Synthesis of Methyl 5-oxo-5-[(tert-butoxycarbonylmethyl)amino]valerate (1). Amide of sarcosine-t-butyl ester with glutaric acid monomethyl ester.
0.1 mEq of sarcosine tert-butyl ester hydrochloride was dissolved in DCM with 0.2 mEq of TEA under stirring at RT. Then 0.1 mEq of glutaric acid monomethyl ester chloride was dissolved in DCM and this solution was added dropwise on the first solution. After 30 min the reaction was complete. It was followed by TLC (DCM:MeOH; 9:1). The crude product was purified by ColCh with a mixture of DCM:MeOH. Final product: meO-glt-Sar-O-tbut. Synthesis of Potassium 5-Oxo-5-(tert-butoxycarbonylamino)valerate (2). Potassium salt of glutaric acid half amide with sarcosine-t-butyl ester.
0.1 mEq of cmpd 1 was dissolved in a mixture of MeOH:water, 9:1. To this solution, 0.1 mEq of KOH was added. The reaction was stirred at RT. After 1 h the reaction was complete. It was followed (Boc UV absorbance) by TLC (DCM:MeOH; 9:1). The solution was dried under vacuum, product in water was extracted in a separation funnel (ether/water), and aqueous phase freeze-dried to give the potassium salt. Final product: KO- glt-Sar-O-tbut.
Synthesis of Methyl 6-oxo-6-[(tert-butoxycarbonylmethyl)amino]hexanoate (3). Amide of sarcosine-t-butyl ester with adipic acid monomethyl ester.
0.1 mEq of glycine tert-butyl ester hydrochloride was dissolved in DCM with 0.2 mEq of TEA under stirring at RT. Then 0.1 mEq of methyl adipoyl acid monomethyl ester chloride was dissolved in DCM and this solution was added dropwise to the first solution. After 30 min the reaction was done. It was followed by TLC (DCM:MeOH; 9:1). The crude product methyl ester was purified by ColCh with a mixture of DCM:MeOH. Final product: meO- adp-Gly-O-tbut.
Synthesis of Potassium 6-Oxo-6-(tert-butoxycarbonylamino)hexanoate (4). Potassium salt of adipic acid half amide with sarcosine-t-butyl ester.
0.1 mEq of cmpd 3 was dissolved in a mixture of MeOH:water 9:1. To this solution, 0.1 mEq of KOH was added. The reaction was stirred at RT. After 1 h the reaction was complete. It was followed (Boc UV absorbance) by TLC (DCM:MeOH; 9:1). The solution was dried under vacuum, product in water was extracted in a separation funnel (ether/water), and aqueous phase freeze-dried to give the potassium salt. Final product: KO- adp-Gly-O-tbut.
Synthesis of Tert-butyl {2-[(4-hydroxypentanoyl)amino]ethyl}carbamate (8). 3- hydroxypentanoic acid amide with mono-Boc-ethylenediamine (a secondary alcohol to which a chromophoric acid can later be attached).
To synthesize a secondary amino alcohol, we have followed the method described by Chalid et al (2011) with some modifications. 0.1 mEq of N-Boc-ethylenediamine was dissolved in 5 mL of methanol in a round bottom flask. Then 0.1 mEq of γ-Valerolactone (Figure S-1) was added to the reaction. The reaction was stirred at RT, and followed by TLC (DCM:MeOH; 9:1). After 16 h the reaction was complete. The mixture was dried under vacuum and purify it by CC (DCM:MeOH). Final produc: Boc-NetN-val-secOH.
O
CH3
O
Figure S-1. Chemical structure of γ-Valerolactone.
There were three compounds (below) for which we used the above alternative synthetic route to attach chromophoric carboxamides to aid in kinetic monitoring of ester hydrolysis, by HPLC.
For example, cmpd 12 is a chromophoric sarcosine amide which can be amide bound to adipic or glutaric esters (left side) of N-blocked amino alcohols to enable facile observation of chromophore detachment following ester hydrolysis (Figure S-11).
The synthesis pathway to achieve the chromophore-sarcosine compound (12) are the following:
Synthesis of Tert butyl (2-[[(3-nitrophenyl)carbonyl]amino]ethyl)carbamate (9). Amide between 3-nitrobenzoic acid and mono-Boc-ethylenediamine.
0.1 mEq of N-Boc-ethylenediamine was dissolved in DCM with 0.1 mEq of TEA under stirring at RT. Then 0.1 mEq of 3-Nitrobenzoyl chloride was dissolved in DCM and this solution was added dropwise on the first solution. After 30 min the reaction was complete. It was followed (nitrophenyl UV absorbance) by TLC (DCM:MeOH; 9:1). The crude product was purified by ColCh with a mixture of DCM:MeOH. Final product: Boc-NetN- ph-NO2.
Synthesis of N-(2-Aminoethyl)-3-nitrobenzamide (10). Removal of Boc group from (9). 0.1 mEq of cmpd 9 was deblocked by dissolution in a mixture of TFA 20 % v/v in DCM at RT. After 17 h the reaction was complete. 20 volumes of toluene were added to the mixture and the sample was dried by rotary evaporation. The reaction was followed by TLC (DCM:MeOH; 9:1). Final product: NetN-ph-NO2.
Synthesis of 2-[N-Methyl(1-tert-butoxyethenyl)amino]-1-[2-(m-nitrobenzoyl-amino) ethylamino]-1-ethanone (11). A chromophoric amide of Boc-Sarcosine.
0.1 mEq of Boc-sarcosine was mixed with 0.2 mEq of TEA and an excess of isobutyl chloroformate in ACN under stirring at RT. On the other hand, after 15 min, 0.1 mEq of cmpd 10 was dissolved in ACN and add it dropwise to the reaction. After 30 min the reaction was complete. It was followed by TLC (DCM:MeOH; 9:1). The crude product was purified by ColCh with a mixture of DCM:MeOH. Final product: Boc-Sar-NetN-ph-NO2.
Synthesis of 2-(Methylamino)-1-[2-(m-nitrobenzoylamino)ethylamino]-1-ethanone (12). Removal of Boc group from (11). The chromophore-Sarcosine compound.
0.1 mEq of cmpd 11 was dissolved in a mixture of TFA 20% v/v in DCM at RT. After 17 h the reaction was complete. 20 volumes of toluene were added to the mixture and the sample was dried by rotary evaporation. The reaction was followed by TLC (DCM:MeOH; 9:1). Final product: Sar-NetN-ph-NO2. (a chromophoric amide of sarcosine)
Synthesis of Methyl 5-(N-methyl{2-[2-(p-nitrobenzoylamino)ethylamino]-2- oxoethyl}amino)-5-oxovalerate (13). Amide of the cmpd 12 with glutaric acid monomethyl ester.
0.1 mEq of cmpd 12 was mixed with 0.2 mEq of TEA in ACN under stirring at RT. Then, 0.1 mEq of glutaric acid monomethyl ester chloride was dissolved in ACN and this solution was added dropwise on the first solution. After 30 min the reaction was complete. The reaction was followed by TLC (DCM: MeOH; 9:1). When the reaction was complete, the mixture was dried under vacuum and extracted with a mixture of water: DCM. The organic phase was collected and dried under vacuum. Final product: meO-glt-Sar-NetN-ph-NO2.