combinatorial chemistry and solid phase synthesis …...department of chemistry and pharmacy -...

Department of Chemistry and Pharmacy - Universität Regensburg

Combinatorial chemistry and solid phase synthesis

seminar and laboratory course

Karoly Agoston

Kruppa Michael

Regensburg 2003

This teaching lab course was developed at the INNOVATEC guest chair “Combinatorial

Chemistry and Solid Phase Synthesis”, which is sponsored by the German Academic

Exchange Service (Deutscher Akademischer Austauschdienst; DAAD). The financial support

is gratefully acknowledged.

Department of Chemistry and Pharmacy - Universität Regensburg 2

Combinatorial chemistry seminar

(room 12.0.16)

List of topics

1.) Principles of combinatorial chemistry

(ref.: I. pp. 1-34)

2.) Solution phase combinatorial chemistry

(ref.: I. pp. 77-123; II. pp. 5-46)

3.) Combinatorial chemistry of multicomponent reactions

(ref.: I. pp. 125-165; IX. pp. 123-131)

4.) Resins and solid phase anchors in the organic chemistry

(ref.: I. pp. 167-228; II. pp. 47-98; V. pp. 465-510)

5.) Solid phase organic reactions, part 1 (C-C bond formation, cyclisation reactions)

(ref.: I. pp. 35-76; II. pp. 99-208, and 329-368; VI. pp. 25-79)

6.) Solid phase organic reactions, part 2 (reduction, oxidation, formation of carbonyl

compounds and derivatives thereof)

(ref.: I. pp. 35-76; II. pp. 99-208, and 329-368)

7.) Synthetic libraries of heterocycles

(ref.: I. pp. 257-290; II. pp. 157-208)

8.) Synthetic libraries of linear oligomers

(ref.: I. pp. 257-290)

9.) Solid phase peptide synthesis, strategies and resins

(ref.: VII. pp. 1-162; IX)

10.) Polymer supported organic reactions

(ref.: II. pp. 209-237; VI. pp. 149-194; III. pp. 83-156; IV. pp. 314-349)

11.) Analytical methods in combinatorial chemistry

(ref.: I. pp. 479-542; V. pp. 247-286)

12.) Illustrative syntheses (one peptide and one non-peptide)

(ref.: VII. pp. 163-191; X; XI)

13.) Industrial concepts of combinatorial chemistry in process development and High-

throughput screening (ref.: VIII. pp 831-884, world wide web)


References:

I. Jung G. (ed.): Combinatorial Chemistry, Synthesis, Analysis, Screening, Wiley-VCH,

Weinheim, 1999 (86/VK5500 J95)

II. Bannwarth W. and Felder E. (eds.): Combinatorial chemistry, A Practical Approach,

Wiley-VCH, Weinheim, 2000 (86/VK 5500 B219)

III. Hodge P. and Sherrington D. C. (eds.): Polymer-supported Reaction in Organic

Synthesis, John Wiley & Sons, New York, 1980 (86/VK 5500 H688)

IV. Schmalz H-G.: (ed.): Organic Synthesis Highlights IV, Wiley-VCH, Weinheim, 2000

V. Jung G. (ed.): Combinatorial Peptide and non Peptide Libraries, A Handbook, Wiley-

VCH, Weinheim, 1996 (86/VK 8560 J95)

VI. Burgess K. (ed.): Solid Phase Organic Synthesis, John Wiley & Sons, New York,

2000 (86/VK 5500 B955)

VII. Atherton E. and Sheppard R. C. (ed.): Solid Phase Peptide Synthesis, A Practical

Approach, Oxford University Press, Oxford, 1989 (86/VK 8560 A868)

VIII. Nicolaou K. C., Hanko R., Hartig W. (ed.): Hanbook of Combinatorial Chemistry

Vol. 1+2, Wiley-VCH, Weinheim, 2002 (86/VC 6250 N637-1 (-2))

IX. Kates S. A., Albericio F.(ed.), Solid-Phase Synthesis, A practical guide, Marcel

Dekker, Inc., 2000 (86/VK 5500 K19)

X. Special Thematic Issue `Combinatorial Chemistry`: Chem. Rev.: Vol. 97, Iss. 2, 347-

510 (1997)

XI. Special Thematic Issue `Combinatorial Chemistry`: Acc. Chem. Res.: Vol. 29, Iss. 3,

111-170 (1996)

Karoly Agoston: room 23.1.25, tel. 4623, [email protected]

Michael Kruppa: room 32.1.22, tel. 4566, [email protected]

Laboratory: 32.0.09 and 32.0.06


Experimental procedures

Solid phase peptide synthesis 1 Synthesis of a pentapeptide on Wang resin with Fmoc strategy

Safety considerations: Protective gloves and safety glasses should be worn at all times.

Be careful working with TFA!

Working with DMF and DCM keep them clean!

After the experiment the miniblock reactors must be cleaned as soon

as possible, TFA is extremely corrosive!

O

O

NH

OHR

O

OOH Wang resin

Fmoc AA1

OO

O

O

NH

R

O

OO

NH2

R

O

O

O

NH

OHR

O

Fmoc AA2

2

11

1

2

1

O

O

NH

R

O OO

N

R

O

2

1

NH

R

O

N

R

O

NH

R

O OO

N

R

ONH2

R

O4

35

2

1

NH

R

O

N

R

O

NH

R

OOH

N

R

ONH2

R

O4

35

Functionalisation of the resin

Deprotection

Coupling

Deprotection-wash-coupling-wash steps

Cleavage


Mission:

You will synthesise a pentapeptide on Wang resin using Fmoc strategy. The synthesis makes

use of functionalisation of the resin, determination of the loading, condensation and

deprotection reactions, resin test, cleavage from the resin, precipitation protocol. Finally you

should analyse your product by NMR, and LC-MS.

Reactions:

Functionalisation of the resin.

Dissolve 0.5 mmol FmocAA1 and 0.49 mmol HBTU in 2 mL DMF then add 1.0 mmol

DIPEA to the mixture. Wait for 2 min, then add the solution to Wang resin (0.1 mmol) and

shake the mixture for 3 h at r.t. (The resin must be preswelled in DMF for 1 h.) Filter the

mixture and wash the residue with DMF (4 × 2 mL), then DCM (4 × 2 mL). Take a sample of

the dry resin (2 × about 1 mg), and determine the yield.

Add 3 mL of piperidine/DMF (2/8) to each sample and mix for 4 min., then put the solvents

into UV kuvette and record the absorbance at 290nm.

Calculated the yield using the following equation:

mmol/g = (Abssolv – Absref) / 1.65 × mg sample.

If the yield is lower than 70% the coupling should be repeated.

Preswell the resin in DCM, then add 0.5 mmol Ac2O and 1.0 mmol pyridine in DCM (2 mL)

and shake the mixture for 30 min. Filter the mixture and wash with DCM (2 × 2 mL) and

DMF (4 × 2 mL). (During this step you will protect the free -OH groups on the resin.)

Deprotection: The Fmoc protecting group should be removed by treatment of the resin with a

solution of piperidine/DMF (2/8, 2 mL) for 5 min then filter the solution of and wash the resin

with DMF (2 mL). Repeat the treatment once more, and wash the resin with DMF (4 × 2 mL)

and DCM (4 × 2 mL). (Finally the piperidine reagent should be removed carefully, otherwise

during the next coupling partial deprotection may occur. At no time during the deprotection is

the resin allowed to become dry.)

Coupling with the second FmocAA2.

Dissolve 0.5 mmol FmocAA2, 0.5 mmol HOBt and 0.49 mmol HBTU in DMF (2 mL) then

add 1.0 mmol DIPEA, mix and add to the resin (preswell in DMF). Shake the mixture for 1 h


then filter the solution and wash the residue with DFM (4 × 2 mL), and DCM (4 × 2 mL). The

reaction should be checked with Kaiser test described below*.



with DMF (2 mL), then repeat the treatment once and wash the resin with DMF (4 × 2 mL)

and DCM (4 × 2 mL).

The cycle of coupling, washing, deprotecting and washing should be repeated until the desired

sequence of amino acids is obtained.**

General coupling: Dissolve 0.5 mmol FmocAA, 0.5 mmol HOBt and 0.49 mmol HBTU in

DMF (2 mL) and then add 1.0 mmol DIPEA mix and add to the resin (preswell in DMF).

Shake the mixture for 1 h then filter the solution and wash the residue with DFM (4 × 2 mL)

and DCM (4 × 2 mL). The reaction should be checked with Kaiser test.

Finally, after the last deprotection step the resin should be washed with DMF (4 × 2 mL),

AcOH (1 × 2 mL), DCM (4 × 2 mL), MeOH (1 × 2 mL), DCM (4 × 2 mL), and dry the resin

under vacuum.

Cleavage: Add TFA (95% and 5% water, 2 mL, (this has to prepared in advance)) to the resin

and shake for 2h (Follow the color of the resin with attention). Then filter the resin and wash

with TFA (95% and 5% water, 2 mL), concentrate the combined filtrate to its half a volume.

The pentapeptide should be obtained by precipitation with cold Et2O. Filter the precipitate and

wash with cold Et2O. (During the evaporation the trap of the rota must be filled with KOH or

NaOH.)

DO NOT DISCARD resin support or ether until peptide analysis is complete. Both can be

stored under nitrogen at 4 oC to prevent oxidation. (If your peptide do not precipitate,

evaporate all of the solvents, dissolve the peptide in DCM (as less as possible) and precipitate

your peptide with hexane)


The product can be lyophilized after dissolving in water and freezing.

Analysis: NMR spectrum (recorded in DMSO d6)

MS-ESI spectrum and if possible LC-MS spectrum should be recorded.

For the LC-MS spectrum: Dissolve 0.5 mg of your compound in MeOH (1

mL), then filter that into a eppendorf tube. Calculate the sumformula and the molecular

weight.

*

Kaiser test:

Prepare the following solutions:

1: 5g Ninhydrin in 100 mL ethanol.

2: 80g of liquefied phenol in 20 mL of ethanol.

3: 2 mL of a 0.001M aqueous solution of KCN in 98 mL pyridine.

Sample of few resin beads and wash several times with ethanol. Transfer to a small glass tube

and add 2 drops of each of the solutions above. Mix well and heat to 120 oC for 4-6 min.

Positive test is indicated by blue resin beads.

**

After each coupling-washing procedure the resin can be stored under DCM in the fridge till

the next lab day.


Solid phase peptide synthesis 2

Parallel synthesis of tripeptide library on Wang resin with Fmoc strategy



Working with DMF and DCM keep them clean!



Mission:

You will synthesize a parallel tripeptide library using solid phase methods and Fmoc

chemistry. The synthesis makes use of functionalisation of the resin, condensation and

deprotection reactions, cleavage protocols. Finally you will have 27 different tripeptide in

your flasks, which will be analysed by NMR, HPLC and MS.

ABCDEF

1 2 3 4 5 6 7 8 9First reactions

FmocAA1

FmocAA2

FmocAA3

FmocAA1 FmocAA2 FmocAA3Second reactions

Third reactions

FmocAA1 FmocAA2 FmocAA3


Reactions:


Place 60-60 mg of Wang resin to 27 reaction vessel and preswell that in DMF for 1 h.

Dissolve 9 × 0.25 mmol FmocAA1 and 9 × 0.25 mmol HBTU in 8 mL DMF and add 9 × 0.5

mmol DIPEA to the mixture. Wait for 2 min, then distribute the solution and add to the resin

(to 9 vessels !! Eg A1, A3, A5, A7, A9, B2, B4, B6, B8). Dissolve 9 × 0.25 mmol FmocAA2

and 9 × 0.25 mmol HBTU in 8 mL DMF and add 9 × 0.5 mmol DIPEA to the mixture. Wait

for 2 min, then distribute the solution and add to the resin (next 9 vessels !! Eg C1, C3, C5,

C7, C9, D2, D4, D6, D8). Dissolve 9 × 0.25 mmol FmocAA3 and 9 × 0.25 mmol HBTU in 8

mL DMF and add 9 × 0.5 mmol DIPEA to the mixture. Wait for 2 min, then distribute the

solution and add to the resin (last 9 vessel!! Eg E and F row) and shake the mixture for 3 h at

r.t. Filter the mixtures and wash the residues with DMF (4 × 1 mL), then DCM (4 × 1 mL). *

Preswell the resins in DCM then add 0.25 mmol Ac2O and 0.5 mmol pyridine in DCM (1 mL)

and shake the mixtures for 30 min. Filter the mixtures and wash with DCM (2 × 1 mL) and

DMF (4 × 1 mL). *

Deprotection: The Fmoc protecting group should be removed by treatment of the resins with

a solution of piperidine/DMF (2/8, 1 mL) for 5 min then filter the solutions of and wash the

resins with DMF (2 mL). Repeat the treatment once more. Then wash the resin with with

DMF (4 × 2 mL) and DCM (4 × 2 mL). (Finally the piperidine reagent should be removed

carefully, otherwise during the next coupling partial deprotection may occur. At no time

during the deprotection is the resin allowed to become dry.)

Coupling of the second FmocAA.

Dissolve 9 × 0.25 mmol FmocAA1, 9 × 0.25 mmol HOBt and 9 × 0.25 mmol HBTU in 8 mL

DMF and add 9 × 0.5 mmol DIPEA to the mixture. Wait for 2 min, then the solution

distribute and add to the resin (to 9 vessel!! Eg A1, A3, B2, C1, C3, D2, E1, E3, F2). Dissolve

9 × 0.25 mmol FmocAA2, 9 × 0.25 mmol HOBt and 9 × 0.25 mmol HBTU in 8 mL DMF and

add 9 × 0.5 mmol DIPEA to the mixture. Wait for 2 min, then the solution distribute and add

to the resin (next 9 vessel!! Eg A5, B4, B6, C5, D4, D6, E5, F4, F6). Dissolve 9 × 0.25 mmol


FmocAA3, 9 × 0.25 mmol HOBt and 9 × 0.25 mmol HBTU in 8 mL DMF and add 9 × 0.5

mmol DIPEA to the mixture. Wait for 2 min, then the solution distribute and add to the resin

(last 9 vessel!! Eg A7, A9, B8, C7, C9, D8, E7, E9, F8) and shake the mixture for 1 h at r.t.

Filter the mixtures and wash the residues with DMF (4 × 1 mL), then DCM (4 × 1 mL).*



with DMF (1 mL), then repeat the treatment once more. Then wash the resin with with DMF

(4 × 2 mL) and DCM (4 × 2 mL).

Coupling of the third FmocAA.


DMF and add 9 × 0.5 mmol DIPEA to the mixture. Wait for 2 min, then the solution

distribute and add to the resin (to 9 vessel!! Eg A1, A7, B4, C1, C7, D4, E1, E7, F4). Dissolve

9 × 0.25 mmol FmocAA2, 9 × 0.25 mmol HOBt and 9 × 0.25 mmol HBTU in 8 mL DMF and

add 9 × 0.5 mmol DIPEA to the mixture. Wait for 2 min, then the solution distribute and add

to the resin (next 9 vessel!! Eg A5, B2, B8, C5, D2, D8, E5, F2, F8). Dissolve 9 × 0.25 mmol

FmocAA3, 9 × 0.25 mmol HOBt and 9 × 0.25 mmol HBTU in 8 mL DMF and add 9 × 0.5

mmol DIPEA to the mixture. Wait for 2 min, then the solution distribute and add to the resin

(last 9 vessel!! Eg A3, A9, B6, C3, C9, D6, E3, E9, F6) and shake the mixture for 1 h at r.t.

Filter the mixtures and wash the residues with DMF (4 × 1 mL), then DCM (4 × 1 mL). *



with DMF (4 × 1 mL), then repeat the treatment once more . Then wash the resin with with

DMF (4 × 2 mL) and DCM (4 × 2 mL).

Finally, after the last deprotection step the resins should be washed with DMF (4 × 1 mL),


under vacuum.


Cleavage: Add TFA (95% and 5% water, 0.5 mL (this has to prepared in advance)) to the

resins and shake for 2h. Then filter the resins and wash with TFA (95% and 5% water, 0.5

mL). Then the solutions should be dried under a vacuum exiccator filled with powdered

KOH. (Close the vessels with parafilm, and make some holes on that with Pasteur pipette.)

The dry samples should be dissolved in water (0.5 mL) deep freeze and lyophilised.

Analysis: ~10 Samples should be selected for LC-MS, and the rest should analysed by

NMR (in DMSO D6).

For the LC-MS you should prepare the samples: take 0.5 mg compound and dissolve that in

MeOH (1 mL), then filter into a eppendorf tube. Calculate the sumformula and the molecular

weight.

*

This point is a convenient place to end the laboratory period. After each coupling-washing

procedure the resin can be stored under DCM in the fridge till the next lab day.


Solid phase peptide synthesis 3

Synthesis of split-mix tripeptide library on Wang resin with Fmoc strategy



Working with DMF and DCM please keep them clean!



Mission:

You will synthesise two tripeptide libraries using split-mix method and Fmoc chemistry. The

synthesis makes use of functionalisation of the resin, condensation and deprotection reactions,

cleavage protocols. Finally you will have 2 tripeptide libraries, which will be analysed by

HPLC and MS.

o o o

o o o

o

o o

o o

o o

derivatization of the resins with the first amino acid

mixing of the resins

distribution of the resins

deprotection-coupling steps untillthe desired sequence is obtained

cleavage from the resin and analysis of the compounds


Reactions:


Place 60-60 mg of Wang resin to 3 reaction vessel and preswell that in DMF for 1 h.

Dissolve 0.25 mmol FmocAA1 and 0.25 mmol HBTU in 1 mL DMF and add 0.5 mmol

DIPEA to the mixture. Wait for 2 min, then add the solution to the first reaction vessel.


DIPEA to the mixture. Wait for 2 min, then add the solution to the second reaction vessel.


DIPEA to the mixture. Wait for 2 min, then add the solution to the third reaction vessel and

shake the mixtures for 3 h at r.t. Filter the mixtures and wash the residues with DMF (4 × 1

mL), then DCM (4 × 1 mL).

Combine the resins into one vessel, mix it and distribute that into two vessel.

Preswell the resins in DCM then add 0.25 mmol Ac2O and 0.5 mmol pyridine in DCM (1 mL)

and shake the mixtures for 30 min. Filter the mixtures and wash with DCM (2 × 1 mL) and

DMF (4 × 1 mL),

Deprotection: The Fmoc protecting group should be removed by treatment of the resins with

a solution of piperidine/DMF (2/8, 1 mL) for 5 min then filter the solutions of and wash the

resins with DMF (2 mL). Repeat the treatment once more, and wash the resin with DMF (4 ×

2 mL) and DCM (4 × 2 mL). (Finally the piperidine reagent should be removed carefully,

otherwise during the next coupling partial deprotection may occur. At no time during the

deprotection is the resin allowed to become dry.)

Coupling the next AA.

Dissolve 0.35 mmol FmocAA4, 0.35 mmol HOBt and 0.35 mmol HBTU in 1 mL DMF and

add 0.75 mmol DIPEA to the mixture. Wait for 2 min, then add the solution to the first

reaction vessel. Dissolve 0.35 mmol FmocAA5, 0.35 mmol HOBt and 0.35 mmol HBTU in 1

mL DMF and add 0.75 mmol DIPEA to the mixture. Wait for 2 min, then add the solution to

the second reaction vessel and shake the mixtures for 1 h at r.t. Filter the mixtures and wash

the residues with DMF (4 × 1 mL), then DCM (4 × 1 mL).*




with DMF (1 mL), then repeat the treatment once more, and wash the resin with DMF (4 × 2

mL) and DCM (4 × 2 mL)..

Coupling the next AA.


DMF and add 2 × 0.75 mmol DIPEA to the mixture. Wait for 2 min, then distribute the

solution into the two reaction vessel and shake the mixtures for 1 h at r.t. Filter the mixtures

and wash the residues with DMF (4 × 1 mL), then DCM (4 × 1 mL).*



with DMF (1 mL), then repeat the treatment once more, and wash the resin with DMF (4 × 2

mL) and DCM (4 × 2 mL).

Finally, after the last deprotection step the resins should be washed with DMF (4 × 1 mL),


under vacuum.

Cleavage: Add TFA (95% and 5% water, 0.5 mL (this has to prepared in advance)) to the

resins and shake for 2h. Then filter the resins and wash with TFA (95% and 5% water, 0.5

mL). Then the solutions should be dried under a vacuum exciccator filled with powdered

KOH. (Close the vessels with parafilm, and make some holes on that with Pasteur pipette.)

The dry samples should be dissolved in water (0.5 mL) deep freeze and lyophilised.

Analysis: The two libraries should be analysed by LC-MS and the results compared with

the Solid phase peptide synthesis 2 experiment .

For the LC-MS you should prepare the samples: take 0.5 mg compound into an eppendorf

tube and dissolve that in MeOH (1 mL), then filter into a eppendorf tube. Calculate the

sumformula and the molecular weight.

*

This is a convenient place to end the laboratory period. After each coupling-washing

procedure the resin can be stored under DCM in the fridge till the next lab day.


Solid supported synthesis of oligoamide

Synthesis of olioamides using PEG monomethylether as a solid support


Working with DMF, DCM and MeOH please keep them clean!

CH3O(CH2CH2O)n-H

O

ClNO2

CH3O(CH2CH2O)n

ONO2

CH3O(CH2CH2O)n

ONH2

O

ClNO2

CH3O(CH2CH2O)n

ONO2

ONH

ONO2

ONHO

NHO


Mission:

You will synthesize a diamide and a triamide using PEG as a solid support. The synthesis

makes use of condensations and reductions, cleavage protocols, and specially work up

protocols for PEG coupled products. Finally you will have oligoamides, which will be

analyzed by NMR and MS.

Reactions:

Functionalisation of the PEG support.

Dissolve 5g of PEG monomethylether (MW:~5000) in DCM (10 mL) and add pyridine (2

ml). Dissolve 5 ekv of p-nitro benzoyl chloride in DMF (2 mL) and add this solution to the

solution of the PEG. Stir the mixture overnight at room temperature. Then isolate your

compound by precipitation with diethyl-ether (400 mL). Dissolve the precipitate in DCM (10

ml DCM per 1 g of precipitate) and precipitate once more with diethyl-ether (400 mL).

Check the yield of the coupling by NMR.

Make the sample in CDCl3 (50-100mg), and compare the integral of the aromatic protons with

the OMe group of the PEG. The functionalisation should be ~100%, otherwise the coupling

must be repeated.

Reduction: Dissolve the functionalised PEG in MeOH (10 mL per 1g compound, if necessary

and small amount of DCM). Add 40 ekv of ammonium formiat and Pd\C (100 mg, per 1 g

compound) and stir the mixture vigorously overnight.

Then filter the mixture through CELITE, then precipitate with diethyl-ether (400 mL),

redissolve the precipitate in DCM, then filter through CELITE and wash with DCM and

finally precipitate once more with diethyl-ether (400 mL). Check the yield of the reaction by

NMR.

Make the sample in CDCl3 (50-100mg), and estimate the yield by the shift of the acylated

CH2 group.

Amide coupling: Dissolve the reduced compound in DCM (10 ml DCM per 1 g compound)

and add pyridine (10 ekv). Dissolve 5 ekv of p-nitro benzoyl chloride in DMF (5 mL per 1 g

of chloride) and add this solution into the solution of the PEG. Stir the mixture overnight at

room temperature. Then isolate your compound by precipitation with diethyl-ether (300 mL).


Dissolve the precipitate in DCM (10 ml DCM per 1 g of precipitate) and precipitate once

more with diethyl-ether (300 mL).

Check the yield of the coupling by NMR.

Make the sample in CDCl3 (50-100mg), and compare the integral of the aromatic protons with

the OMe group of the PEG, and check the shift of the acylated CH2 group. The

functionalisation should be ~100%, otherwise the coupling must be repeated

Cleavage: Procedure I: Dissolve the compound in MeOH, and add NaOMe until the pH 9 is

reached. Stir the reaction mixture overnight, then dilute with water and EtOAc, extract and

isolate the organic phase, dry, filter and concentrate.

Procedure I: Dissolve the compound in 2N KOH and stir it at r. t. for 2 h. Neutralize with HCl

and add EtOAc and water, extract and isolate the organic phase, dry, filter and concentrate.

.

Check the diamide by NMR and MS.

Preparation of triamide:

Repeat the reduction and the condensation step once more.

Reduction: (Same as before) Dissolve the functionalised PEG in MeOH (10 mL per 1g

compound, if necessary and small amount of DCM). Add 40 ekv of ammonium formiat and

Pd\C (100 mg, per 1 g compound) and stir the mixture vigorously overnight. Isolate the

compound by precipitation the same manner as after the first reduction.

Check the yield of the reaction by NMR; make the sample in CDCl3 (50-100mg), and

estimate the yield by the shift of the acylated CH2 group.

Amide coupling: (Same as before) Dissolve the reduced compound in DCM (10 ml DCM per

1 g compound) and add pyridine (10 ekv). Dissolve 5 ekv of p-nitro benzoyl chloride in DMF

(5 mL per 1 g of chloride) and add this solution into the solution of the PEG. Stir the mixture

overnight at room temperature. Then isolate your compound by precipitation with diethyl-

ether (250 mL). Dissolve the precipitate in DCM (10 ml DCM per 1 g of precipitate) and

precipitate once more with diethyl-ether (250 mL).

Check the yield of the coupling by NMR; make the sample in CDCl3 (50-100mg), and

compare the integral of the aromatic protons with the OMe group of the PEG, and check the


shift of the acylated CH2 group. The functionalisation should be ~100%, otherwise the

coupling must be repeated

Cleavage: Procedure I: Dissolve the compound in MeOH, and add NaOMe until the pH 9 is

reached. Stir the reaction mixture overnight, then dilute with water and EtOAc, extract and

isolate the organic phase, dry, filter and concentrate.

Procedure I: Dissolve the compound in 2N KOH and stir it at r. t. for 2 h. Neutralize with HCl

and add EtOAc and water, extract and isolate the organic phase, dry, filter and concentrate.

Check the diamide by NMR (in DMSO d6) and MS.


Synthesis with polymer supported reagents


Working with DMF, DCM and MeOH please keep them clean!

OH

O

NH2

N

NH

NH2

NO2

O

Cl

N

NO2

O

Oxidation: TEMPO-resin

Reductive amination: CNBH3-resin

Remove the excess of amine by using4 benzyloxybenzaldehyde resin.

Acylation using piperidinomethyl-PS resin as a base

Remove the excess of acid chloride usingaminomethylated PS.

NO2

O

Cl


Mission:

You will synthesize an acylated secondary amine in solution phase. The synthesis makes use

of resins as catalyst or promoters for organic reactions. Other resins will be used for removing

the excess of reagent you applied. Finally your compound will be analyzed by NMR and MS.

Reactions:

Oxidation with TEMPO polystyrene.

Dissolve N-chlorosuccinimide (524 mg, 3.92 mmol) in DCM containing 4N HCl in dioxane

(1.2 mL, 4.7 mmol). After 5 min add the solution to TEMPO polystyrene (0.5 g, 0.87 mmol)

preswollen in DCM. Agitate the mixture for 15 min, and then isolate the resin by filtration,

wash with dry DCM.

Add this material to a solution of benzyl alcohol (16 µL, 0.157 mmol) dissolved in DCM.

Agitate the solution for 1 h at rt. Then remove the resin by filtration, wash with DCM.

Evaporate the combined filtrates to dryness provide benzaldehyde.

Record the 1H NMR spectrum of the compound in CDCl3. (Recover the compound after the

measurement.)

Reductive amination:

Dissolve N-hexylamine (0.314 mmol) in a 1:10 mixture of AcOH : DCM (2 mL) and shake at

rt. Add benzaldehyde (prepared in the step before), followed by (polystyrylmethyl)ammonium

cyanoborohydride (0.4 mmol) and shake the reaction mixture for 16h at rt. Filter the resin and

wash with DCM and dry the combined filtrates provide the crude reaction mixture.


measurement.)

Remove the excess of amine by 4-benzyloxybenzaldehyde polystyrene.

Add the resin (3 eq. Relative the excess of amine) to the crude reaction mixture in DCM.

Stir for 3 h at 50 oC, and the reaction may be catalyzed by the addition of AcOH.

Remove the resin by filtration and wash with DCM (3x).


measurement.)


Acylation of the secondary amine using piperidinomethyl polystyrene as a base:

Stirr the mixture of benzylhexylamine (the product you prepared before, 0.157 mmol) and

piperidinomethyl polystyrene (6 eq.) in DCM. Add p-nitro benzoylchloride (3 eq.) to the

mixture and stir for o/n. Remove the resin by filtration and wash with DCM. Evaporate the

combined filtrates to yield the crude mixture.

Remove the excess of acyl chloride using aminomethyl polystyrene:

Add the amine resin (6eq. to the excess of acyl chloride) to the crude reaction mixture in

DCM. Agitate gently for 4 h at rt. Remove the resin by filtration and wash the resin with

DCM. Evaporate the combined filtrates to dryness.

Check your compound by NMR (in CDCl3) and MS.


Chemical Reactions in a PASSflow-apparatus The basis of this new technology are monolithic microreactors, which allow to perform

chemical reactions with immobilised reagents in flow through processes.

The proprietary composites are loaded with reagents or catalysts (C). Chemical reactions (A=

starting material) take place on the surface leaving used reagents in the reactor. The product

(B) passes the column, i.e. no work up is necessary (e.g. without extraction nor filtration).


1) Reductive Amination with NaBH4

OH

H

H

H

OH

H

H

H

ONH

+NMe3BH4-

MeOH, BnNH2

a) Microreactor Preparation

The microreactor has a total capacity of approximately 0.4 mmol. In order to generate the

chloride form of the microreactor, preparation should be done by the following washing

procedure (flow rate 5 mL/min, check the flow direction):

1. washed with 15 mL of ethanol

2. washed with 15 mL of water

3. washed with 15 mL of 1N NaOH


5. washed with 15 mL of 1N HCl



b) Loading of the microreactor with BH4-

The microreactor (Cl--Form) was loaded with 50 mL of an 0.25 M aqueous solution of

sodium borohydride (flow rate 7 mL/min). The reactor was then washed with 10 mL of water

and 15 mL of methanol.

c) Reaction

A solution of 5α-Androstan-17β-ol-3-one(41.6 mg, 0.125 mmol) and benzylamine (16.4 µL,

0.15 mmol) in methanol (40 mL) was pumped in a cycle mode through the BH4--loaded

microreactor for 12 hours at room temperature. After the complete conversion, the

microreactor was rinsed with methanol (20 mL) and the combined organic mixtures were

concentrated under vacuum. (yield: 85 %, (β/α = 4:1)), (small amounts of reduced alcohol

were found as well.)

After evaporation the product is analyzed by NMR.


d) Regeneration of the microreactor

In order to regenerate the chloride form of the microreactor again, regeneration should be

done by the following washing procedure(flow rate 5 mL/min) should be carried out:









2) Reduction of aldehydes with NaBH4

CHO OH

OMeOMe

EtOH, r.t.

+NMe3BH4-

a) Microreactor Preparation

The microreactor has a total capacity of approximately 0.4 mmol. In order to generate the

chloride form of the microreactor, preparation should be done by the following washing

procedure(flow rate 5 mL/min, take care of the flow direction):







7. washed with 15 mL of ethanol b) Loading of the microreactor with BH4

-

The microreactor (Cl--Form) was loaded with 50 mL of an 0.25 M aqueous solution of

sodium borohydride (flow rate 7 mL/min). The reactor was then washed with 10 mL of water

and 15 mL of methanol.

c) Reaction

A solution of the aldehyde (0.25 mmol) in 10 mL of ethanol was pumped in a cycle mode

through the BH4--loaded microreactor at room temperature with a flow rate of 4 mL/min.

After the complete conversion, the microreactor was rinsed with ethanol (20 mL) and the

combined organic mixtures were concentrated under vacuum.

After evaporation the product is analyzed by NMR.


d) Regeneration of the microreactor

In order to regenerate the chloride form of the microreactor following washing

procedure(flow rate 5 mL/min) should be carried out:









Discovery of novel catalyst for allylic alkylation with a visual colorimetric

assay

Transition metal catalysed allylic alkylation represents a powerful tool for the formation of

carbon-carbon bonds. Next to all investigations focused on palladium complexes as catalytic

active species, other transition metals such as nickel, rhodium, iron, molybdenum, ruthenium

etc. have also shown promise.

Therefore novel catalysts for allylic alkylation in neutral medium should be discovered with

the aid of a fast, parallel colorimetric screening.

Catalytical studies on the reaction allylic alkylation

O

O

O OO

R

O

R

OH

O

R

O

R

O

R

O

R

Cl

NN

OH

+MLn

Cl

NN+

Cl

CO2

MLn

-


Mission:

You will study the reaction allylic alkylation in catalytical point of view. The strategy for the

detection of allylic alkylation in based on the fact that colorless 1-naphtol will react with the

diazonium salt to give a color product. Under these reaction conditions the starting allyl-

naphtyl carbonate does not react with the Fast Red diazonium salt. You will use different

substrates and allyl scavenger, and you have to find the right metal salt – ligand combination

for the reaction.

Reactions:

Prepare the following solutions:

A:

50 mL 0.1 M naphtyl-allyl carbonate in THF.

B:

B1: 20 mL 0.4 M DABCO (1,4-diazabicyclo[2.2.2] octane) in THF.

B2: 20 mL 0.4 M dimedon (5,5-dimethyl-1,3-cyclohexanedione) in THF.

B3: 20 mL 0.4 M diethyl malonate in THF.

B4: 20 mL 0.4 M pyridine

C:

C1: 10 mL 0.001 M FeCl3 in THF.

C2: 10 mL 0.001 M NiCl2 in THF.

C3: 10 mL 0.001 M PdAc2 in THF.

D:

D1: 10 mL 0.001 M PPh3 in THF.

D2: 10 mL 0.001 M P(OEt)3 in THF.

D3: 10 mL 0.001 M P(o-Tol)3 in THF.

Solution of Fast Red: 50 mg of fast red dissolved in THF (2 mL) and add water (0.1 mL )

At the end of this handout you find a matrix for each reaction. So you don`t have to label each

reaction tube.


B1:

Take 9 reaction tubes and add 0.5 mL of solution A and B1 into each tubes. Then add 0.5 mL

of C1 to the first 3 tubes C2 to the second 3 tubes and C3 to the third 3 tubes, and 0.5 mL of

D1 to the first, 4th and the 7th tubes, D2 to the 2nd, 5th and the 8th tubes, D3 to the 3rd, 6th, and

9th tubes. Close the tubes and let them stand for 2 h, and keep them dry as possible. Then add

a solution of Fast Red in wet THF (THF/H2O 20/1), and check the change of the color.

*

B2:






*

B3:






*

B4:






If necessary Finally blind samples should be prepared using 0.5 mL pure THF instead of the

use of the solutions of the metal salts, or the ligands. This is necessary to show, that ligands,

or metal salts do not generate color on treatment with Fast Red. For the preparation of blind

samples you can only use diethyl malonate as substrate.


Parallel Combinatorial Esterification and Identification of Different

Odours

The success of combinatorial experiments always depends on general and efficient reaction

procedures. Simple and practical screening methods are also necessary to analyze the large

number of synthesised compounds. Therefore simple biochemical analytical tests are often

used to identify the molecule of interest in a very short and fast way.


Use the assigned syringe for each vessel. Each syringe has to be

cleaned with water and aceton after each use!

Mission:

In this experiment you will learn how to create a small library of eight esters by sulfuric-acid

catalyzed Fischer esterification in a parallel way. After a short work-up (also done in parallel)

a distinct odour should be identified.

Reactions:

All esterifications are performed in a heating-block with a special cooling system.


The reaction tubes should be labeled as shown below:

A B The two columns A and B are filled with two different alcohols. A1-A4 and B1-B4 represent

acids used in the different reactions.

Each reaction tube should be filled with the carbonic acid first followed by the necessary

amount of alcohol and sulfuric acid. If you have to work with anthranilic acid the whole

mixing procedure should be performed in a ice bath (exothermic reaction).

Once all the reactants have been added, the cooling system has to be plugged into each tube

and the heating block turned on (maximum temperature setting). Each reaction should reflux

for approximately 30 minutes.

Each group only has to identify one of the characteristic odours (wintergreen, banana or pear).

a) Identification of Wintergreen (tooth paste, chewing gum)

A: Methanol

A1: 6 mmol anthranilic acid 50 mmol MeOH 40 drops H2SO4

A2: 6 mmol benzoic acid 24 mmol MeOH 15 drops H2SO4

A3: 6 mmol salicylic acid 24 mmol MeOH 15 drops H2SO4

A4: 12 mmol propionic acid 24 mmol MeOH 15 drops H2SO4

A1

A2

A3

A4

B1

B2

B3

B4


B: Ethanol

B1: 6 mmol anthranilic acid 50 mmol EtOH 40 drops H2SO4

B2: 6 mmol benzoic acid 24 mmol EtOH 15 drops H2SO4

B3: 6 mmol salicylic acid 24 mmol EtOH 15 drops H2SO4

B4: 12 mmol propionic acid 24 mmol EtOH 15 drops H2SO4

b) Identification of Banana

A: 1-Methylpropanol

A1: 12 mmol butyric acid 24 mmol 1-Methylpropanol 15 drops H2SO4

A2: 6 mmol benzoic acid 24 mmol 1-Methylpropanol 15 drops H2SO4

A3: 6 mmol salicylic acid 24 mmol 1-Methylpropanol 15 drops H2SO4

A4: 24 mmol acetic acid 24 mmol 1-Methylpropanol 15 drops H2SO4

B: 3-Methyl-1-butanol

B1: 12 mmol butyric acid 24 mmol 3-Methyl-1-butanol 15 drops H2SO4

B2: 6 mmol benzoic acid 24 mmol 3-Methyl-1-butanol 15 drops H2SO4

B3: 6 mmol salicylic acid 24 mmol 3-Methyl-1-butanol 15 drops H2SO4

B4: 24 mmol acetic acid 24 mmol 3-Methyl-1-butanol 15 drops H2SO4

c) Identification of Pear

A: Propanol

A1: 12 mmol butyric acid 24 mmol Propanol 15 drops H2SO4

A2: 6 mmol benzoic acid 24 mmol Propanol 15 drops H2SO4

A3: 6 mmol salicylic acid 24 mmol Propanol 15 drops H2SO4

A4: 24 mmol acetic acid 24 mmol Propanol 15 drops H2SO4


B: Isopropanol

B1: 6 mmol anthranilic acid 50 mmol Isopropanol 40 drops H2SO4

B2: 6 mmol benzoic acid 24 mmol Isopropanol 15 drops H2SO4

B3: 6 mmol salicylic acid 24 mmol Isopropanol 15 drops H2SO4

B4: 12 mmol propionic acid 24 mmol Isopropanol 15 drops H2SO4

Work-up: After the reaction mixtures have cooled down to room temperature, a 20 ml syringe, labeled

according to the reaction tube, is used to get the mixture out of the tube. The tube is washed

with 3 ml ether, 3 ml water and 3 ml ether and all liquids are collected in the same 20 ml

syringe.This 20 ml syringe is then used for extraction. The extraction procedure can be

performed for all eight reactions at the same time.

The organic layer is extracted twice with 5% NaHCO3 solution. Each ether phase is then filled

into a small glass vial (which is labeled!!) and closed with a plastic cap.

To identify the odour of each ester a piece of paper towel is dipped into the vial.

Note: You should be able to identify more than just the odour required!


B1

D1

D2

D3

C1

C2

C3


B2

D1

D2

D3

C1

C2

C3


B3

D1

D2

D3

C1

C2

C3


B4

D1

D2

D3

C1

C2

C3

combinatorial chemistry and solid phase synthesis …...department of chemistry and pharmacy -...

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