Microwave Heating and Solid Phase Microwave Heating and Solid Phase ChemistryChemistry

(i). Peptides(i). Peptides(ii).(ii). PeptoidsPeptoids(iii). PNA(iii). PNA(iv).(iv). TriphosphatesTriphosphates(v). Dyes(v). Dyes(vi). O(vi). O--AlkylAlkyl IsoureasIsoureas

(i). Solid Phase Peptide Synthesis(i). Solid Phase Peptide Synthesis -- TheThe FmocFmoc/Bu/Butt

RouteRoute

Base/Weak Acid Base/Weak Acid OrthogonalityOrthogonality

3 Steps:3 Steps:••PiperidinePiperidine (to remove (to remove FmocFmoc groups)groups)

••Coupling of new amino acid to growing Coupling of new amino acid to growing peptide chainpeptide chain

••TFA for cleavage from the resin and side TFA for cleavage from the resin and side chainchain deprotectiondeprotection!!

OO

O

O

Fmoc-NH(i) 20% Piperidine

TFA Cleavage

(ii) Fmoc-Lys(Boc)-OH / Coupling reagent

OHO

HN

OH2N

NH2HO

PS

OO

O

O

NH

PSOFmoc-NH

Boc-NH

(i). Solid Phase Peptide Synthesis (i). Solid Phase Peptide Synthesis –– Which Steps Which Steps Might Benefit from Microwave Heating?Might Benefit from Microwave Heating?

OO

O

O

Fmoc-NH(i) 20% Piperidine

TFA Cleavage

(ii) Fmoc-Lys(Boc)-OH / Coupling reagent

OHO

HN

OH2N

NH2HO

PS

OO

O

O

NH

PSOFmoc-NH

Boc-NH

PiperidinePiperidine (to remove (to remove FmocFmoc groups)groups)

TFA for cleavage from the resin and TFA for cleavage from the resin and side chain side chain deprotectiondeprotection!!

Coupling of new amino acid to growing Coupling of new amino acid to growing peptide chainpeptide chain

(i). Solid Phase Peptide Synthesis (i). Solid Phase Peptide Synthesis –– Which Steps Which Steps Might Benefit from Microwave Heating?Might Benefit from Microwave Heating?

OO

O

O

Fmoc-NH(i) 20% Piperidine

TFA Cleavage

(ii) Fmoc-Lys(Boc)-OH / Coupling reagent

OHO

HN

OH2N

NH2HO

PS

OO

O

O

NH

PSOFmoc-NH

Boc-NH

PiperidinePiperidine (to remove (to remove FmocFmoc groups)groups)No need!No need!

Coupling of new amino acid to growing Coupling of new amino acid to growing peptide chainpeptide chain

TFA for cleavage from the resin and TFA for cleavage from the resin and side chain side chain deprotectiondeprotection!!

(i). Solid Phase Peptide Synthesis(i). Solid Phase Peptide Synthesis –– Which StepsWhich StepsMight Benefit from Microwave Heating?Might Benefit from Microwave Heating?

OO

O

O

Fmoc-NH(i) 20% Piperidine

TFA Cleavage

(ii) Fmoc-Lys(Boc)-OH / Coupling reagent

OHO

HN

OH2N

NH2HO

PS

OO

O

O

NH

PSOFmoc-NH

Boc-NH

iperidineiperidine (to remove (to remove FmocFmoc groups)groups) ––No need!No need!

Coupling of new amino acid to growing Coupling of new amino acid to growing peptide chain peptide chain –– YesYes –– Main cause of Main cause of peptide synthesis failurepeptide synthesis failure

TFA for cleavage from the resin and TFA for cleavage from the resin and side chain side chain deprotectiondeprotection!!

Solid Phase Peptide SynthesisSolid Phase Peptide Synthesis –– Which Steps MightWhich Steps MightBenefit from Microwave Heating?Benefit from Microwave Heating?

OO

O

O

Fmoc-NH(i) 20% Piperidine

TFA Cleavage

(ii) Fmoc-Lys(Boc)-OH / Coupling reagent

OHO

HN

OH2N

NH2HO

PS

OO

O

O

NH

PSOFmoc-NH

Boc-NH

PiperidinePiperidine (to remove (to remove FmocFmoc groups)groups) ––No need!No need!

Coupling of new amino acid to growing Coupling of new amino acid to growing peptide chain peptide chain –– YesYes –– Main cause of Main cause of peptide synthesis failurepeptide synthesis failure

TFA for cleavage from the resin and TFA for cleavage from the resin and side chain side chain deprotectiondeprotection!!AvoidAvoid –– Hazardous!! Hazardous!!

(i). Peptide Chemistry and Microwave Heating(i). Peptide Chemistry and Microwave Heating

•• FmocFmoc group stability and DMF decompositiongroup stability and DMF decomposition

•• Many coupling agents have short halfMany coupling agents have short half--lives once added lives once added to a reaction mixture at 25to a reaction mixture at 25ooC (see C (see AlbericioAlbericio, JOC, , JOC, 1998 e.g. HATU and 1998 e.g. HATU and PyBOPPyBOP –– tt1/21/2 = 40 min at 25= 40 min at 25ooCC –– sosoat 65at 65ooC, tC, t1/21/2 = 2.5 min).= 2.5 min).

•• Avoid the use of exotic (and expensive) coupling Avoid the use of exotic (and expensive) coupling regentsregents –– side reactions, short lived side reactions, short lived

••Do not heat Do not heat FmocFmoc groups above 65groups above 65ooCC••DMF, DMA or NMP as solvent DMF, DMA or NMP as solvent

••Use DIC/Use DIC/HOBtHOBt for couplingfor coupling

(i). Peptide Chemistry and Microwave Heating (i). Peptide Chemistry and Microwave Heating -- FRET PeptidesFRET Peptides

10 minute couplings: 6010 minute couplings: 60ooC, DMF, DIC/C, DMF, DIC/HOBtHOBtAllAll FluorophoresFluorophores coupled easily (10 minutes).coupled easily (10 minutes).

NH

NH-RinkO

O

O

Fmoc-HN

NH

HN

O

NH O

HN

O

OtBu

NH O

HN

O

NH O

O

X-HN

O

HN

O-tBu

X'

X = Dabcyl; X' = FAM X = Fmoc-3-(NO2)Tyr; X' = 2-Abz

TG

NH2

min2 4 6 8 10 12 14

mAu

5500

6000

6500

7000

7500

8000

8500

9000

9500

ADC1 A, ADC1 (0508\05080052.D)

7.7

22

DabcylDabcyl/FAM Peptide/FAM Peptide(>90% purity)(>90% purity)

(ii). Peptoid Chemistry and Microwave(ii). Peptoid Chemistry and MicrowaveHeatingHeating

NOH

ONHBoc

Fmoc

O

N

NHBoc

H6

NH RINK

O

N

NH2

CargoNH2

7

TFA/TIS

PS

6

6H2N RINK PS

O

N

NHBoc

Fmoc6

n

NH RINK PS

Repeat Coupling

-Fmoc, + Cargo

+ Coupling Reagent-Fmoc Grou[p

NN--AlkylglycinesAlkylglycines for cell for cell deliverydelivery

Secondary amine makes Secondary amine makes coupling difficultcoupling difficult

(ii). Peptoid Chemistry and Microwave Heating(ii). Peptoid Chemistry and Microwave Heating

N OH

ONHBoc

FmocO

N

NHBoc

Fmoc 6

7

NH RINK PS

6

m/z200 400 600 800 1000

0

20

40

60

80

100

*MSD1 SPC, time=0.523:1.447 of 0508\05080078.D API-ES, Pos, Scan, 70

Max: 712697 445

.5

334

.4

667

.6

min0 2 4 6 8

Norm.

0

2500

5000

7500

10000

12500

15000

17500

ADC1 A, ADC1 (0509\05090229.D)

3.19

7

PMP1, Solvent B

Microwave heating: 60 Microwave heating: 60 ooCC, 10 minute couplings, 10 minute couplingsDIC/DIC/HOBtHOBt (0.1M, 3equiv.)(0.1M, 3equiv.)

20%20% PiperidinePiperidine –– 10 minutes 10 minutes

98% purity, ELS Detection98% purity, ELS DetectionSingle couplingsSingle couplings

(ii). Cellular Delivery (ii). Cellular Delivery -- Cell Permeable Cell Permeable PeptoidsPeptoids

Oligomer n = 3

% Incorporation 99

n = 5 n = 5 n = 7

93 (25oC)97 (37oC)99

FlowFlow cytometrycytometry analysisanalysisHEK293THEK293T

PerettoPeretto, I.; Sanchez, I.; Sanchez--Martin, R. M.; Wang, X.Martin, R. M.; Wang, X.--h.;h.; EllardEllard, J.; , J.; MittooMittoo, S.; Bradley, M. , S.; Bradley, M. Chemical Communications Chemical Communications 20032003, 2312, 2312--23132313

NNH2

NH2

O6

ONH 5

n

Fluo

HEK293T (human embryonic HEK293T (human embryonic kidney) cells, general kidney) cells, general

transmission image at low transmission image at low magnificationmagnification

1010 M, 37ºC for 6h, M, 37ºC for 6h, fluorescent image at low fluorescent image at low

magnificationmagnification

(ii). Cell Permeable (ii). Cell Permeable PeptoidsPeptoids -- UptakeUptake

Comparative uptake of cells Comparative uptake of cells with oligomers (n=1,3,5,7,9) with oligomers (n=1,3,5,7,9)

at 10 at 10 µµMM

K562K562

HeLaHeLa

L929L929

(ii). Cell Permeable (ii). Cell Permeable PeptoidsPeptoids

Cellular uptake of Cellular uptake of peptoid ( n = 7) by a peptoid ( n = 7) by a

hippocampalhippocampal sliceslicecultureculture

ThioninThionin stainedstainedhippocampalhippocampal sliceslice

showing all the cell showing all the cell layerslayers

(ii). Directed Nuclei (ii). Directed Nuclei LocalisationLocalisation

Delivery within 30 Delivery within 30 minsmins

FluoresceinFluorescein--PPKKKRKVKKKRKV--PeptoidPeptoid (5(5 mermer))

(DAPI stained (DAPI stained nuclei)nuclei)

Nuclei labelling in live cellsNuclei labelling in live cells

DeliveryDeliveryNLSNLS

--MSHMSH :: aa--MelanocyteMelanocyte Stimulating HormoneStimulating Hormone( Ac( Ac--SYSMEHFRWGKPVSYSMEHFRWGKPV--NHNH22 ))

•• Cell impermeable.Cell impermeable.•• Binds the Binds the MelanocortinMelanocortin 1 receptor ( MC1R).1 receptor ( MC1R).•• Stimulates the pigmentation of Stimulates the pigmentation of melanocytesmelanocytesand melanoma cells.and melanoma cells.

(ii).(ii). --MSHMSH

(ii). Peptide and Peptoid Chemistry and (ii). Peptide and Peptoid Chemistry andMicrowave HeatingMicrowave Heating

ON

NH2

O

HN

6

7

5 NH2MSHO

NH

FAM-NH 5

Addition of 17Addition of 17--mer peptide (mer peptide ( MSHMSH))

Microwave heating: 60 Microwave heating: 60 ooCC, 10 minute couplings, 10 minute couplingsDIC/DIC/HOBtHOBt (0.1M, 3equiv.)(0.1M, 3equiv.)

Coupling with Coupling with 5(6)5(6)--carboxyfluoresceincarboxyfluorescein (again 10 (again 10 minutes and quantitative)minutes and quantitative)

Isolated yield of peptide = 45% (>98% per step)Isolated yield of peptide = 45% (>98% per step)

(ii). Delivery of (ii). Delivery of --MSHMSH into Cells into Cells PigmentationPigmentation AssayAssay

S91 melanoma S91 melanoma cellscells in culture in culture

(ii). Delivery of (ii). Delivery of --MSH into MSH into Human SkinHuman Skin

FluoFluo-- --MSHMSH--PeptoidPeptoid (99) (3h, 10(99) (3h, 10--33 M)M) FluoFluo-- --MSHMSH (3h, 10(3h, 10--33 M)M)

(ii). Macrophage Recruitment to LPS (ii). Macrophage Recruitment to LPS Inflamed Lungs Inflamed Lungs

Cy 7 labelled macrophages (9Cy 7 labelled macrophages (9--mer peptoid) mer peptoid) instilled in 10 million cells in 200instilled in 10 million cells in 200 ll

Imaged for 5 Imaged for 5 secssecs

(i) & (ii): Peptide Chemistry and Microwave(i) & (ii): Peptide Chemistry and MicrowaveHeatingHeating

6060ooC is optimal (rapid couplings and no byC is optimal (rapid couplings and no by--products)products)

Single couplings needed in all cases tested to dateSingle couplings needed in all cases tested to date

DIC/DIC/HOBtHOBt performed excellently under these conditionsperformed excellently under these conditions

Avoids the use of exotic coupling reagents with their Avoids the use of exotic coupling reagents with their problems and sideproblems and side--reactionsreactions

Excellent method for coupling poorly reactive acids such Excellent method for coupling poorly reactive acids such asas fluorophoresfluorophores

Time needed reduced form 1 week to 1 day with PeptoidTime needed reduced form 1 week to 1 day with Peptoid--Peptide conjugatePeptide conjugate

ArrheniusArrhenius basedbased accelerationacceleration

N

N

N N

N

O

OPO

O

-O

N

NN

ON

N

O

OPO

O

-O

N

N

N N

N

O

OPO

O

-O

N

NN

ON

N

O

OPO

O O

-O

N

NO

O N

NN

O N

NO

ON

NN

O

H

H

H

HH

H

H

H

H

H

HH

H

HH H

N N N N

HN

HN

O O

HN

O

HN

OOO O O

(iii). Peptide Nucleic Acid (PNA)(iii). Peptide Nucleic Acid (PNA)

(Nielsen et al. Science, (Nielsen et al. Science, 19911991))

(iii). About PNA (iii). About PNA

Chemically robust Chemically robust

Nuclease and protease resistant.Nuclease and protease resistant.

WatsonWatson--CrickCrick--base pairing.base pairing.

The neutral backbone provides stronger binding The neutral backbone provides stronger binding and greater specificity of interaction.and greater specificity of interaction.

PNA/DNA mismatch’s more destabilizing than PNA/DNA mismatch’s more destabilizing than DNA/DNA.DNA/DNA.

N

NO

O N

NN

O N

NO

ON

NN

O

H

H

H

HH H

N NN N

HN

HN

O O

HN

O

HN

OOO

O O

NN

OO N

NN

O

H

HH

N NProtecting group-NH Protecting group-NH

O OOO

OH OH

(iii). PNA (iii). PNA -- PeptidePeptide--like Synthesislike Synthesis

(iii). Microwave Mediated PNA Synthesis(iii). Microwave Mediated PNA Synthesis

•• Polystyrene resin downloaded to 0.06mmol/g prior Polystyrene resin downloaded to 0.06mmol/g prior to PNA synthesisto PNA synthesis

•• 3 couplings: single peak in HPLC and desired mass3 couplings: single peak in HPLC and desired mass

•• 5 couplings or more: multiple peaks in HPLC and 5 couplings or more: multiple peaks in HPLC and desired mass + smallerdesired mass + smaller

H2N-Rink

NH

N OHO

Dde

O Base

NH-RinkHN

NO

Dde

OBase

2. 20% imidazole/NH2OH.HCl

NMP

60o C, 25 min

PEGAPEGA

1. PyBOP; NEM; NMP

60o C, 20mim

(iii). Microwave Peptide(iii). Microwave Peptide--PNA synthesisPNA synthesis

RPRP--HPLC of HPLC of ArgArg--GlyGly--GluGlu––12mer12mer--PNAPNASynthesized under acidic Synthesized under acidic DdeDde--deprotectiondeprotection conditionsconditions(20% NH(20% NH22OH·HCl/imidazole 1:0.75 in NMP/DMF 5:1).OH·HCl/imidazole 1:0.75 in NMP/DMF 5:1).

Arg-Gly-Xa.a.

NHFAM TATC TGTT TCAA

O

NH2

OHN

ONH

The Bradley GroupThe Bradley Group

•• AntonioAntonio FaraFara•• Dr Juan José Dr Juan José DíazDíaz--MochónMochón•• DrDr AsierAsier UncitiUnciti--BrocetaBroceta•• Geraldine EscherGeraldine Escher•• DrDr KevKev DhaliwalDhaliwal (Dr John Simpson)(Dr John Simpson)•• NinaNina SvensenSvensen•• AlessandraAlessandra ChighineChighine•• DrDr BronoBrono LinclauLinclau•• StefanoStefano CrosignaniCrosignani•• MariaMaria LopalcoLopalco•• Dr Jin Ku Dr Jin Ku ChoCho•• Dr Ivan Dr Ivan TrkuljaTrkulja

MRC; EPSRC, BBSRCMRC; EPSRC, BBSRC