biology - chemistry frontier. part 1. roger adams award ... · biology - chemistry frontier. part...
TRANSCRIPT
Biology - Chemistry Frontier. Part 1.
Roger Adams Award Lecture
Samuel Danishefsky, Memorial Sloan-Kettering Cancer Center
Biology - Chemistry Frontier. Part 1.
Roger Adams Award Lecture
Samuel Danishefsky, Memorial Sloan-Kettering Cancer Center
ColleaguesMultivalent Antitumor VaccineJennifer AllenStacy KedingQian Wan
Erythropoietin / Peptide LigationGong ChenJiehao ChenZihao HuaCindy KanZhongping TanJohn TrzupekQian WanDavid WarrenBin WuYu Yuan
Grandisine ADavid Maloney
MigrastatinChristoph Gaul Jon NjardarsonLucy Perez
IsomigrastatinIsaac KraussMihir Mandal
EpothilonesAlexey RivkinYoung Shin ChoFumihiko Yoshimura
CycloproparadicicolXudong GengKana YamamotoZhi-Quang Yang
ColleaguesMultivalent Antitumor VaccineJennifer AllenStacy KedingQian Wan
Erythropoietin / Peptide LigationGong ChenJiehao ChenZihao HuaCindy KanZhongping TanJohn TrzupekQian WanDavid WarrenBin WuYu Yuan
Grandisine ADavid Maloney
MigrastatinChristoph Gaul Jon NjardarsonLucy Perez
IsomigrastatinIsaac KraussMihir Mandal
EpothilonesAlexey RivkinYoung Shin ChoFumihiko Yoshimura
CycloproparadicicolXudong GengKana YamamotoZhi-Quang Yang
Acknowledgement
I dedicate this Roger Adams Award Lecture toSarah Danishefsky for a lifetime of creative support,encouragement and leadership.
Acknowledgement
I dedicate this Roger Adams Award Lecture toSarah Danishefsky for a lifetime of creative support,encouragement and leadership.
ChemistryBiology
Intellectualization at the level of function
Mechanisms: Identification of all components required to attain and maintain biological function → reconstitution!
The power of bioreplicative synthesis
Intellectualization at the level of structure
Mechanisms: Pathways of chemical transformations usually involving breaking and making of covalent bonds → prediction of new reactions!
The power of unencumbered synthesis
Biology - ChemistryFrontier
ChemistryBiology
Intellectualization at the level of function
Mechanisms: Identification of all components required to attain and maintain biological function → reconstitution!
The power of bioreplicative synthesis
Intellectualization at the level of structure
Mechanisms: Pathways of chemical transformations usually involving breaking and making of covalent bonds → prediction of new reactions!
The power of unencumbered synthesis
Biology - ChemistryFrontier
On the Value of Natural Products in Pharma Discovery
The de novo discovery of a new molecular agent of value in medicine is a daunting task. The risks are virtually incalculable. The study of small molecule natural products (SMNPs) may allow for entry into the discovery progression at a more advanced stage than the screening of medicinal chemistry sample collections, let alone standard diversity libraries. If properly exploited, this advantage may well compensate for the difficulties of exploration, collection maintenance, isolation, proof of structure and more complicated follow up synthesis studies which are often cited against the natural products based discovery route.
Highly Successful Small Molecule Natural ProductDerived Blockbuster Drugs (Partial List)
(a) Antibiotics: β-lactam, aminoglycosides, macrolides
(b) Statins: Zocor, Pravastatin, Lipitor
(c) Steroids: Population control, antiinflammatories,cardiotonics, antiestrogens, antiandrogens, dermatological applications, etc.
(d) Alkaloids: Antidepressants, antitumor agents (Vinca, thecins)
(e) Terpenoids: Taxol
(f) Polyketies: Anthracyclines
Natural Products as Lead Candidates:The Statin Class of Antilipidemic Drugs
OH
OO
OHO
Lovastatin (Mevacor®)Natural Product
(Isolated from A. terreus)
OH
OO
OHO
Simvastatin (Zocor®)Natural Product-Derived
OHCO2H
HO
Atorvastatin (Lipitor®)Natural Product-Inspired
NF
NHPh
O
Why Have Natural Products Been a Rich Source of Drug Discovery?(a) Wisdom of the ages: While the developmental justifications
for the biosynthesis of secondary metabolites are often mysterious, in many instances there could well be some overall rationale which can be exploited.
(b) The small molecule natural product has possibly been designed for its ability to bind to biomacromolecules.
(c) Presumably the biosynthesis of the natural product was under enzymatic control. At its “launching,” the natural product had been contained and extruded from some protein pocket.
(d) There is often a built in advantage in that the natural product has already been maintained in some living host, without unmanageable toxicity.
Why Chemical Synthesis of Complex Molecules?
1. The sheer challenge!
2. A setting for the evaluation of the scope of current methodology.
3. A setting for the evaluation of “problem areas”.
4. Incitement to strategy level and new methodologycentered creativity.
5. A medium for the discovery and development of new drug possibilities through diverted total synthesis.
6. To provide material for pre-clinical and clinical investigation.
7. Molecular editing and diverted total synthesis.
Diverted Total Synthesis
Allow for the exploration of chemical space not available directly from the natural product.Through the process of Diverted Total Synthesis, it is possible to manipulate chemical functionality that can not be altered through manipulation of the natural product itself.
Starting Materials
ChemicalSynthesis
AdvancedIntermediate
Total Synthesis Natural Product Biosynthesis
Analogs
AnalogsDTS
reducecomplexity
addcomplexity
DTS = DIVERTED TOTAL SYNTHESIS
DTS
Summary of the Epothilone Program: Molecular Editing through Total Synthesis
O O
OOH
Me
OHH
S
NO O
OOH
Me
OHH
S
N
O O
OOH
F3C
OHH
S
N
Epothilone B (EpoB) dEpoB (KOS-862)Phase II
9,10-dehydro-dEpoB (KOS-1584)Phase I
Fludelone (KOS-1591)Preclinical
Remove Epoxide
Decrease toxicity
Install Unsaturation
Improve Potencyand Biological Stability
Install Trifluoro Group
Decrease Toxicity andBroaden Therapeutic Index
S
NO O
OHO
Me
OHO
O O
OOH
F3C
OHH
ON
Iso-Fludelone (KOS-1803)Preclinical
Modify Heterocyclic Sector
Increase Efficacy andStability
Iso-Fludelone: Therapy of Extra Large Tumors
Day 25 Day 39
Day 53Therapeutic effect of Iso-fludelone against
extra-large MX-1 tumors
(30 mg/kg, Q12Dx4,6hr-infusion, N=4)
The Radicicol ProgramRadicicol and Cycloproparadicicol
Mode of Action: Potent inhibition of Hsp90.The first total synthesis of radicicol was completed in our laboratory in 2001. As reported, radicicol was confirmed to be a potent inhibitor of the molecular chaperone, Hsp90.
Radicicol was largely inactive in mouse xenograft studies. We suspected that the epoxide moiety was responsible for the failure to translate protein inhibition to in vivo efficacy.
Cycloproparadicicol was synthesized in our laboratory and shown to be an effective inhibitor of Hsp90. Importantly, this fully synthetic analog is active in in vivo settings.
HO
OH
O O
OCl
H
H
Cycloproparadicicol
HO
OH
O OO
OCl
H
H
RadicicolIsolated from M. nordinii
Total Synthesis of Cycloproparadicicol
HO
Me
+
H
H1) n-BuLi; CO2
47% (2 steps)Me
OTBS
2) PPh3, DIADMe
OTBS
O O
Me
H
H Co2(CO)8
100%
Ring-ClosingMetathesis
(CO)3Co
OO
Me
OTBS
MeMe
OTMS
TMSO
140єC, then silica gel
Me
Me
H
HO
H
MeMe
OH
OTBS(CO)3Co
1) Grubbs' 2nd
gen. cat.57%
2) I2, 69%
Diels-AlderCycloaddition
75%
OO
OTBS
HO
OH
Me
H
H
OO
O
HO
ClOH
Cycloproparadicicol
Me
H
H
The Radicicol ProgramRadicicol and Cycloproparadicicol
Total Synthesis of the Tumor Cell Migration Inhibitor Migrastatin
O
OMeOH
O
O NH
O
O
Migrastatin
OMe
O
OMe
OTMS
O
OOMe
H2O
HO
OMeOTBS
O
OMeOH
O
O NH
O
O
Diverted Total Synthesis: Migrastatin
Wound Healing Assay
No Serum Serum
Serum + 200 nM Core Serum + 200 nM Migrastatin
OMeOH
O
O
O NH
O
O
MigrastatinOMeOH
O
O
Migrastatin Core
Diverted Total Synthesis: Migrastatin
Inhibition of Metastasis in Mouse Breast Tumor (4T1) Model
OMeOH
O
NH
Lactam
OMeOH
O
Ketone
Tumor size on day 21
0
2
4
6
8
10
12
14
16
contr
olKeto
ne(10
mg/kg)
Ketone
(20mg/k
g)La
ctam(10
mg/kg)
Lacta
m(20mg/k
g)
Dia
met
er o
f tum
or (m
m)
Clonogenecity Assay
-100
0
100
200
300
400
500
600
700
800
900co
ntrol
Ketone
(10mg/k
g)Keto
ne(20
mg/kg)
Lacta
m(10mg/k
g)La
ctam(20
mg/kg)
Num
ber o
f col
onie
s
Anti-Metastatic Activity of Migrastatin Analogs (MDA-MB-231 cells)
2,3-dihydro-migrastatin ether (ME)
Collaboration with Moore Lab at MSKCC
control group ME (pre)
control group ME (post) ME (pre+post)
Migrastatin
Serum
Serum + 10µM ME
No Serum
MeOH
OMe
MeO
HN
O
O
3Me
OOMe OH
OMe
Me
O
Post-surgery:
Pre-surgery:
Migrastatin - Isomigrastatin Family
Isolated from S. Platensis
O
Me OH
OMe
Me
O NH
O
O
O 3
Me OH
OMe
MeO
HN
O
O
3
OO
Isomigrastatin Migrastatin
IsolationIsomigrastatin:Woo, E. J.; Starks, C. M.; Carney, J. R.; Arslanian, R.; Cadapan, L.;Zavala, S.; Licari, P. J. Antibiot. 2002, 55, 1411.
Migrastatin:(a) Nakae, K.; Yoshimoto, Y.; Sawa, T.; Homma, Y.; Hamada, M.;Takeuchi, T.; Imoto, M. J. Antibiot. 2000, 53, 1130. (b) Nakae, K.;Yoshimoto, Y.; Ueda, M.; Sawa, T.; Takahashi, Y.; Naganawa, H.;Takeuchi, T.; Imoto, M. J. Antibiot. 2000, 53, 1228.
Instability of Isomigrastatin
Isomigrastatin
SN2’
addition-elimination
water-assisted 3,3-rearrangement
O
Me
HOOMe
O
Me
HOOMe
O
O
H2O
H2O
Me
O
R
NH
O
O
R =
O
Me
HOOMe
HO
Me
O
R
OH
O
Me
HOOMe
HO
Me
O
R
OH
MeR
O
H2O Dorrigocin Aepi-Dorrigocin A
Dorrigocin B
Migrastatin
Ju, J.; Shen, B. et. al. JACS 2005, 1622
Unsuccessful Early Strategies to Isomigrastatin
OO
OMe
MeOP
R
O
O
OP
Me
OMe
R
OO
OMe
MeOP
R
OO
OMe
MeOP
R
(R)(R)
trans-trans-dienolide
2,3-cis-6,7-trans-dienolide
Total Synthesis of Isomigrastatin – Fragment Union
O
MeOMeO
O
OH
MeOMe
MOMO
O
MeOMe
O
MeOTMS
OMe
OMe
O+
NH
O
O
O Ph3P PPh3
O
NH
O
O
2) H2, Pd/C
1)
OPh3P
A
B
A + B
OMe
OR
OMe OMOM
OMe
OR
OTBSMe OMOM
OMe
HO R
OTBSMe OMOM
MeLiMeCuCN
= R
N BO
PhPhH
Me
BH3
Krauss, I. J., Mandal, M. , Danishefsky, S. J. Angew. Chem. Int. Ed. Engl. 2007, ASAP
Delayed Implementation of Dienolide Core – Completion of Synthesis
(+)-Isomigrastatin21 % trans36 % cis(separable)
>8:1 d.r. at C2
OMe
HO
TBSOMe OMOM
Me
OMe
O R
OTBSMe OMOM
Me= R O
PhSe
OHOPhSe(±)
EDCI/DMAPkineticr esolution
xs.
OMe
O R
OMe OH
MeO
PhSe
OMe
O R
OMe OH
MeO
PhSe
Grubbs IIOMe
O R
OMe OH
MeO
either C2epimer
mCPBA
NH
O
O3
O
O
OH
Me
OMe
R
O
Me
cat. PMe3
Krauss, I. J., Mandal, M. , Danishefsky, S. J. Angew. Chem. Int. Ed. Engl. 2007, ASAP
• displays affinity for the human δ-opioid receptor (2.7 µM).
• µ and κ agonist have been associated with adverse sideeffects when administered.
• in animal models, δ-opioid agonists have been well tolerated.
Total Synthesis of Grandisine A (background)
Grandisine A
rel. energyGrandisine A 0 kcal/mol
8-epi ~-6 kcal/mol9-epi ~-4 kcal/mol
J. Org. Chem., 2005, 70, 1889
O
N
H H
H
H
O
O
H
9
8
γ-pyridone
Total Synthesis of the Grandisine A
NCbz NCbz
OTIPS
NCbz
O
O
H
H
H
HO
N
H H
HO
O
H
H
OH3C
NCbz
OTIPS
OH
H
H
+
BF3OEt2-78 oC
TBAF
AcOH/THF
O
+NCbz
OTIPS
OH
H
H~5-10% from opposite face
(+)-Grandisine A
H HNCbz
O
NCbz
O
OH
H
Nuc..
E+
endo
ax. eq.
H
O OSi(Et)3
HO
O
O
NCbz
H H
H
H
LHMDS
OTES
ax.
unpublished results
Carbohydrate-Based Antitumor Vaccines: Rationale
• Cell surface (tumor) antigens are carbohydrate structures commonly expressed in glycoproteins, mucins, and glycosphingolipids.
• Antibodies that recognize these glycoconjugates can, on occasion, be found in human sera. It has been postulated that these antibodies are part of an immune response to the tumor state.
• Antibody formation is provoked by suitable glycoconjugates and not by the oligosaccharides alone. Having been elicited in this way, most antibodies are primarily sensitive to the structure of the carbohydrate domain.
• Vaccination with carbohydrate vaccines may provide immunologicalprotection against micrometastases and circulating tumor cells.
• The possibility of an immune response to cancer could have an enormous impact on the in vivo diagnosis and therapy of cancer.
Oligosaccharide Synthesis: How We Got Started
O
HO
HO
HO
Glycal Assembly Approach
O
R2
OMeR1
R3SiO R3
O
R2
OMeR1
R3SiO R3
O
R2
R1
HO R3
Lewis Acid
O
HO
HO
HO
OH
OH
Classic Approach
Oligosaccharide Synthesis: The Glycal Assembly Method
O
O
O
OO
O XR
OH
O O
OH
O
OO O
OH
OOO I+ I+
PhSO2NH2 O
NHSO2Ph
I
O
NHSO2Ph
SEt
O
NSO2PhO
NHSO2Ph
OO
I+
OI
O
RO
RO
O
O
RO
RO
HO
O
RO
RO
O
O
RO
RO
O
OH
O
RO
RO
O
O
RO
RO
O
OHO
RO
RO
O
OH
O
Oligosaccharide
OO
OI
OOI
OO
OHO
HOO
OP
P
P
O
RO
RO
RO
O
RO
RO
RO
E+
glycal
O
RO
RO
RO
E
OR'
O
RO
RO
RO
E
X
E+
in situ generatedglycosyl donor
glycosyl donor
Carbohydrate Cancer Vaccine Program
Angew. Chem. Int. Ed. 1996, 35, 1380
Breast Cancer-Associated Antigen: MBr1 Antigen
OOTIPSO
OO
OTIPSAcO
O
O
OBnOBn
OBn
O
O O
O OBn
BnOOBn
O
OBnOBn
HOBnO
OOBn
BnO
OOHHO
HO O
O
HOOH
OH
O
OOHHO
AcNH
OOHHO
OHO
O O
OH
HOOH
O O
OH
HO OHO
HN
OH
OC25H51
C13H27
Globo-H
O
NHSO2Ph
SEt
OO
Clinical Trials with Carbohydrate-Based Antitumor Vaccines
OO
O
O
O OH
HOOH
HO
HO OH HO OH
NHAcO
OOH
HO
HO
OO
O O
OH OH
OHOHOOH
HOLinker KLH
Globo-HSCLC (Phase I)
Prostate (Phase I)Breast (Phase I)
HOO
HO OH
AcHN
Linker KLHAcHN
HN
NHO
O
O
O
O
O
HOO
HO OH
AcHN
HOO
HO OH
AcHN
Tn(c)Prostate (Phase I)
OO
O
OHHO
HO OH HO OH
AcHN
Linker KLHAcHN
HN
NHO
O
O
O
O
O
OO
O
OHHO
HO OH HO OH
AcHN
OO
OHO OHOHHO
HOOH AcHN
TF(c)Prostate (Phase I)
OO
O
O OH
HOOH
HO
HO OHO
OO
O
O
OHOH
OH
OH OH
NHAc OHO
HO
Linker KLH
LewisY
Ovarian (Phase I)
OO O
O
O
OO
O
O
HO2CO O
OHHO
HO
Me
OH OHOH
OH OH
AcHN
OH
NHAc
HO
HOHO
OH
OH
HOOH
O
OH
Linker KLH
Fucosyl-GM1SCLC (Phase I)
Flexible Accesses to Glycosyl-amino Acids
Protectedcarbohydrate O
X O X
X = CH2 X = Glycosyl DonorX = O
CO2Bn
NHFmocPO
CO2R
NHBoc
MeOMeO
O
Horner-Emmons
CO2R
NHBocO CO2Bn
NHFmocO CO2Bn
NHFmoc
HO CO2Bn
NHFmocGlycosylation
(L.A.)
H2/Pt-CH2/Pt-CH2(S, S)-Et-DuPHOS-Rh
pentenyl glycosides allyl and pentenyl glycosides glycosyl donnors
n
n = 1 or 3
CrossMetathesis
n
O CO2Bn
NHFmocn
Protectedcarbohydrate
Protectedcarbohydrate
Protectedcarbohydrate
Protectedcarbohydrate
Protectedcarbohydrate
Protectedcarbohydrate
Unimolecular Multivalent Vaccine for Prostate Cancer:A Construct Containing Five Different Antigens
OOAcHN
HOO
OHHO
HOOH
OH
OHO
HOAcHN
O
OAcNH OH
CO2HHO
HO OH
OO
OO
OOH OH OHOHOHOH
HONAcH
HO
O OH
HO
Me
OH
O
OOO
HO
OHHO
O
OH OHHO
HAcNHN
NH
HN
NHO
O
O
O
O
OHO
OHHO
AcHNOO
O
O
HN
O
Tn
STn
Globo-H
TF
Linker KLH
OOO
OH
OH OHHO
OH
O
OH
NHAc
HO
HO2C
HOHO
HOO
O
HO OH
AcHNO
GM2
A fully synthetic, unimolecular pentavalent vaccine incorporating five of the known antigens associated with breast cancer has been synthesized and evaluated in preclinical settings. This compound is scheduled to enter Phase I clinical trials at MSKCC in fall / winter 2007.
Erythropoietin (EPO): A Multiply Glycosylated Protein
24
38
83
126
Sialic acidGalactoseMannose
FucoseN-Acetylglucosamine
161
7
29
33
Cysteinepair
Cysteinepair
166-Residue glycoproteinN-Glycosylated at Asn24, Asn38, and Asn83
O-Glycosylated at Ser126
Two cysteine pairs: Cys7-161 and Cys29-33
Heterogeneous glycoprotein
Hematopoietic growth factorTreatment of anemiaVarious glycoforms have different biological properties
Erythropoietin (EPO): A Multiply Glycosylated Protein
EPO is a heterogeneous glycoprotein that may exist as a number of glycoforms. The amino acid sequence is highly conserved among isoforms.Current manufacturing processes yield inseparable mixtures of glycoforms, which can lead to complications at the regulatory level.
Could we use total synthesis to gain access to single glycoforms of erythropoietin?
We elected to synthesize the EPO isoform containing the consensus amino acid sequence and displaying highly branched and sialidated carbohydrate sectors. Incorporation of these types of carbohydrates is known to convey biostability. We will evaluate whether our fully synthetic erythropoietin is able to fold properly and whether it possesses EPO-like biological activity.The successful completion of this undertaking would require the development of some new synthetic methodologies to allow for the preparation and merging of very large and complex glycopeptide fragments.
Toward Erythropoietin (EPO): The Development of Novel Methods for Glycopeptide Ligation
I. A Cysteine-Based Glycopeptide Ligation
XO
Glycan
Peptide1
Glycan
Peptide2
Glycan
Peptide2NH
OGlycan
Peptide1H2NO
R
O
R
Glycopeptide Ligation
The Challenge
OGlycan
Peptide1 O
SSEt Glycan
Peptide2H2NO
SStBu
OGlycan
Peptide1 O
HS
Glycan
Peptide2H2NO
HS
OGlycan
Peptide1 S
HO
OGlycan
Peptide1Glycan
Peptide2H2NO
S
Glycan
Peptide2NH
OGlycan
Peptide1O
HS
Reduce S-S
Toward Erythropoietin (EPO): The Development of Novel Methods for Glycopeptide Ligation
Dr. Gong Chen
II. A Cysteine-Free Ligation Model
Glycan
Peptide2H2NO
ROS
OO
Glycan
Peptide1
PGlycan
Peptide2HNO
R
SO
OGlycan
Peptide1
P
Glycan
Peptide2HNO
R
SHO
OGlycan
Peptide1
Glycan
Peptide2NO
R
SHHO
OGlycan
Peptide1
S O
Glycan
Peptide2NH O
ROGlycan
Peptide1
Removing P
S N
Glycan
Peptide2HNO
R
SHO
OGlycan
Peptide1
Toward Erythropoietin (EPO): The Development of Novel Methods for Glycopeptide Ligation
III. A Cysteine-Free Glycopeptide Ligation
OGlycan
Peptide1 O
SSEt
Glycan
Peptide2H2NO
R
MeOOMe
OMe
OSPGlycan
Peptide2HNO
R
MeOOMe
OMe
SR'S
OGlycan
Peptide1
Glycan
Peptide2HNO
R
MeOOMe
OMe
S
Glycan
Peptide2NH
OGlycan
Peptide1O
R
OGlycan
Peptide1
Glycan
Peptide2NO
R
MeOOMe
OMe
SH
Philip Dawson
Toward Erythropoietin (EPO): The Development of Novel Methods for Glycopeptide Ligation
IV. A Cysteine-Free Glycopeptide Condensation
OGlycan
Peptide1 O
SSEt Glycan
Peptide2H2NO
R
Glycan
Peptide2NH
OGlycan
Peptide1O
R
Gly / Pro
Lys and Cys Protected
AgClHOOBt, DIEA
DMSO, RT
OGlycan
Peptide1 O
SSEt Glycan
Peptide2H2NO
R
Glycan
Peptide2NH
OGlycan
Peptide1O
R
Gly / Pro
Lys and Cys Protected
TCEPHOOBt, DIEA
DMSO, RT
S OO O
S OO O
Saburo Aimoto
Toward Erythropoietin (EPO): The Development of Novel Methods for Glycopeptide Ligation
V. Free Radical Desulfurization: Cys to Ala
Glycan
Peptide2NH
OGlycan
Peptide1O
SH Glycan
Peptide2NH
OGlycan
Peptide1O
TCEP, tBuSH, VA-50H2O, RT
N NHN
H2N NH2
NHHCl
VA-50
P(CH2CH2COOH)3TCEP
S-H SIn. .
PR
RR CH2
S PR
RR
.CH3
H-S
FmocN
SSMe
S O
O O
SAcm
PeptidePeptide Peptide
S PR
RR
.
Peptide Peptide
tolerated
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
O
BnOOBn
OBnF
OOO
PMBOPh OBn
SO Ph
HOOTIPSO
BnOSEtO
BnOAcO
BnOPhSO2NH
OO
HOO
BnOAcO
BnOPhSO2NH BnO
O OO
PhSO2NH
O
BnOOBn
OBn
OBnOHO
BnOPhSO2NH
BnO
OTBS
O OO
PhSO2NH
O
BnOOBn
OBn
OBnO
OBnO
PhSO2NHBnO
OBnOHO
OBn
OTBS
HO
OBzOBnO
BnOBnO
SEt2 xOH
OBnOBnO
BnO
OBnO
BnOBnO
OH
O OO
PhSO2NH
O
BnOOBn
OBn
OBnO
OBnO
PhSO2NHBnO
OBnOO
OBn
OTBS
O
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
O OBnO
OBn
SEtNPht
OOBnHO
HOBnO
OAcHNAcO
AcO OAcOAc
CO2Me
OP(OBn)2
OAcHNAcO
AcO OAcOAc
CO2Me
O O OAcO OBn
OOBn
BnO
OBn
NPht
SEt
HO OBnO
OBn
SEtNPht
OAcO OAc
AcOPivOO
NH
CCl3
OOBnO
OO
HO OOBn
BnO
O O TMSESO2NH2EtSH
O OBnO
OBn
SEtNPht
OOBnHO
HOBnO
O OOBn
BnOO
OBnO
OBnO
O
+
+ O OBnO
OBn
SEtNPht
OAcO OAc
AcOPivO
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
OAcHNAcO
AcO OAcOAc
CO2Me
O O OAcO OBn
OOBn
BnO
OBn
PhtN
SEt
2 x
OHOBnO
BnOBnO
OBnO
BnOBnO
OH
O OO
PhSO2NH
O
BnOOBn
OBn
OBnO
OBnO
PhSO2NHBnO
OBnOO
OBn
OTBS
O
OAcHNAcO
AcO OAcOAc
CO2Me
O O OAcO OBn
OOBn
BnO
BnO
PhtN
O
OBnOBnO
BnO
OBnO
BnOBnO
O OO
PhSO2NH
O
BnOOBn
OBn
OBnO
OBnO
PhSO2NHBnO
OBnOO
OBn
OTBS
O
OAcHNAcO
AcO OAcOAc
CO2Me
O O OAcO OBn
OOBn
BnO
OBn
PhtN
O
+
1. Global Deprotection2. Kochetkov Amination
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
O OO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHNH2
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHN
O
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHNO
OHOHOHO
OHOOHO
O OHO
AcHN
OHOOHO
AcHNHO
OHOO
OHNH2
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHN
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHN
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
OO
O
OTIPSO
O
O
OTIPS
N3
O CCl3
NH
OO
O
OH
N3ONHFmoc
COOBnO
OP(OBn)2
AcOAcHN
AcOOAc
OAc
COOMe
OOAc
O
O
COOMe
AcOAcHN
AcOOAc
OAc
O
N3
FmocHNOBn
O
HO
OOH OTIPS
HO
O
AcOAcHN
AcOOAc
OAcO
O
OO OTIPS
O
AcOAcHN
AcOOAc
OAc
O
O
OO
OAc
OAc
SEt
O
OP(OBn)2
AcOAcHN
AcOOAc
OAc
COOMe
OOAc
O
O
COOMe
AcOAcHN
AcOOAc
OAc
O
NHAc
FmocHNOBn
O
OO
AcOAcHN
AcOOAc
OAc
O
O
OO
OAc
OAc
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
O OO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHNH2
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHNO
Ala Glu Asp Ile Thr Thr Gly
O
OH
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
O OO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHHN
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHNO
+O
O
SSEt
Ala Glu Asp Ile Thr Thr Gly O
O
SSEt
Cys Ala Glu His Cys Ser Leu Asn Glu
HS
H2N
Dmab
Dmab
NCL
Aspartylation
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
O OO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHHN
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHNO
Ala Glu Asp Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu
EPO 22-37
Dmab
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
OOO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHNH2
O
OAcHNHO
HO OHOH
CO2H
O O O
OH OH
OOH
HO
OH
AcHNO
+
AspartylationThrLys Glu Ala Glu Asp Ile GlyThr
Allyl Allyl
ivDde
ProAla Pro Arg Leu Ile Cys Asp Ser AlaLeuValArg Glu Arg Tyr Leu Glu O
O
SSEt
S
O O
O
O
OH
FmocHN
Removing Fmoc
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
OOO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHHN
O
OAcHNHO
HO OHOH
CO2H
O O O
OH OH
OOH
HO
OH
AcHNO
ThrLys Glu Ala Glu Asp Ile GlyThr
Allyl Allyl
ivDde
S
O O
O
O
H2N TCEPFragment
Condensation
Acm
+
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
ProAla Pro Arg Leu Ile Cys Asp Ser AlaLeuValArg Glu Arg Tyr Leu Glu
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
O OO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHHN
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHNO
ThrLys Glu Ala Glu Asp Ile GlyThr
Allyl Allyl
ivDde
S
O O
O
O
Acm
?
EPO 1-28
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
Ala
Ser
Ala Ala Asp Pro Pro Ser Ile Ala NHFmoc
OO
SSEt
Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg
Lys leu Lys Gly Arg Leu
HN
Asn
Phe
O
OAc
O
OCOOMe
AcOAcHN
AcOOAc
OAc
O
AcNHO
AcOAcHN
AcOOAc
OAc
O
O
OO
OAc
OAc
O Ala Glu Lys Gln
Val Tyr Ser
Cys Ala Glu Gly Thr Tyr LeuArg Asp Gly Thr Arg
SH
MeOOMe
OMe
ivDdeivDde
ivDde
ivDde
Acm
+
AuxiliaryLigation
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
Ala
Ser
Ala Ala Asp Pro Pro Ser Ile Ala NHFmoc
Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg
Lys leu Lys Gly Arg Leu
Asn
Phe
O
OAc
O
OCOOMe
AcOAcHN
AcOOAc
OAc
O
AcNHO
AcOAcHN
AcOOAc
OAc
O
O
OO
OAc
OAc
O Ala Glu Lys Gln
Val Tyr Ser
Cys Ala Glu Gly Thr Tyr LeuArg Asp Gly Thr Arg
ivDdeivDde
ivDdeivDde
Acm
EPO 114-166
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
Ala
Ser
Ala Ala Asp Pro Pro Ser Ile Ala NHFmoc
O
OSSEt
Ala Pro
Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg
Lys leu Lys Gly Arg Leu
Asn
Phe
O
OAc
O
OCOOMe
AcOAcHN
AcOOAc
OAc
O
AcNHO
AcOAcHN
AcOOAc
OAc
O
O
OO
OAc
OAc
O Ala Glu Lys Gln
Val Tyr Ser
Cys Ala Glu Gly Thr Tyr LeuArg Asp Gly Thr Arg
ivDdeivDde
ivDde
ivDde
Acm
TCEPFragment
Condensation
H2N--
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
Ala
Ser
Ala Ala Asp Pro Pro Ser Ile Ala NHFmoc
Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg
Lys leu Lys Gly Arg Leu
Asn
Phe
O
OAc
O
OCOOMe
AcOAcHN
AcOOAc
OAc
O
AcNHO
AcOAcHN
AcOOAc
OAc
O
O
OO
OAc
OAc
O Ala Glu Lys Gln
Val Tyr Ser
Cys Ala Glu Gly Thr Tyr LeuArg Asp Gly Thr Arg
ivDdeivDde
ivDdeivDde
Acm
EPO 114-166
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
O OO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHNH2
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHNO
Pro
Gln
Ser Ser Asp Val Leu Leu Ala Gln NHFmoc
O
O
OH
O
SSEt
Pro
Gln
Ser Ser Asp Val Leu Leu Ala Gln NHFmoc
O
O
O
SSEt
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
O OO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHHN
O
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHNO
+
Aspartylation
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
O OO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHO
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHN
O
Pro
Gln
Ser Ser Asp Val Leu Leu Ala Gln NHFmoc
O
OHN
O
SSEt
Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg
Gly Leu Ala Arg Leu Leu Thr
H2N
Ser
Thr
+
TCEPFragment
Condensation
SO
OO
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
O OO
AcHN
O
HOOH
OH
OHOOHO
AcHNHO
OHOO
OHO
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHN
O
Pro
Gln
Ser Ser Asp Val Leu Leu Ala Gln NHFmoc
OHN
Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg
Gly Leu Ala Arg Leu Leu Thr
Ser
Thr
SO
O
EPO 78-113
O
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
HO
AcHN
O
OHOHO
HO
OHOHOHO
O OO
AcHN
O
OHOH
OH
OHOOHO
AcHNHO
OHOO
OHO
OAcHNHO
HO OHOH
CO2H
O O OOH OH
OOH
HO
OH
AcHN
O
Pro
Gln
Ser Ser Asp Val Leu Leu Ala Gln NHFmoc
OHN
Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg
Gly Leu Ala Arg Leu Leu Thr
Ser
Thr
Ala
Ser
Ala Ala Asp Pro Pro Ser Ile Ala
Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg
Lys leu Lys Gly Arg Leu
Asn
Phe
O
OAc
O
OCOOMe
AcOAcHN
AcOOAc
OAc
O
AcNHO
AcOAcHN
AcOOAc
OAc
O
O
OO
OAc
OAc
O
Ala Glu Lys Gln
Val Tyr Ser
Cys Ala Glu Gly Thr Tyr LeuArg Asp Gly Thr Arg
ivDdeivDde
ivDdeivDde
Acm
E
AgClFragment
Condensation
EPO 78-166
EPO 78-113 Gly S O
O O
NH2- Ala EPO 114-166
Saburo Aimoto
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
Pro Gln Ser Ser Asn Val Leu Leu Ala GlnTrpGluProLeuGlnLeuHisValAspLys
Ala
ValSer Gly Leu Arg
GlyLeuAlaArgLeuLeuThrSer Thr
Ala Ser Ala Ala Asp Pro Pro Ser Ile
Ala
AlaProLeuArg
Thr
Ile
Thr
Ala
Asp
Thr
Phe
Arg
Lys
Leu
Phe
Arg
LysleuLysGly
Arg
Leu
Asn
Phe
Ala GluLys
Gln
Val
Tyr
Ser
CysAlaGluGlyThrTyrLeu ArgAspGlyThrArg
Leu
Gln Gln Ala Val Glu Trp Gln Gly leu AlaGlyVal
ValProAspThrLysAsp
Lys
Val
SerTrp Lys Arg Met Glu
Thr Ile AsnGluAsn Leu Ser
ProAla Pro Arg Leu Ile Cys Asp Ser
Serleuleu
Glu
Gly ArgLeu
Val
GluLysAla
Ala
Thr Thr Ile
Glu
Cys His Glu Ala Cys Gly
LeuValArg Glu Arg Tyr Leu Glu
Ala
Asn
EPO 78-166
EPO 1-28
EPO 29-77
Summary
We close this review with some thoughts concerning life on the chemistry–biology frontier. In this account, we have shown by historical progression how our laboratory, starting with fascinating problems in the field of “small molecule” natural products, has become involved in issues of tumor expression and tumor immunology. One of the singular contributions which we and like-oriented research groups bring to such coalitions is a sensitivity for precisely defined structures. When collaborating with biologists in identifying bioactive compounds and charting their functions, the chemist insists that the compounds in question be demonstrably pure and that the structural assignments, down to each stereogenic center, be corroborated. But chemistry’s contribution to the enterprise is certainly more than restraints arising from insistence on thoroughness and intellectual exactitude. Methodological building upon the principles of our science leads to the magic of synthesis – with its unique capability to prepare molecules of virtually any shape and juxtaposition of functional groups. Creative synthesis is the indispensable talent that the chemist will bring to the many exciting struggles and opportunities in the future.
Angew. Chem. Int. Ed. 1996, 35, 1380
Thank you!