Chemistry from Nature
˜ the Application of
Evolutional Principals˜

Literature Seminar 3
11/4/26
Yutaka Saga (D1)

Today’s Contents

0. Introduction
1. Genetic Algorithms
2. Dynamic Combinatorial Chemistry
3. DNA-Templated Synthesis
4. Future Direction

0. Introduction

Traditional Chemical Approaches

Chemists specifically have been interested in the discovery offunctional molecules (catalysts, drugs, materials etc).
Iterated cycle
design, synthesis, activity assay, SAR(structure activity relationship)
Z.J.Gartner et al. Pure Appl. Chem., 2006, 78, 1

Chemistry of GluCAPO & FujiCAPO
Ar
Ar
P
O
HO

ScreeningO(metal, solvent, temp etc)

O

X

HO

Y

Cyanosilylation of ketons
Strecker Reactions
Conjugate Addition of CN to N-acyl Pyrroles
Ring-Opening of meso-Aziridines

Ar
Ar
P
O

Ring-opening of meso-Aziridines
Conjugate Addition of CN to Enones
Diels-Alder Reactions
Protonation Reactions

HO
O

X

HO

Y

SAR

Ph
Ph
P
O

Protonation Reactions

HO

design

N

X

HO

Y

Me

Ar Ar
O

Conjugate Addition of CN to Enones

HO

Synthesis

O

X

HO

Y

M. Shibasaki, M. Kanai. Org. Biomol. Chem., 2007, 5, 2027

Overcoming the Limitations (1)HTS (High-Throughput Screening) assays
HTS of Heck reactions by using FRETJ. F. Hartwig et al. JACS., 2001, 123, 2677

With HPLC, MS, colorimetric, IR ,etc

In Situ Enzymatic Screening

D.B. Berkowitz et al. ACIE., 2002, 41, 1603
J. F. Hartwig et al. Curr. Opin. Chem. Biol, 2003, 7, 420

Overcoming the Limitations (2)Diversity-Oriented Synthesis

S.L. Schreiber et al.
Science., 2000, 287, 1964
ACIE., 2006, 45, 3635

Simple SM

Complexity,
Diversity

Overcoming the Limitations (3)Self-Assembled Libraries

N.H. Reek et al. Nat. Chem., 2010, 2, 615
B.Breit et al. ACIE., 2005, 44, 1640
Dr. Shimizu’s Lit. Seminar (D3)

> large catalyst libraries> just mixing components> Not covalently bonded,with multiple weak interactions
Also see: T. Ooi et al. Science., 2009, 326, 120

Toward Evolutional Approaches (1)Chemists' Approches

Nature's Approches> limited diversities and complexities> vast magnitude of diversities and complexities> individually evaluated (homogeneous)> in one-pot evaluated (heterogeneous)> spatial separation> without spatial separation> high conc

> very low conc
> new reaction discovery ???
Z.J.Gartner et al. Pure Appl. Chem., 2006, 78, 1

Toward Evolutional Approaches (2)???

Z.J.Gartner et al. Pure Appl. Chem., 2006, 78, 1

1. Genetic Algorithms

GA (Genetic Algorithm)> a search heuristic that mimics the process of natural evolution.
> computationally mimic Darwinian evolution> generate solutions to optimization problems inspired by natural evolution> first pioneered by Holland in the 1970s> utilized for numerous fields (computer, mathmatics, biology, economics etc)

> adjusting several parameters for the evolution process(G0, R, RE, RM, RR, NI etc)

J.H.Holland Adaptation in Natural and Artificial Systems
Univ.of Michigan Press, 1975.
L. Weber et al. Curr. Opin. Chem. Biol, 1998, 2, 381

GA (Genetic Algorithm) for Japanese Gamethe player cultivating vegetables on a fictional, futuristic star system.
the objective is to win vegetable contests.
pest called Baboo undergo the evolution based on ‘genetic algorithm’Ambitious Application of GA (1)O
R1 N C+

Compounds

40

×

gene

0010

R2

H
40

01100

R1

R2 O

H
N
O

N R4
R3

+

×

O

NH2

+

10

×

R3

011

R4

OH
40

0100111600000010/ 01100/ 011/ 010011

target : serine protease thrombin
Crossover
0010/ 01100/ 011/ 010011

0010/ 01100/ 011/ 100011

0010/ 01100/ 011/ 100011

0010/ 01100/ 011/ 010011

Mutation
0010/ 01100/ 011/ 010011

0010/ 01100/ 011/ 000111

Replication
0010/ 01100/ 011/ 010011

0010/ 01100/ 011/ 010011
L. Weber et al. ACIE, 1995, 34, 2280

Ambitious Application of GA (2)best 20 coumpounds × 20 generations

S
Me

O

H
N

N

> the best inhibitor exhibited Ki < 1 µΜ
> only 400 reactions out of 160000 reactionsO> totally adjusting GA principals for targeting
HN

NH2> but nonpeptidic (no structural biases)

L. Weber et al. ACIE, 1995, 34, 2280

Ambitious Application of GA (3)O
R1 N C

+

16

×

0010

R2

H
80

01100

+

×

R3

NH2

12
011

R1

R2

H
N
O

NH
R3

15360
0010/ 01100/ 011> chosing the amines with a structural bias toward binding pocket(hydrogen-donor, hydrogen-acceptor etc)
in a manner analogous to proteins or nucleotic acids> 15360 reactions were actually performed to provide library.
> IC50 values were evaluated to be coded by spectral colors> IC50 < 1µM: 0.059 %, 1~10µM: 0.352 %, 10~100µM: 4.395 %simulate GAs to optimize the various parameters
L. Weber et al. Chem. Biol., 2000, 7, 433

Ambitious Application of GA (4)Optimization of generation size

Optimization of the mutation rates
and cross-over strategy

traditional
screening> P (the average activity) =(mGA/NGA)/(mrondom/Nrondom)(mGA, mrondom = the slopes of the performance curves) > c = crossover only between starting materials
n = DNA-like crossover at any bit> P =23.6, 14.1, 10.3, 3.1 (N = 5, 10, 20, 80)> P×N = 118, 141, 206, 248 (N = 5, 10, 20, 80)> small N learns more per individual> large N learns faster> judge the efficiency of GAs or other methods> c excerts a little better peformance despite of lower diversity> increasing the mutation rate deteriorated the performance→ mutation destroyed the acquired knowledges

L. Weber et al. Chem. Biol., 2000, 7, 433

Various Applications of GAs (1)> identification of peptides for the protease stromolysin> only 300 synthesis out of 64000000 hexapeptides

J. Singh et al. JACS., 1996, 118, 1669

Various Applications of GAs (2)Selective Hydrogen Oxidation Catalysts via GAs

Asymmetric Hydrogenation Catalysts via GAs

A: chiral amino alcohol × 11 B: aldehyde × 30
C: metal (Ru, Ir) × 6

Ce1-x-yM1xM2yO2 dopants
over 17000 combinations
from 26 metals in 5 cat amounts> the best catalyst found by evaluating only 10 % of the whole> a time reduced to 30 % compared to the traditional methods> development of double algorithm

J. Beckers et al. Adv. Synth. Catal., 2008, 350, 2237

O. Riant et al. Chem. Eur. J., 2009, 15, 6267

Features of GAs

Advantages> mimicking the evolutional cycle in Nature→ realizing the diversity close to Nature> a small fraction of the 'virtual liblary'→ not time-consuming> equally applied to synthetic molecules as to DNA or proteins

Disadvantages> the need to synthesize and assay indivisual molecules→ What's the difference from traditional methods?
> insufficient size and complexity> beneficial only for the known developed systems→ known things of which parameters set in advance2. Dynamic Combinatorial
ChemistryDynamic Combinatorial Chemistry@ Fischer's study on carbohydrates
in 19th century
in equiribrium

thermodynamicalbinding

template@ Watson & Crick's
DNA helix templates
in 1950s@ Busch's first example
in 1960's

no formation
witout metal
D. H. Busch et al. JACS. 1962, 84, 1762

> the composition is determined by the themodynamic stability, in equilibrium.
> upon the addition of templates, desired molecules can form the stable complex.
> not screen, but selection→ evaluated in mixtures, not indivisually> for the discovery of small molecules, catalysts, and materials
J.K.M. Sanders, S. Otto et al. Chem Rev., 2006, 106, 3652
D.R.Liu et al. Curr. Opin. Chem. Biol., 2007, 11, 259

Reversible Reactions Used for DCC> weak and labile noncovalent bonds to achieve rapid equilibriumvscovalent bonds to ensure thermodynamic stability
J.K.M. Sanders, S. Otto et al. Chem Rev., 2006, 106, 3652

DCC for Amplification of Molecules (1)small peptide-hydrazone
building blocks
3

2

4

unpredictable structures
O

NMe3

+ ACh

O
J. K. M. Sanders et al. Org. Biomol. Chem. 2003, 1, 1625

@ With the addition of ACh, accessing unpredictable structures ???
J.K.M. Sanders, S. Otto et al. Science., 2005, 308, 667

DCC for Amplification of Molecules (2)HPLC traces> first kinetically favoured product was observed. (blue)> afterwards thermodynamically favoured product wasobserved. (red)→ [2]catenane> [2]catenane was amplified at the expense of all theother materials.
> determined also by NMR analysis and MS analysis> only one diastereoisomer in 67 % yield> complex functions from small and simple SMs
J.K.M. Sanders, S. Otto et al. Science., 2005, 308, 667

DCC for Catalyst Discovery (1)DCL with anmonium ion
S. Otto et al. Science., 2002, 297, 590

What cat ???

> disulfide chemistry for DCLs of macrocycles

Disulfide chemistry
for DCL> with high affinity for cationic hydrophobic molecules→ the Diels-Alder reaction

J.K.M.Sanders et al. JACS, 2000, 122, 12063

S. Otto et al. ACIE., 2003, 42, 1270

DCC for Catalyst Discovery (2)HPLC analysis of a DCL
without template 6

with template 6> 1, 2 and 3 mixed in water, under O2> With template, macrocycles 7 and 8
were obtained in the mixture.

ITC analysis of the thermodynamic datas

SM
diene

TM

> 8 binds more strongly with 4> 7 binds more strongly with 6→ incorporation of 5 into 7•4 complex→ 7 would be catalytically active

S. Otto et al. ACIE., 2003, 42, 1270

DCC for Catalyst Discovery (3)Observed rate constant for the Diels-Alder reactionin the presence of 7

> indeed 7 induced a modest acceleration> 8 turned out to be catallytically inactive(not shown)> 7 seemed to leave enough space to binddienophile 5in the absence of 7

> the efficiency of 7 was moderate compared withother catalysts@ In the mixture, the reacton reversibly discovered the efficient catalysts
S. Otto et al. ACIE., 2003, 42, 1270

DCC for Functional Materials (1)Supramolecular formationin non-covalent fashion

Na, K, NH4 etc
hydrogels> undergoing the association into G-quartets in the presence of cations> with hydrogel formations
J. M. Lehn et al. PNAS., 2005, 102, 17

DCC for Functional Materials (2)
O
AcHN
10

N
H

NH2

in covalentfashion> A = G-quartets + 6, B = G-quartets + 7> C = 9 + 6, D = 9 + 8, E = 10 + 6, F = 10 + 8> 1H NMR determined the equilibrium> At higher temp (~ 80 ºC), the selectivity got worse due to the melting of hydrogels> with the thermodynamic gelation event, 1 preferencially scavenged 8.

J. M. Lehn et al. PNAS., 2005, 102, 17

DCC for Functional Materials (3)Non-covalentfashioncovalent
fashion> the lability of noncovalent interactions + reversible covalent bonds→ multistep self-assembly for DCLs
> with themoreversible hydrogels> demonstrating DCC can be applied to a supramolecular scale→ discovery of materials with desired bulk properties
J. M. Lehn et al. PNAS., 2005, 102, 17

Features of DCC
Advantages> reversible and thermodynamical process in equilibrium> desired = amplified, undesired = removed→ 'automatically' selection + amplification> applications to small molecules, catalysts, materials

Disadvantages> limited diversity of available building blocks for appropreate targets> difficulty of deconvoluting the complex mixtures> Life can be thought of as 'a kinetic state of matter'→ systems biology and systems chemistry ???

3. DNA-templated
Synthesis

DNA for Evolutional Approaches
Chemists' approach to control reactivity

GAs and several methods canaccerelate the cycle.

Nature's approach to control reactivity

DNA as a toolfor evolutional approaches
D. R. Liu et al. ACIE., 2004, 43, 4848

> in one-pot fashion> reactions are randomly performed> at much lower concentrations> ideally suited to perform the iterative operation(translation, selection, amplification and diversification)→ power of PCR method> with high information density> with chemical stability> with the ease of manipulation
D. R. Liu et al. Curr. Opin. Chem. Biol., 2007, 11, 259

Several Methods
Encoding Chemical Libraries Using DNA > identification of DNA-limked small molecules

pioneered by Brerner et al using amino acid
PNAS. 1992, 89, 5381

Also see:
D. Neri et al. Nat. Biotechnol., 2004, 22, 568

DNA Library > reaction of DNA-linked small molecules individuallylibrary synthesis of unnatural pentapeptides
P. B. Harburry et al. PLos Biol. 2004, 2, 1022

D.R.Liu et al. Curr. Opin. Chem. Biol., 2007, 11, 259

DNA-Templated Synthesis> spatial separation is not required> DNA annealing controlls reactivity→ all templates and reagents coexist in a single solution> Using complementary DNA-linked reagents via Watson-Crick base-pairing
D.R.Liu et al. ACIE., 2004, 43, 4848
D.R.Liu et al. Curr. Opin. Chem. Biol., 2007, 11, 259

David R. Liu as a Key Person

Pioneering Work (1)Michael-type reactions directed by DNA templates@ Early work: Synthesis of oligonucleotide analogue

PNAS. 1968, 59, 726;
J. Mol. Biol, 1968, 38, 381

H product> H and E DNA architectures exhibited similar reactivities.
E product> no products were observed by using mismatched DNAs.
> DNA-templated reactions proceeded even the products
differed from the natural backbones.

control

Various reactions directed by DNA templates> Other reaction systems could be applied for (SN2 substitutions, additions to αβ -unsaturated carbonils etc).

D.R.Liu et al. JACS., 2001, 123, 6961

Pioneering Work (2)Distant-independent (DTS) DNA-templated synthesis

Evaluation of possible contribution> possible contributions were evaluated.
(conformational prefecences, interbase interactions,backbone hydrophilicity etc)
> demonstrating remarkable distance independence.
> Even in 30 bases distances, efficiently reacted.
(through transition state 200 membered ring !!! )> sharply contrast with traditional organic synthesis> the backbone structural elements were not responsible.
> complementary DNAs to interventing region
significantly reduced the products.
→ the flexibility of the region is likely to crucial.

@ The fact that DNA annealing became the RDS would explane the distant independence.
(decreasing the conc. resulted in a marked reduce in the reaction rate)
D.R.Liu et al. JACS., 2001, 123, 6961

Expansion to the Reaction Generality> the first useful C-C bond forming ractions (nitro-aldol, Wittig, Heck coupling etc)
D.R.Liu et al. ACIE., 2002, 41, 1796
D.R.Liu et al. ACIE., 2003, 42, 1370

New Modes of Controlling Reactivity (1)traditional chemistry> different type of reactions were conducted in one solution.
> the different length of DNAs enabled easier detection.
> selective 3 reactions took place to afford 7, 8, 9 exclusively.
(from 9 possible reactions)> other possible products were obtained in < 5 %.

D.R.Liu et al. ACIE., 2002, 41, 4140

New Modes of Controlling Reactivity (2)biotynylated template

12 reactantsinone solution

before purification with beads after purification with beads> the reactions were performed containing
6 templates and 6 reactants in one solution.
> 6 templates with biotinylation> biotynylated templates could be purified withmagnetic beads.
> only the single product was obtained
pragrammed by DNA sequences.

@ DTS exhibited the ability to direct different types of reactions
in one solution, incompatible under traditional reaction conditions.
D.R.Liu et al. ACIE., 2002, 41, 4140

Applications of DTS ~ Selection of a Library (1)D.R.Liu et al. Science., 2004, 305, 1601

Applications of DTS ~ Selection of a Library (1)A

D.R.Liu et al. Science., 2004, 305, 1601

Applications of DTS ~ Selection of a Library (2)> multistep synthesis afforded 65 macrocycles.
> each step was monitored by PAGE and TOF-MS
D.R.Liu et al. JACS., 2002, 124, 10304
D.R.Liu et al. Science., 2004, 305, 1601

Applications of DTS ~ Selection of a Library (3)> a pilot liblary of 65 macrocycles was utilized for selective protein binding affinity (carbonic anhydrase).
> 2 selection rounds enriched the single molecule containing sulfonamide group.

@ DTS coupled with in vitro selection enables the translation, selection, amplification of DNA
encoding synthetic small molecules not biological molecules.

→ the combined ability of synthetic chemistry and PCR method !!!

D.R.Liu et al. Science., 2004, 305, 1601

Applications of DTS ~ Reaction Discovery (1)the product of cycloaddition> identifying all substrate pairs capable of forming a covalentbond→ n× m discrete pairs> also n× m discrete pairs were mixed in one solution> first the cycloaddition of azide and alkyne in the presence ofCu was performed to test the ability.
> the successful detection despite of DNA degradation by Cu→ the extreme sensitivity of PCR amprification

D.R.Liu et al. Nature., 2004, 431, 545

Applications of DTS ~ Reaction Discovery (2)> 168 reactions in the presence of PdII were evaluated.
> green/red ratio → bond-formation

D.R.Liu et al. Nature., 2004, 431, 545

Applications of DTS ~ Reaction Discovery (3)> characterizion of array positives

???

Heck

D.R.Liu et al. Nature., 2004, 431, 545

Applications of DTS ~ Reaction Discovery (4)> demonstrating the 'discovering reaction' in organic solventsthe first example !!!

@ the value of searching a large number ofsubstrate combinations for unexpected reactions
D.R.Liu et al. Nature., 2004, 431, 545

D.R.Liu et al. JACS., 2007, 129, 2230

Applications of DTS ~ Reaction Discovery (5)RDPCR (Reactivity-Dependent PCR)

Reaction discovery of mild azide reduction
O
R
N3

O

Ru cat, hv

R
H2 N

D, Liu et al. JACS. 2009, 131, 9189

Reaction discovery compatible with organic solvent
Me
Au/Ag cat+> in orgnic or aqueous solvent, open to air, at r.t

N
PG

+
N
PG

R1

R2

R3

H

cat TfOH

D, Liu et al. JACS. 2007, 129, 14933

R1

R2 R3
H

> compatiable with various functional groups(alcohols, phenpols, acids, alkenes, aldehydes etc)
> on oligonucleotides, oligo saccharides, protein
D, Liu et al. Nat. Chem. 2011, 3, 146

Features of DNA-Templated Synthesis
Advantages> genuine evolutional cycle (selection, translation, amplification)> with the synthetic molecules not amplifiable information (RNA etc)
> in a single solution→ the enormous magnitude of diversity, easiness, new reactivity> reactivity controlled by DNA annealing→ selectivity

Disadvantages> the need to prepare DNA-linked reagents> under conditions that support DNA hybridization(in the aqueous solvent, moderate temp., neutral pH etc)
> just the combination of reactants in 'rediscovering reactions'→ the ability to develop conceptually new methodologies ???

4. Future Direction

Toward Future Methodologies

New methods supported
by chemical modifications(HTS etc)

Traditional
chemical approaches> reductionistic approaches> time-economic, easy screening> beneficial for 'optimization screening'
1990

Evolutional Approaces(GA, DCC, DTS)

What is thenext generation???

> new concept inspired by Nature> huge diversity treated in the mixture> new reactivity for 'rediscovering reactions'
2000

2010

Key Words> kinetic and thermodynamic combined system> auto-catalytic> self-replicating> feedback> oscillation> systems chemistry> application to medicine
Also see: Mr. Sato’s Lit. Seminar (B4)

@ multiple component as a whole system

Molecular Networks under Kinetic Control
pDCL (pseudo-Dynamic Combinatorial Library)
the most rapidly
synthesized

irreversible system> synthesis

irreversible system> destruction
the tightest
binding> biology: balance between several processes that are kinetically controlled→ drug discovery ???
J. L. Gleason et al. ACIE., 2004, 43, 2432

Self-Replicating (1)> 2 cross-replicating RNA enzymes> Self-sustained amplification without other proteins
G. F. Joyce et al. Science., 2009, 323, 1229
Also see: Mr. Sato’s Lit. Seminar (B4)

Self-Replicating (2)irreversible
cycloaddition

dynamic libraryself-replicating
D. Philip et al. ACIE., 2006, 45, 6344
D. Philip et al. ACIE., 2008, 47, 9965

Self-Replicating (3)> the catalytic [3•5•trans 7] complex accelerated own formation
D. Philip et al. ACIE., 2006, 45, 6344 D. Philip et al. ACIE., 2008, 47, 9965

Self-Replicating (4)complementary
recognition sitebond formation

D. Philip et al. ACIE., 2006, 45, 6344 D. Philip et al. ACIE., 2008, 47, 9965

Ideal System

An

+

Bm

An

feedback+

Bm

Cu, Ni, Fe, Co....

AnBmfeedback

Cu

Fe

Pnm

Pnm

+

Pnm

Pnm

Pnm

@ Can we construct the similar system
as 'organic chemists of organometallics' ???

Application to Medicine (1)activation of 5FU etc

prodrug

catalyst

triggering sequence> rational concept design for 'triggered catalytic drug release'> the desease state (binding by catalyst) triggered the release of drugs
J. S. Taylor et al. PNAS., 2000, 21, 11159

Application to Medicine (2)trigger

catalyst> only in the presence of a catalyst and a trigger, drug release proceeded> enzyme-like behavior following Michaelis-menten kinetics> sensitive to mismatch sequences → SNP ???

J. S. Taylor et al. PNAS., 2000, 21, 11159

Application to Medicine (2)Phostoswithcable molecular glue for DNA@ Without using external switch, can we develop the original methodologies ???
→ New systems with 'oscillation' in time (rhythms) and space (patterns)
K. Nakatani et al. JACS., 2007, 129, 11898