3.6 2.3 3.7

0.3
37 25 1.6 9.4 8.1 -52 14 9.6 13 9.4 3.9 5.1

0.26
6.8 0.7

33 34 35

0.25
2.7 -2.3 2.3 4 3.3

0.83
6.8 5.1 3 5 4 14 1.2 7 6

0.75
3.8 3.2

R=

0.32
1 0.7 4.6 1.7 2 7.6 1.6 4.5

HO HO H 2N

36
O HO O HO NH2 O H2N

HO H2N

NH2 O HO

37 38 39 40 41 42 43

0.28
12 2.6 3.8 3.5 3.1 9.5

0.7
5 4 8.5 6.4 3.5 14

34
HO O OH OH NH2 O HO

35
H2 N O HO HO HO O H 2N O HO NH2

0.42
8

0.67
2.5 1.7

0.53
2 1

HO O H 2N

37
NH2

39

HO

HO O NH2

O NH2

EcTG: E. coli. transglycosidase; HCV2b: HCV IRES domain IIb; HCV3d: HCV IRES domain IIId; HIV-FSS: HIV frameshift signal; HIV-PAS: HIV protease active site; Bcr-Abl: Human oncogenic Bcr-Abl mRNA; hTSulT: Human tyrosine sulfotransferase

OH OH

41

Liang, F. S. et al., Angew. Chem. Int. Ed. Engl. 2004, 43, 6496.

Surface Plasmon Resonance Studies: 2 Equiv. Neamine Bound

Semilogarithmic Plot
AS U1406A U1495A

Scatchard Plot

Equiv. Bound / [neamine]

0.2

Equiv. bound

Kd = 4M Kd = 10M

0.15

HO HO
0.1

NH2 O H2N H2N O HO OH

NH2

0.05

Neamine

0 1

Kd = 40M
10

0 0 0.5 1 1.5 2 2.5 3 3.5

[neamine] M
Semilogarithmic Plot Equiv. Bound / [neo B] M
3 4 3.5 3 2.5 2 1.5 1 0.5 0 0 0.5
AS U1406A U1495A

Equiv. Bound
Scatchard Plot
NH2 O NH2 OH

Equiv. bound

Kd = 0.67M

Kd = 0.12M
1

H2N H2N O O HO O OH NH 2 H2N O OH O OH

HO HO

0 0.1 1

Kd = 0.25M
10

Neomycin
1 1.5 2 2.5 3

[neomycin B] M

Equiv. Bound

Dimeric Aminoglycoside Antibiotics

J. Am. Chem. Soc. 2000, 122, 5230
A)
Biotin 5' G G

Mode of Action of Aminoglycosides C C

G

C
G
1406 U

RO O

C U 1495

OH n H N N O N H H N

OR O H2N H O P RO OR O H O OH n

C G A A C GA 1410 A U 1490 C G C G G U U C

OH H3N N N N O N

H H

B)

bacterial 16S RNA A-site

Linker

GC base Pair
H2N

phosphodiester
HO HO NH2 O NH2 HO

OH O O NH2 OH OH HO N

H2N O H2N

H2N OH O O

OPT-11, Kd = 40 nM Ki (APH 2") = 0.8 M

NH2

DNA Array Analysis for Specificity vs Whole Transcriptome

Method II
mRNA pool Reverse transcription

Method I
Mix & wash

In vitro transcription labeling

w/ or w/o small molecule

Hybridization on gene array

bound RNA

DNA Array Analysis

DNA Array Analysis w/ or w/o Adding Small Molecule

Probe Set ID Gene Title Genes

with decreased hybridization

Gene Symbol level when

5 M neomycin B added

1552988_at 1555141_a_ at 1556192_x_ at 203564_at 208701_at 211027_s_at 212214_at 219986_s_at 224004_at 227253_at 227679_at 229845_at 230171_at 232101_s_at 235520_at 235866_at 237953_at 238838_at 239595_at 239935_at 243327_at 244043_at

hypothetical protein MGC33948 CG10806-like Metastasis suppressor 1 Fanconi anemia, complementation group G Amyloid beta (A4) precursor-like protein 2 inhibitor of kappa light polypeptide gene enhancer in Bcells, kinase beta optic atrophy 1 (autosomal dominant) acyl-Coenzyme A dehydrogenase family, member 10 zinc finger protein 226 Ceruloplasmin (ferroxidase) Histone deacetylase 11 mitogen-activated protein kinase associated protein 1 Ubiquitin-conjugating enzyme E2D 3 (UBC4/5 homolog, yeast) phosphatidylinositol glycan, class N suppressor of hairy wing homolog 3 (Drosophila) chromosome 9 open reading frame 85 Dipeptidylpeptidase 4 (CD26, adenosine deaminase complexing protein 2) Transcription elongation regulator 1 Glutathione peroxidase 2 (gastrointestinal) MAM domain containing 1 Epidermal growth factor receptor Transcription factor Dp-2 (E2F dimerization partner 2)

MGC33948 LOC150159 MTSS1 FANCG APLP2 IKBKB OPA1 ACAD10 ZNF226 CP HDAC11 MAPKAP1 UBE2D3 PIGN SUHW3 C9orf85 DPP4 TCERG1 GPX2 MAMDC1 EGFR TFDP2

Human mRNA vs
Affymetrix Human Genome U133 Plus 2.0 Array (cover over 47,000 genes)

26 genes 172 genes

New method for analysis at the level individual probe is under developme Other RNA-binding small molecules will also be tested

1.05 Structure of WT DERA In 1.1 Structure of K201L DERA Carbinolamine Complex with DRP In Schiff Base Complex with DRP

Refined Residues 503. Refined Water Molecules 742. Refined Substrate Atoms 26. Resolution Range in Refinement 30.0-1.05 . r.m.s. bond length deviation 0.016 . r.m.s. bond angle deviation 2.2. Average B-value protein 17.6,19.2 2.

Refined Residues 503. Refined Water Molecules 557. Refined Substrate Atoms 24. Resolution Range in Refinement 8.0-1.10 . r.m.s. bond length deviation 0.014 . r.m.s. bond angle deviation 2.2. Average B-value protein 13.0,13.8 2.

2-Deoxy-ribose-5-phosphate-Aldolase
Lys 201 Lys 201 Lys 201

NH3

H O

NH2 H

H H

NH2 H2O

H H

Asp 102

Asp 102
CH3

HO
O O

Asp 102

HN

CH3

N
OH

CH3

H NH
O

Lys 167 Lys 167 Lys 201 Lys 201

Lys 167

NH3

H HH N

H OH Ser-238, 239 Gly-205 OPO3= H OH H2O Thr-170 Lys-172 Hydrophilic site H2O
O

NH3

H H

Asp 102

H O O

H O OPO3= OH

Asp 102

HN

CH2

Lys 167

Hydrophobic site

Lys 167

Lys 201
H H O OH OPO3= OH CH3 + OH O OPO32DERA H OH O OH OPO32-

Asp 102
O O

NH3

O O

NH2

Lys 167

Science. 2001, 294, 369

Stereochemistry of Pd-Mediated Reductive Amination

OH HO N (OH)n OH
H2 H2/Pd-C

OH
H N

(OH)n

Trans product
avoid tortional strain

OPO32HO N (OH)n OH
H2 H2/Pd-C

CH3 H N

(OH)n

OH R N (OH)n
H2/Pd-C R=OH, OPO32-

R OH
H N

Cis product
steric effect

(OH)n
H2 R=OH, H

O O
2-

E
OPO32OH

O
2-

OH OPO3 OH OH
2-

O3PO

OH

O3PO

E: FDP Aldolase

HO O O
22-

OH N3 OH OH

O3PO

1) Phosphatase OH 2) H2/ Pd OH

H N

O3PO

OH FDP Aldolase

OH HO O
2-

O H OH N3 O3PO OH OH OH N3 1) Phosphatase 2) H2/ Pd

H N OH HO OH

3 equiv

Pederson, King, Wong, Tet. Lett. 1988, 29, 4645 Ziegler, Straub, Effenberg, Angew. Chem. 1988, 100, 737