Lecture 5' An Overview of Solid Phase Organic Synthesis

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Lecture 5. An Overview of Solid Phase Organic
Synthesis

• Outline
• Solid Phase Synthesis
• An Overview of Solid Supports
• Linkers
• Parallel Synthesis and Split-Pool Synthesis
• Characterization and Analysis
• Encoding
• Solid Phase Reagent Scavenger Resins

Young-Kwon Kim Ryan Spoering Gojko Lalic
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Why Use Solid Phase Synthesis?

• Purification of compounds bound to the solid
  support from those in solution is accomplished by
  simple filtration
• This allows the use of a large excess of
  reagents, improving the efficiency of many
  transformations
• The solid support can be used to compartmentalize
  library members, permitting the use of split-pool
  synthesis

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Synthesis of Functionalized Polystyrene Resin

• Polymerization of styrene can be conducted with
  functionalized monomers
• Alternatively, polystyrene can be functionalized

4
Effects of Crosslinking

• Cross-Linking imparts mechanical stability and
  improved diffusion and swelling properties to the
  resin

Without cross-linking, each polymer chain can
dissolve under thermodynamically favored
conditions Cross-linking can induce some
sites of permanent entanglement maintaining
structural integrity
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Swelling of Polymer by Solvent
Shrunken state
Swollen state Permeable to solvent and reagent
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Common Solid Support Resins

• Cross-Linked Polystyrene
• Swells in methylene chloride, toluene but cannot
  swell in methanol, water
• Tolerant of a wide rangeof reaction conditions

• Polyamide resin (Sheppards resin)
• Developed for peptide chemistry

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Common Solid Support Resins

• Polystyrene-Poly(ethylene glycol) graft
  (TentaGel)
• Swells in a wider variety of solvents (e.g.
  water, methanol)
• Usually shows lower loading and is less robust
  under mechanical stress than crosslinked PS resin

• Controlled pore glass
• Used for automated DNA synthesis
• Diffusion occurs through the rigid pore structure
• Reaction happens only at solvated surface
• Lower loading

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Practical Considerations in Choosing a Solid
Support

• Mode of attachment and cleavage of materials from
  the resin (linker)
• Compatibility of the chemistry planned for the
  library synthesis
• The amount of material desired (loading level)
• Size - affects efficiency of diffusion within the
  polymer (reaction rates!)

90 ?m (TentaGel) 0.75 mmol/ g 350 pmol/ bead Ca.
180 ng/ bead
200 ?m (PS) 1.05 mmol/ g 4 nmol/ bead Ca. 2 ?g/
bead
500 ?m (PS) 1.05 mmol/ g 60 nmol/ bead Ca. 30
?g/ bead
Diffusion Efficiency
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Linkers Introduction

• A linker covalently connects molecules to the
  solid support, and should provide a means for
  their chemical attachment and cleavage
• Stability of the linker affects the scope of the
  chemistry that can be employed in the library
  synthesis
• Many linkers are adapted from protecting group
  chemistry
  

Synthetic Steps
X
Attachment
Resin
Linker
Molecule
Resin
Linker
Cleavage
Molecule
Resin
Linker
Molecule
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Alkylsilyl Linker - Fluoride Labile

• Mild cleavage conditions compatible with various
  functional groups
• Designed for attachment through an alcohol
• Compatibile with strong anionic, cationic,
  oxidative, and reductive conditions

Ellman J. et al. JOC, 1997, 62, 6102. Foley MA
et al. J. Comb. Chem. 2001, 3, 312.
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Acid Labile Linkers

• Many historically important resins (Merrifield,
  Wang, Sasrin, Sieber, Rink resins) have linkers
  that are cleaved under acidic conditions
• Acidic conditions were intended to prevent
  racemization of amino acids during solid phase
  peptide synthesis

X H, Wang linker X OMe, Sasrin
linker Sieber linker
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Nucleophile Labile Linkers

• Kaiser Oxime linker
• Advantage Introduction of diversity in cleavage
  step
• Difficulty Often too reactive for common
  nucleophilic reaction conditions

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Safety-catch linker

• Kenners sulfonamide linker
• A safety-catch linker can solve the reactivity
  problem with a two step cleavage
• 1) An activation step that is orthogonal to
  common functional groups
• 2) Cleavage of the activated linker under mild
  conditions

Ellman J. et al. JACS, 1996, 118, 3055.
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Traceless Linkers

• This type of linker creates a C-C or a C-H bond
  at the site of cleavage
• C-H bond generation Si-Ge linker (protonolysis
  or radical reduction)
• C-C bond generation

Ellman J. et al. JOC, 1995, 60, 6006.
Nicolaou KC et al. ACIEE, 1997, 36, 2097.
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Photo-labile linker

• Photolytic conditions can be very mild and
  selective
• Dimerization of the support-bound nitroso
  by-product sometimes hampers further cleavage
• Aryl nitro group is incompatible with some
  organometallic chemistry

Krafft GA et al. JACS, 1988, 110, 301.
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Parallel Library Synthesis

• Optimization of 12 reactions provides 9 compounds
• The library members are spatially separated, so
  this technique can be used for solution as well
  as solid phase synthesis

diversification reaction
diversification reaction
divide
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Split-pool Synthesis

• Optimization of 6 reactions leads to 9 compounds
• Each library member must be compartmentalized
  (each compound on its own bead) to allow pooling
  of the library

split
pool and split
diversification reaction
diversification reaction
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An Example of Split-Pool Synthesis
split
pool
diversification reaction
Ellman, J. et al. J. Am. Chem. Soc., 1995, 117,
3306.
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An Example of Split-Pool Synthesis
split
diversification reaction
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Overview of the Entire Split-Pool Library
Ellman, J. et al. J. Am. Chem. Soc., 1995, 117,
3306.
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Example of the Efficiency of the Split-pool
Strategy

• Optimization of 154 reactions affords 105
  amplification in the number of compounds

Schreiber SL et al. JACS, 1998, 120, 8565.
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Structural Characterization Direct Methods

• Off-bead Analysis
• Cleavage, then use of analytical techniques used
  in TOS (e.g. LC, MS, NMR)
• Requires high sensitivity and high throughput
  format
• Example LC-UV/ MS

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Structural Characterization Direct Methods

• On-bead Analysis I
• Can be used to monitor the progress of a reaction
  
• MAS-NMR ( Magic angle spinning NMR ) is necessary
  due to polymer
• Magic angle rotor (left), rotor
  spinning at the magic angle (right)
• MAS- NMR spectrum (600 MHz)

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Structural Characterization Direct Methods

• On-bead Analysis II
• Example Single-bead FT-IR microspectrometry
• Can be used to monitor the progress of a reaction

Beads in IR cell
Wavelength (cm-1)
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Structural Characterization Indirect Methods

• Deconvolution
• Screen as a mixture ofcompounds then
  re-synthesize and re-assaypossible
  candidatesin active pools
• Drawbacks
• Interference byunwanted propertiesof other
  compounds(e.g. cytotoxicity)
• Possible synergisticinteraction of
  multiplecompounds
• Sub-library synthesisis cumbersome

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Encoding

• Encoding should provide a fast and simple way to
  identify the structure of all library members
• Classification
• Spatial encoding position of the compound
  provides the information about its structure
  (possible only in parallel synthesis)
• Graphical encoding bar codes or other graphical
  tags are displayed on the solid support used in
  the library synthesis
• Chemical encoding every reaction used in the
  library synthesis is recorded on the solid
  support by the chemical attachment of a tag
• binary coding (presence or absence of a tag) or
  polymer based (polypeptide, DNA)
• Spectrometric encoding using a spectrometric
  technique (NMR, MS, Fluorescence microscopy, NMR
  etc.) to read tags directly from the solid
  support
• Electronic encoding radio frequency memory chip
  attached to the solid support records and emits
  coded information

A. C. Czarnik Current. Opp. Chem. Biol. 1997, 1,
60
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Encoding in Split-pool Synthesis

• Optimization of 6 reactions leads to 9 compounds
• Each library member must be isolated on its own
  bead to allow pooling of the library

How do you know what compound is on a given bead
after a pool step?
split
pool and split
diversification reaction
diversification reaction
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Chemical Encoding in Split-Pool Synthesis

• Every diversification reaction is followed by a
  tagging reaction in which a tag(s) that codes for
  a particular transformation is covalently
  attached to the solid support

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Decoding

• Every bead has tags that provide information,
  once cleaved, about the chemical history of that
  bead
• Conditions for cleavage of compound and tags have
  to be orthogonal

compound cleavage
tag cleavage
small molecule
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Binary Chemical Coding
11
10
011
110
101
111
1
Building blocks
00
01
001
010
100
Binary (base 2) codon
0
Tags
2 digit codon 22 4 max
3 digit
1 digit
11
110
1
n binary tags code for 2n building blocks
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Another Example
10
011
0
11
10
011
110
101
111
1
Building Blocks
0
00
01
001
010
100
Tags
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Halogenated Aromatics As Tags

• Small amount of tag can be reliable detected
  (0.5-1 pmol/bead) using easily automated electron
  capture GC in the mixture of tags based on
  different retention times
• Inert under most reaction conditions

W. C. Still et al. J. Org. Chem. 1994, 59, 4723
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Attachment and Cleavage of Tags

• Tags are attached using rhodium carbene-insertion
  chemistry and can be cleaved using (NH4)Ce(NO3)6
  (CAN)

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Binary Chemical Encoding of a Peptide Library

• A library of decapeptides was synthesized and
  screened for binding to 9E10 mAb.
• 7 Amino acids were used at each position (S, I,
  K, L, Q, E, D)
• Every amino acid was assigned a 3 digit binary
  codon (001S, 010I, 011K, 100L, 101Q, 110E,
  111D) where 1presence of a tag and 0absence
• For each step in the library synthesis there are
  3 tags designated nX (total of 18 tags for a
  library of 117,649 members, maximum encodable is
  218 262,144)

EQKLISEEDL known to bind 9E10
110 101 011 100 010 001 identified as the best
binder
W.C. Still et al. PNAS 1993, 90, 10923
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Solid Phase Reagent and Scavenger Resins

• Attaching reagents to the solid phase instead of
  substrates provides similar advantages
• Ease of purification allows the use of excess
  reagents
• Excess reagents can be removed by use of a solid
  phase-bound scavenger that reacts with or binds
  the excess reagent

Reagent

Reagent
Filter
Starting Material
Clean Product
Product
Excess Reagent
Scavenger
1)

Reagent
Clean Product
Starting Material
Product
2) Filter

Scavenger
Reagent
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An Example of Solid Phase Reagents and Scavengers

• An extremely efficient three step reductive
  amination and triflation is accomplished by the
  use of solid phase reagents and scavengers

Ley SV et al. J. Chem. Soc. Perkins. Trans. I
1999, 63, 6625.