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8th December 2011 @ 08:26
*PS = Pictet-Spengler

Pictet-Spengler (PS) cyclisation to give racemic PZQ (KAB3-3), the dimethoxy N-benzoyl PZQ analogue (KAB8-1), the dimethoxy PZQ analogue (KAB1-2) and the N-benzoyl PZQ analogue (KAB7-1).




Scheme



Hazard and Risk Assessment
Hazard and Risk Assessment



Pre-Procedure

08/12/11
Mass spectra (MS) recorded for all four intermediates prior to PS reaction. The MS of the dimethoxy and dimethoxy N-benzoyl intermediates corresponded to the expected m/z of the molecular ion and daughter ions.
KAB5-1 and KAB6-1 appeared anomalous. Expected molecular ion m/z was ~377. These were absent, but both showed very strong peaks with m/z at 427 and 420, respectively. These correspond to the ethoxy-acetal. Consequently, notes for KAB5-1 and KAB6-1 were noted to be corrected.
The starting materials were deemed to be adequate for the following reaction.
MS KAB5-1
MS KAB6-1


Procedure
The starting materials for the reaction were added to 100 mL round bottom flasks then dissolved in toluene. Following the addition of methanesulfonic acid (MSA), the reaction mixtures were reflux heated at 70C.
Five 0.1 mL aliquots were extracted from each of the 4 reaction mixtures over the duration of the reflux. The aliquots were quenched in saturated sodium bicarbonate (2 mL) and EtOAc (1 mL). The reaction progress was monitored by TLC of the aliquots against the corresponding starting materials (EtOAc/hexane, 3:1, KMnO4 stain).

KAB3-3:
Structure KAB3-3

Starting material (KAB5-1) added - 2.3 g, 5.7 mmol
MSA added - 0.8 mL, 5.7 mmol
Toluene - 20 mL, ~50 eq.
2:15 pm - acid added
2:28 pm (+13 min) - reached 70.0C
2:49 pm (+34 min) - first aliquot taken
3:05 pm (+50 min) - second aliquot taken
3:21 pm (+66 min) - third aliquot taken
3:32 pm (+77 min) - fourth aliquot taken
3:42 pm (+87 min) - fifth aliquot taken
4:04 pm (+109 min) - taken off heat

KAB8-1:
Structure KAB8-1

Starting material (MNR10-1) added - 2.2 g, 4.8 mmol
MSA added - 0.7 mL, 5 mmol
Toluene - 20 mL, ~50 eq.
2:15 pm - acid added
2:23 pm (+ 8 min) - reached 70.0C
2:37 pm (+ 22 min) - first aliquot taken
2:47 pm (+32 min) - second aliquot taken
3:04 pm (+49 min) - third aliquot taken
3:20 pm (+65 min) - fourth aliquot taken
3:40 pm (+85 min) - fifth aliquot taken
4:04 pm (+109 min) - taken off heat

KAB1-2
Structure KAB1-2

Starting material (MNR8-1) added - 1.29 g, 2.78 mmol
MSA added - 0.4 mL, 3 mmol
Toluene - 18 mL, ~50 eq.
2:15 pm - acid added
2:34 pm (+19 min) - reached 66.5C
2:50 pm (+35 min) - first aliquot taken
3:06 pm (+51 min) - second aliquot taken
3:22 pm (+67 min) - third aliquot taken
3:33 pm (+78 min) - fourth aliquot taken
3:44 pm (+89 min) - fifth aliquot taken
4:04 pm (+109 min) - taken off heat

KAB7-1
Structure KAB7-1

Starting material (KAB6-1) added - 2.2 g, 5.6 mmol
MSA added - 0.8 mL, 5.6 mmol
Toluene - 21 mL, ~50 eq.
2:15 pm - acid added
2:35 pm (+20 min) - reached 63.6C
2:51 pm (+36 min) - first aliquot taken
3:07 pm (+52 min) - second aliquot taken
3:22 pm (+67 min) - third aliquot taken
3:33 pm (+78 min) - fourth aliquot taken
3:45 pm (+90 min) - fifth aliquot taken
4:04 pm (+109 min) - taken off heat

Reaction Setup


Final TLC KAB3-3
Final TLC KAB8-1
Final TLC KAB1-2
Final TLC KAB7-1

Final extracted aliquots of all reaction mixtures against corresponding starting materials. Plates from L to R: KAB3-3, KAB8-1, KAB1-2, KAB7-1

All aliquots of all products and starting materials

Plates from L to R: KAB3-3, KAB8-1, KAB1-2, KAB7-1. On each plate from L to R: (1)1st aliquot (2)2nd aliquot. (3)3rd aliquot. (4)Starting material. From plates L to R, KAB5-1, MNR10-1, MNR8-2, KAB6-1. (5)4th aliquot. (5)5th aliquot. First, second and fourth plates (L to R) in EtOAc/hexane, 3:1, v/v. Third plate was eluted with X, as the substrate and product had very similar retention. All were stained with KMnO4.

All reactions had gone to completion by 109 minutes following the addition of MSA. The reaction mixtures were allowed to cool to room temperature. Each reaction mixture was diluted with EtOAc (50 mL) then washed with saturated sodium bicarbonate (50 mL) to quench the acid. After the organic fraction was separated, the aqueous layer was extracted with EtOAc (3 x 50 mL). The organic fractions were combined, dried over magnesium sulfate, filtered, then concentrated under reduced pressure to give the crude Pictet-Spengler cyclised products.

Crude KAB3-3

Crude KAB3-3: 1.834 g (Flask: 148.141 g)

Crude KAB8-1

Crude KAB8-1: 948 mg (Flask: 159.360 g)

Crude KAB1-2

Crude KAB1-2: x g (Flask: 123.733 g)

Crude KAB7-1

Crude KAB7-1: 2.217 g (Flask: 152.838 g)

NOTE: Following workup of KAB8-1, the reaction mixture round bottom flask contained a red oily substance. This remained in the flask after the reaction mixture was removed by pouring to the separating funnel. After standing at room temperature for ~20 min, the oil had formed crystals, which were store at room temperature then at 4C.


09/12/11
Samples to be purified by column chromatography.

TLC comparison of the final aliquots of all four products vs PZQ

Crude KAB3-3 vs PZQ

This is the second TLC that was performed, to confirm the first. Both showed the same result, suggesting the product formed from the PS of KAB5-1 was not racemic PZQ.
Crude product was a dark reddish-brown solid

Crude KAB8-1 vs PZQ


Crude KAB1-2 vs PZQ


Crude KAB7-1 vs PZQ
Crude KAB7-1 vs PZQ (2)
Crude KAB7-1 vs PZQ (3)

Three TLCs were run containing the 5th aliquot of KAB7-1. It initially appeared as though something was eluting with the same retention as PZQ, which could have explained the absence of the PZQ spot in KAB3-3. i.e. possible mix-up when weighing out KAB5-1 and KAB6-1. It was difficult to resolve by TLC. Both KAB3-3 and KAB7-1 were processed by column chromatography.

Fractions 7-8, flask 50.9868 g
Fractions 9-18, 0.568 g as a yellow oil (flask 152.838 g)

1:52 pm - KAB2-1 and KAB8-1 were removed from the freezer and left at room temperature from 8:30 am. Formation of crystals.
Murray recrystallised both samples.

12/12/11

Purification crude KAB1-2 (~820 mg) by silica gel column chromatography, 20 cm, 4 cm diameter, EtOAc/hexane 60-100%(v/v). Collected 76 fractions, monitored by TLC (EtOAc/hexane, 75% (v/v), KMnO4 stain).

TLC of columned KAB1-2, fraction number indicated in TLC lane
KAB1-2 combined fractions 40-70


Combined fractions.
1-39 (4.6 mg): Recorded NMR. As expected, the NMR showed a mix of products. (UPLOAD NMR)
40-70 (~700 mg): Concentrated under reduced pressure, NMR pending.
71-76:
Approximate total recovery ~80% (not inc. 71-76).

Murray columned non-crystallised KAB8-1 (948 mg).
NMR of the crystallised KAB8-1 suggested the product was not clean ---> column the crystals?

The NMR of KAB7-1, fractions 9-18, suggested a mixture of the cyclisation intermediate and product: N-benzoyl PZQ analogue. Integration values calibrated to the two doublets corresponding to the (tautomerised imine?) indicate the majority of the fraction was the intermediate.
The NMR of fractions 6-8 contained clean peaks corresponding to adjacent CH2, CH3 (ethoxy?), presumably the leaving group from the partial/complete Pictet-Spengler cyclisation.
Analysis ongoing.

15/12/11
In order to account for mass, the aqueous layer of KAB8-1 was re-extracted. The pH of the aqueous layer of KAB8-1 was 9. After extracting with dichloromethane (DCM, 3 x 30 mL), the strong yellow colour of the aqueous fraction faded to a pale yellow. The final DCM extraction fraction was almost colourless. The pH was adjusted to 5 with hydrochloric acid (1 M) and the aqueous fraction was further extracted with DCM (2 x 30 mL). The combined organic fractions were dried over magnesium sulfate, filtered, then concentrated under reduced pressure. The amount of product obtained from the re-extraction procedure with DCM across neutral pH was less than 5 mg. Due to the low yield, no further extractions were performed on the aqueous solutions of KAB3-3, KAB7-1 or KAB1-2. All aqueous fractions were retained.
Re-extraction of KAB8-1 aqueous layer (1)
Re-extraction of KAB8-1 aqueous layer (2)
Re-extraction of KAB8-1 aqueous layer (3)


16/12/11
Mass spec of red crystals:
MS of red crystal by-product

1H-NMR
13C-NMR
The 1H-NMR spectrum of the N-benzoyl PZQ analogue (KAB7-1) suggested a mixture of PS cyclised product, the enamine intermediate and the starting material (KAB6-1). The starting material and the product have similar Rf in eluent compositions of EtOAc/hexane, 50-100%, v/v. Separation by silica gel column chromatography was difficult and the major spot (on the TLC) contained all three stages of the reaction.

The dimethoxy PZQ analogues (KAB8-1 and KAB1-2) were solids. 1H-NMR of the purified samples indicated clean samples, however the samples were too dilute ----> need to re-run NMR and obtain C-NMR. Rough yield for both products >500mg (still contained some solvent).
HNMR KAB1-2 (1)


02/01/12
Simon and Goodman proposed mechanism for the action of chiral phosphoric acid involves formation of iminium-catalyst adduct, although not regarding PS reaction (10.1021/ja808715j).
The PS mechanism for the cyclisation of the peptide acetals:
PS mechanism

(concentrating on the by-product of the cyclisation to form KAB8-1)
Possible formation of methanesulfonic acid-ethoxy adduct from steps a to b and g to h. Mass Spec of red crystals recorded on 16/12/11 contained a peak at m/z 164.73. The m/z of methanesulfonic acid-ethoxy adduct, M, has MW of 141.17. The m/z of [M + Na]+ is 164.16.
Peaks at 441 and 991 still unknown.

03/01/12
H-NMR of the columned KAB8-1 and KAB1-2 were re-recorded:
HNMR KAB8-1
H-NMR of KAB1-2

Raw H-NMR KAB8-1
Raw H-NMR KAB1-2

Both contained some solvent peaks (EtOAc and H2O) and were placed under high vacuum at 9:30 am.

TLC of the red crystals (see final NOTE made on 08/12/11 above).
TLC comparison of KAB8-1 red crystals

From left to right: (1)Red crystals, (2)2nd aliquot taken of KAB8-1 reaction mixture, (3)Co-spot, (4)Product (used MW54 instead of KAB8-1 as it was drying under vacuum).

No useful information from this TLC - was trying to resolve co-spots, possibly visualize MS peaks.

KAB7-1 and KAB3-3 were removed from the fridge and stored at rt out of light.

05/01/12
Further analysis of KAB8-1 red product was performed:
MS KAB8-1 red product (re-run)
IR spectrum of KAB8-1 red product
TLC of KAB8-1 red product



--------------------------------------------
Notes:
Crude product of incomplete reactions were brown/red in colour (no dimethoxy). Completed reaction crude products were yellow in colour (dimethoxys).

The reaction is difficult to monitor, particularly of the substituents without the dimethoxy group. So far, TLC (EtOAc/hexane, 3:1, v/v with KMnO4 stain) seems to be the most efficient way of monitoring the reaction progress. Both the product and the starting material have similar Rf. The enamine intermediate resulting in the partial PS cyclisation followed by (deprotonation?) then tautomerisation of the iminium ion has a clearly larger Rf value.
I'm thinking of monitoring the reaction progress by TLC and waiting until the starting material has been completely consumed and leaving it until it seems that the starting material has reappeared, at least by Rf values.
The reaction set-up was good. Use of the two necked flasks during the reflux meant that I could easily obtain samples (via syringe through the septum) without disrupting the reaction conditions - i.e. removing the condensing tube.

02/01/12
Not sure how the MSA can form an adduct with the ethoxy group through the hydrogen. But is the mass spec peak coincidence?
I think there may be two or three products in the "red crystal mix." When I got back to the oil, it had crystallised, but it was difficult to see if the crystals were colourless in some red liquid or if the crystals were red. May try recrystallising the sample.
If the peak at 164 corresponds to the adduct, then it seems as though (some or all?) the acid is not regenerated, as it should be if the reaction proceeds via the PS.
It must form some kind of adduct (associative transition state?) as this forms the basis of enantioselectivity of BINOL acids.

05/01/12
- Re-recorded the mass spectrum, H-NMR, TLC for the red KAB8-1 crystals and recorded IR. With the exception of the MS, all analyses indicate that the major constituent is KAB8-1 itself (i.e. dimethoxy N-benzoyl PZQ analogue). Previously recorded NMR was different to latest recorded NMR of the red product. Similarly, the recorded MS is different, with differently fragmented ions. I can't recall if the red product was quenched with NaHCO3, which may explain why it appears to be KAB8-1 after 2 weeks, though did not appear to be product immediately following the reaction workup.
- Processing C-NMR of both dimethoxy analogues.
- Both dimethoxy analogues are as clean as possible. Samples put under high vacuum in attempt to remove solvent peaks from the H-NMR.

Future Considerations:
USE MOLECULAR SIEVE to remove ethoxy leaving group. 3-5 A sieve? Ref Hiemstra 2007 and 2008.

Conclusion
The reaction went to completion to give both dimethoxy PZQ analogues. It is estimated the procedure went to completions in less than 30 minutes of refluxing at 70 C. The final products of KAB3-3 and KAB7-1, PZQ and the N-benzoyl PZQ analogue, both contained a mixture of the enediamide intermediate and the starting materials.
Formation of the enediamide intermediate and complete consumption of the starting material for both non-dimethoxy products occurred in less than 1 hour.
The yield was low (~23 %) at best (KAB8-1).

--------------------------------------------


Related Experiments
Acid-catalyzed Pictet-Spengler reaction with methanesulfonic acid (MW56-5 to MW56-8)
Acid-catalyzed Pictet-Spengler reaction with binaphthalenedisulfonic acid (MW56-1 to MW56-4)
Acid-mediated Pictet-Spengler of MW29 to give rac-PZQ MNR13-1
Continuation: Acid-catalyzed Pictet-Spengler reaction with methanesulfonic acid (MW56-9 to MW56-14)
Acid-mediated Pictet-Spengler of the N-benzoyl-Ugi intermediate (MW53-1 to MW53-3)
Acid-mediated Pictet-Spengler reaction to give PZQ (KAB3-4) and the N-benzoyl PZQ analogue (KAB7-2)
Acid-mediated Pictet-Spengler reaction to give PZQ (KAB3-5)

Links
Informal commentary and additional photographs here.
Linked Posts
Attached Files
Hazard and Risk Assessment
Scheme
Table
Final TLC KAB3-3
Final TLC KAB8-1
Final TLC KAB1-2
Final TLC KAB7-1
Reaction Setup
All aliquots of all products and starting materials
Structure KAB3-3
Structure KAB8-1
Structure KAB1-2
All four products
Crude KAB3-3 vs PZQ
Crude KAB8-1 vs PZQ
Crude KAB1-2 vs PZQ
Crude KAB6-1 vs PZQ
Crude KAB7-1 vs PZQ (2)
Crude KAB7-1 vs PZQ (3)
Crude KAB7-1 vs PZQ
KAB1-2 combined fractions 40-70
TLC of columned KAB1-2, fraction number indicated in TLC lane
MS KAB5-1
MS KAB6-1
HNMR KAB1-2 (1)
Structure KAB7-1
Crude KAB1-2
Crude KAB3-3
Crude KAB8-1
Crude KAB7-1
Re-extraction of KAB8-1 aqueous layer (1)
Re-extraction of KAB8-1 aqueous layer (2)
Re-extraction of KAB8-1 aqueous layer (3)
MS of red crystal by-product
PS mechanism
H-NMR of KAB1-2
HNMR KAB8-1
Raw H-NMR KAB8-1
Raw H-NMR KAB1-2
TLC comparison of KAB8-1 red crystals
TLC of red crystals
MS KAB1-2
MS KAB7-1 fractions 45-49
MS KAB8-1 red product (re-run)
IR spectrum of KAB8-1 red product
TLC of KAB8-1 red product