Synthesis of Mono-and Dithiols of Tetraethylene Glycol and Poly ( ethylene glycol ) s via Enzyme Catalysis

This paper investigates the transesterification of methyl 3-mercaptopropionate (MP-SH) with tetraethylene glycol (TEG) and poly(ethylene glycol)s (PEG)s catalyzed by Candida antarctica Lipase B (CALB) without the use of solvent (in bulk). The progress of the reactions was monitored by 1H-NMR spectroscopy. We found that the reactions proceeded in a step-wise manner, first producing monothiols. TEG-monothiol was obtained in 15 min, while conversion to dithiol took 8 h. Monothiols from PEGs with Mn = 1000 and 2050 g/mol were obtained in 8 and 16 h, respectively. MALDI-ToF mass spectrometry verified the absence of dithiols. The synthesis of dithiols required additional fresh CALB and MP-SH. The structure of the products was confirmed by 1H-NMR and 13C-NMR spectroscopy. Enzyme catalysis was found to be a powerful tool to effectively synthesize thiol-functionalized TEGs and PEGs.


Introduction
Poly(ethylene glycol) (PEG) is the most frequently used polymer for biomedical research and applications because it is soluble in organic as well as aqueous media [1], is not cytotoxic and immunogenic [2], and is easily excreted from living organisms [3].Click chemistries and Michael addition reactions are often used for PEGylation of drugs to make them more water soluble [4].Thiol-functionalized PEGs have an important role in these reactions [5][6][7][8][9][10] and can be used as a 'Michael donor' or in thiol-ene click reactions to synthesize conjugates for targeted drug delivery [7].Other uses include the following: An anti-fouling biosensor coating [8], to stabilize gold nanorods used to test water for chemical pollutants [9], and to stabilize gold nanoparticles used as drug delivery vehicles [10].Thiol-functionalized PEG is also a favorite to make self-assembling monolayers on gold surfaces [8].Mahou et al. [11] reported the single synthetic strategy to obtain PEG-monothiol.They tosylated one hydroxyl end-group of the PEG-diol using p-toluenesulfonyl chloride in the presence of silver oxide and potassium iodide catalyst and toluene solvent.The tosylated PEG was then reduced with sodium hydrosulfide at 60 • C to yield PEG-monothiol with 84% yield.PEG-dithiols have been synthesized by various methods.In one method, the hydroxyl end groups were reacted with allyl bromide at 120 • C, followed by a radical-mediated addition of thioacetic acid and subsequent reduction to thiol using sodium hydroxide/sodium thiomethoxide, with a 56% yield [12][13][14].Another route reported tosylation of the hydroxyl end groups, followed by a reaction with a xanthate and de-protection with an alkyl amine that gave 98% yield [15,16].The simplest method used esterification of mercapto-acids in toluene at 120 • C using p-toluenesulfonic acid or sulfuric acid as catalysts: An example is shown in Figure 1 [17][18][19][20][21][22][23][24][25].
These methods employ acid catalysts; and hence are not "green".Against this background, we investigated the synthesis of thiol-functionalized tetraethylene glycol (TEG) and PEGs by transesterification of methyl 3-mercaptopropionate (MP-SH) under solvent-less conditions using a heterogeneous catalyst, namely, Candida antarctica Lipase B (CALB).CALB-catalyzed functionalization of low molecular weight polymers was first reported by our research group yielding pure products with high efficiency [26][27][28][29][30][31][32].For example, halogen-functionalized PEGs were made by the transesterification of halo-esters with PEG monomethyl ether under solvent-less conditions at 65 °C for 4 hours under vacuum (70 milliTorr) [31].Methacrylate, acrylate, and crotonate functionalization of PEGs was also achieved under solvent-less conditions within 4 hours at 50 °C by reacting PEG with the corresponding vinyl esters (vinyl methacrylate, vinyl acrylate, and vinyl crotonate) in the presence of immobilized CALB [32].
Precise thiol-functionalization of TEG and PEGs by enzyme catalysis has not been reported previously in the literature.This study presents the first examples of precision synthesis of TEG and PEG monothiols and dithiols.In this study, two types of PEGs (Mn = 1000 g/mol and Mn = 2050 g/mol) were used to evaluate the effect of PEG chain length on the kinetics of the CALB-catalyzed transesterification reaction of methyl 3-mercaptopropionate (MP-SH) with PEGs at 50 °C, an optimum temperature for CALB-catalyzed reactions [33].MP-SH was selected because of its low cost and the convenient removal of the methanol side product by vacuum.CALB supported on various carriers were reported to be more effective than the native enzyme [34][35][36] and depending on the specific conditions were shown to be recyclable four [37] or twenty times [36].We have been using the only commercially available CALB (20 wt% immobilized on a macroporous acrylic resin, Novozyme® 435).

CALB-Catalyzed Transesterification of MP-SH with TEG.
First, MP-SH was transesterified with TEG under solvent-less conditions using CALB as the catalyst.The catalytic triad for transesterification of CALB was shown to consist of serine (Ser105), histidine (His224), and aspartate (Asp187) [38].Figure 2 illustrates our rendition of the mechanism of transesterification of MP-SH by TEG [33].The top (dark shaded) portion of the enzyme is the socalled "carbonyl pocket" while the bottom (lighter shaded) is the "hydroxyl pocket".First, the nucleophilic serine (Ser105) in the free enzyme interacts with the carbonyl group of the thioester, forming the first tetrahedral intermediate (THI-1) that is stabilized by the so-called oxyanion hole (three hydrogen bonds: One from glutamine (Gln106) and two from threonine (Thr40)) [38].In the second step, the ester bond in THI-1 is cleaved to form an acyl-enzyme complex (AEC) that releases the first product, methanol in this case, which is removed due to the applied vacuum (420 Torr), making the reaction irreversible.In the third step, the HO-group of the diol positioned in the These methods employ acid catalysts; and hence are not "green".Against this background, we investigated the synthesis of thiol-functionalized tetraethylene glycol (TEG) and PEGs by transesterification of methyl 3-mercaptopropionate (MP-SH) under solvent-less conditions using a heterogeneous catalyst, namely, Candida antarctica Lipase B (CALB).CALB-catalyzed functionalization of low molecular weight polymers was first reported by our research group yielding pure products with high efficiency [26][27][28][29][30][31][32].For example, halogen-functionalized PEGs were made by the transesterification of halo-esters with PEG monomethyl ether under solvent-less conditions at 65 • C for 4 h under vacuum (70 milliTorr) [31].Methacrylate, acrylate, and crotonate functionalization of PEGs was also achieved under solvent-less conditions within 4 h at 50 • C by reacting PEG with the corresponding vinyl esters (vinyl methacrylate, vinyl acrylate, and vinyl crotonate) in the presence of immobilized CALB [32].
Precise thiol-functionalization of TEG and PEGs by enzyme catalysis has not been reported previously in the literature.This study presents the first examples of precision synthesis of TEG and PEG monothiols and dithiols.In this study, two types of PEGs (M n = 1000 g/mol and M n = 2050 g/mol) were used to evaluate the effect of PEG chain length on the kinetics of the CALB-catalyzed transesterification reaction of methyl 3-mercaptopropionate (MP-SH) with PEGs at 50 • C, an optimum temperature for CALB-catalyzed reactions [33].MP-SH was selected because of its low cost and the convenient removal of the methanol side product by vacuum.CALB supported on various carriers were reported to be more effective than the native enzyme [34][35][36] and depending on the specific conditions were shown to be recyclable four [37] or twenty times [36].We have been using the only commercially available CALB (20 wt% immobilized on a macroporous acrylic resin, Novozyme ® 435).

CALB-Catalyzed Transesterification of MP-SH with TEG
First, MP-SH was transesterified with TEG under solvent-less conditions using CALB as the catalyst.The catalytic triad for transesterification of CALB was shown to consist of serine (Ser105), histidine (His224), and aspartate (Asp187) [38].Figure 2 illustrates our rendition of the mechanism of transesterification of MP-SH by TEG [33].The top (dark shaded) portion of the enzyme is the so-called "carbonyl pocket" while the bottom (lighter shaded) is the "hydroxyl pocket".First, the nucleophilic serine (Ser105) in the free enzyme interacts with the carbonyl group of the thioester, forming the first tetrahedral intermediate (THI-1) that is stabilized by the so-called oxyanion hole (three hydrogen bonds: One from glutamine (Gln106) and two from threonine (Thr40)) [38].In the second step, the ester bond in THI-1 is cleaved to form an acyl-enzyme complex (AEC) that releases the first product, methanol in this case, which is removed due to the applied vacuum (420 Torr), making the reaction irreversible.In the third step, the HO-group of the diol positioned in the hydroxyl pocket interacts with the carbonyl group of the AEC, forming the second tetrahedral intermediate (THI-2), which is also stabilized by the oxyanion hole.In the last step, the enzyme is deacylated to form a TEG-monothiol that is released from the THI-2 and the enzyme is regenerated.
The second -OH group of the TEG-monothiol will then be converted to thiol in a second cycle in a similar manner as the first cycle as shown in Figure 3.However, the first and second cycle may proceed simultaneously in a competitive reaction between the hydroxyl groups of unreacted TEG and TEG-monothiol.Thus, we first studied the kinetics of CALB-catalyzed transesterification of MP-SH with TEG.

88
The second -OH group of the TEG-monothiol will then be converted to thiol in a second cycle in    We theorize that in the second cycle the carbonyl group of the free MP-SH competes with the carbonyl group of the TEG-monothiol for complexation in the carbonyl pocket of CALB, thereby slowing down the second cycle of the reaction.Another reason might be the deactivation of CALB by the methanol released in the reactions that is not completely removed in the vacuum.Thus, the reaction proceeds sequentially in a consecutive manner.

Synthesis of TEG-monothiol and TEG-dithiol
Figure S1 shows the 1 H-and 13 C-NMR spectra of TEG-monothiol synthesized with a reaction time of 15 min after filtering the enzyme and removing the excess thioester but without further purification (93% reaction yield because some material is lost with the enzyme).In the 1 H-NMR spectrum of the monothiol (Figure S1A), the ratio of the integral of the methylene protons next to the SH group in the product at 2.75 ppm (b') and δ = 2.64 ppm (c') to the integral of the methylene protons next to the carbonyl group at δ = 4.23 ppm (e') is 4.00:2.00,indicating the formation of the TEG-monothiol with 100% conversion.In the 13 C-NMR spectrum of the monothiol (Figure S1B), signals corresponding to the carbons in the thiol end group (B, C, D, E' and F') and the carbons next to the -OH end group (E and F) appears distinctly, that demonstrates the formation of the TEG-monothiol.
Figure S2 shows the1 H-and 13 C-NMR spectra of the TEG-dithiol that was synthesized with a reaction time of 7. In summary, CALB-catalyzed transesterification of MP-SH with TEG in bulk yielded TEG-monothiol in 15 min with 93% reaction yield, and TEG-dithiol in 7.5 h with 88% reaction yield and 100% conversion without purification, such as column chromatography.

Kinetics of CALB-catalyzed transesterification of MP-SH with PEG
MP-SH was reacted with PEG 1000 using enzyme catalysis, and the reaction was monitored over 24 h with 1 H-NMR spectroscopy (see Figure 5).Because low molecular weight PEGs (<3000 g/mol) are liquid at the reaction temperature and are miscible with MP-SH, no solvent was necessary as a medium for the reaction.The main chain protons (g) and the methylene protons next to the -OH (e and f) and the thioester (f') appear at δ = 3.61 ppm.For PEG 1000 -monothiol, the new methylene protons next to the carbonyl group (e') appear at δ = 4.23 ppm, which makes the integral value of internal protons of PEG 1000 (g, e, f, and f'): 88 − 2 = 86.Therefore, the integral value of the internal protons was set to 86 for calculating the extent of the reaction.Based on the integral ratio of (g, e, f, and f'): (e'), about 60% of the PEG 1000 was converted to PEG 1000 -monothiol in 60 min (Figure 5).Then the reaction slowed down, and it took 8 h to convert all PEG 1000 into monothiol.Dithiol was not detected even after 24 h.The mechanism presented in Figure 2 for TEG also applies for PEG.Thus, we theorize that in the second cycle the carbonyl group of the free MP-SH competes with the carbonyl group of the PEG-monothiol for complexation in the carbonyl pocket of CALB, thereby slowing down the second cycle of the reaction.In addition, the CALB may be deactivated by the methanol released in the reactions that is not completely removed by the vacuum.

143
MP-SH was reacted with PEG1000 using enzyme catalysis, and the reaction was monitored over 144 24 hours with 1 H-NMR spectroscopy (see Figure 5).Because low molecular weight PEGs (<3000  protons was set to 86 for calculating the extent of the reaction.Based on the integral ratio of (g, e, f, 151 and f'): (e'), about 60% of the PEG1000 was converted to PEG1000-monothiol in 60 minutes (Figure 5).

152
Then the reaction slowed down, and it took 8 hours to convert all PEG1000 into monothiol.Dithiol was 153 not detected even after 24 hours.The mechanism presented in Figure 2 for TEG also applies for PEG.MP-SH was also transesterified with PEG 2050 and the reaction was monitored over 24 h by 1 H-NMR spectroscopy (not shown).Similarly to the PEG 1000 , only monothiol was obtained.In addition, complete conversion to monothiol was achieved in 16 h, which suggests higher molecular weight required longer reaction time.

Synthesis of PEG 1000 -monothiol
The 1 H-NMR of the PEG 1000 -monothiol is shown in Figure 6A.The integral ratio of (b') + (c') to the methylene protons in the new ester bond at δ = 4.23 ppm is 4:00:1.86,indicating the formation of PEG 1000 -monothiol.Figure 6B shows the 13 C-NMR spectrum of PEG 1000 -monothiol.Signals corresponding to the carbons next to the thioester (E' and F') and -OH end groups (E and F) appear simultaneously, indicating the formation of monothiol.distribution of peaks appearing under the doubly charged Na complex distribution corresponds to traces of unreacted PEG1000 from the reaction mixture (<5%) that could not be detected by NMR.Thus, based on the MALDI mass spectrometry data, over 95% conversion of one of the OH groups to thiol was achieved in 24 hours.No traces of PEG-dithiol were found.Therefore, it can be concluded that the product was exclusively PEG1000-monothiol with no traces of dithiol, with 100% yield.The product was further analyzed by MALDI-ToF mass spectrometry and Figure 7 shows the spectrum.There are two major distributions of peaks (Figure 7A), each separated by 44 m/z units (Figure 7B). .The small distribution of peaks appearing under the doubly charged Na complex distribution corresponds to traces of unreacted PEG 1000 from the reaction mixture (<5%) that could not be detected by NMR.Thus, based on the MALDI mass spectrometry data, over 95% conversion of one of the OH groups to thiol was achieved in 24 h.No traces of PEG-dithiol were found.Therefore, it can be concluded that the product was exclusively PEG 1000 -monothiol with no traces of dithiol, with 100% yield.

199
PEG2050 mono-and di-thiols were also synthesized as described in the Experimental section.The

Synthesis of PEG 1000 -dithiol
PEG 1000 -dithiol was obtained by reacting PEG 1000 -monothiol with fresh MP-SH and CALB for 24 h under solvent-less conditions.Figure 8 shows the 13 CNMR spectrum of the PEG 1000 -dithiol.The disappearance of the signals (F and E, Figure 6) at δ = 72.66ppm and δ = 61.72 ppm, corresponding to the methylene protons next to the hydroxyl end-groups from the PEG 1000 -monothiol indicates full conversion to PEG 1000 -dithiol in 24 h with 85% reaction yield.PEG2050 mono-and di-thiols were also synthesized as described in the Experimental section.The 1 H-NMR spectra shown in Figure S3 verified the structure of the PEG2050 mono-and di-thiols that were obtained with 100% and 94% reaction yield.

Materials
Candida antarctica Lipase B (CALB, 33273 Da, 20 wt% immobilized on a macroporous acrylic resin Novozyme® 435) was obtained from Sigma Chemicals (St. Louis, MO, USA).Poly(ethylene PEG 2050 mono-and di-thiols were also synthesized as described in the Experimental section.The 1 H-NMR spectra shown in Figure S3 verified the structure of the PEG 2050 mono-and di-thiols that were obtained with 100% and 94% reaction yield.

2.
Synthesis of TEG-monothiol TEG (3.8805 g, 20 mmol) was dried under vacuum (Schlenk line) at 65 • C and 0.2 Torr until bubble formation ceased.It was then mixed with MP-SH (7.4007 g, 61.6 mmol) at 50 • C and 420 Torr in the presence of CALB (0.4912 g resin @ 20 wt% enzyme, 0.0029 mmol).After 15 min, the reaction mixture was diluted with 3 mL of dried THF, filtered over a Q5 filter paper and then dried under vacuum (Schlenk line) at 50 • C for two hours.The product was then dried in a vacuum oven for further analysis (4.1685 g, 93% reaction yield).

3.
Synthesis of TEG-dithiol TEG (1.9782 g, 10.2 mmol) was dried under vacuum (Schlenk line) at 65 • C and 0.2 Torr until bubble formation ceased.It was then mixed with MP-SH (3.6204 g, 30.1 mmol) at 50 • C and 420 Torr in presence of CALB (0.2549 g resin @ 20 wt% enzyme, 0.0015 mmol).After 450 min, the reaction mixture was diluted with 3 mL of dried THF, filtered over a Q5 filter paper and then dried under vacuum (Schlenk line) at 50 • C for two hours.The product was then dried in a vacuum oven for further analysis (3.5327 g, 88% reaction yield).C and 420 Torr in the presence of CALB (0.0977 g resin @ 20 wt% enzyme, 0.00058 mmol).After 1 min, the vacuum was removed and N 2 gas was passed through the system and an aliquot was collected.The vacuum was reinstated, and the procedure was repeated to collect aliquots at 3, 5, 10, 15, 30, 60, 120, 240, 360, 480, 600, 720, 960, and 1440 min. 1 H-NMR spectroscopy was used to check the extent of the reaction.

•
PEG 2050 PEG 2050 (4.6117 g, 2.25 mmol) was dried under vacuum (Schlenk line) at 65 • C and 0.2 Torr for 16 h.It was then mixed with MP-SH (1.6550 g, 13.7 mmol) and reacted at 50 • C and 420 Torr in the presence of CALB (0.1134 g resin @ 20 wt% enzyme, 0.00068 mmol).After 1 min, the vacuum was removed and N 2 gas was passed through the system and an aliquot was collected.The vacuum was reinstated, and the procedure was repeated to collect aliquots at 3, 5, 10, 15, 30, 60, 120, 240, 360, 480, 600, 720, 960, and 1440 min. 1 H-NMR spectroscopy was used to check the extent of the reaction.

Synthesis of PEG-monothiols
• PEG 1000 -monothiol PEG 1000 (6.0882 g, 6.16 mmol) was dried under vacuum (Schlenk line) at 65 • C and 0.2 Torr for 16 h.It was then mixed with MP-SH (2.1793 g, 18.13 mmol) and reacted at 50 • C and 420 Torr in the presence of CALB (0.1501 g resin @ 20 wt% enzyme, 0.00090 mmol).After 24 h, the reaction mixture was diluted with 3 mL of dried THF, filtered over a Q5 filter paper and then dried under vacuum (Schlenk line) at 50 • C for 16 h.The product was then dried in a vacuum oven for further analysis (6.0967 g, ~100% reaction yield).

•
PEG 2050 -monothiol PEG 2050 (4.6117 g, 2.25 mmol) was dried under vacuum (Schlenk line) at 65 • C and 0.2 Torr for 16 h.It was then mixed with MP-SH (1.6550 g, 13.7 mmol) and reacted at 50 • C and 420 Torr in the presence of CALB (0.1134 g resin @ 20 wt% enzyme, 0.00068 mmol).After 24 h, the reaction mixture was diluted with 3 mL of dried THF, filtered over a Q5 filter paper and precipitated in 100 mL of diethyl ether.The precipitate was then dried in a vacuum oven for further analysis (4.2034 g, ~100% reaction yield).

3.
Synthesis of PEG-dithiols • PEG 1000 -dithiol PEG 1000 -monothiol (2.1005 g, 1.95 mmol) was dried under vacuum (Schlenk line) at 65 • C and 0.2 Torr for 16 h.It was then mixed with MP-SH (3.1031 g, 25.8 mmol) at 50 • C and 420 Torr in presence of CALB (0.0940 g resin @ 20 wt% enzyme, 0.00056 mmol) for 24 h.After 24 h of reaction time, the reaction mixture was diluted with 3 mL of dried THF, filtered over a Q5 filter paper and then dried under vacuum (Schlenk line) at 50 • C for 16 h.The product was then dried in a vacuum oven for further analysis.(2.1461 g, 85% reaction yield).

•
PEG 2050 -dithiol PEG 2050 -monothiol (4.000 g, 1.87 mmol) was dried under vacuum (Schlenk line) at 65 • C and 0.2 Torr for 16 h.It was then mixed with MP-SH (2.7125 g, 22.5 mmol) at 50 • C and 420 Torr in the presence of CALB (0.2274 g resin @ 20 wt% enzyme, 0.0013 mmol) for 24 h.After 24 h, the reaction mixture was diluted with 3 mL of dried THF, filtered over a Q5 filter paper and precipitated in 100 mL of diethyl ether.The precipitate was then dried in a vacuum oven for further analysis (3.7791 g, 94% reaction yield).

Nuclear Magnetic Resonance (NMR) Spectroscopy
Varian Mercury 300 MHz and 500 MHz spectrometer (Palo Alto, CA, USA) was used to record the 1 H-NMR and 13 C-NMR spectra in CDCl 3 at 20 mg/ml and 60 mg/ml respectively with the following parameters: 10 second relaxation time, 128 scans (5000 scans for 13 C-NMR) and 90 • angle.The internal reference for chloroform was δ = 7.26 ppm ( 1 H-NMR) and δ = 77 ppm ( 13 C-NMR).

Figure 4 .
Figure 4. 1 H-NMR monitoring of the kinetics of the transesterification of MP-SH with TEG [15 min:

122 2 . 1 . 2 .Figure 4 .
FigureS1shows the 1 H-and13 C-NMR spectra of TEG-monothiol synthesized with a reaction 5 h (88% reaction yield).The ratio of the integral values of signals (b') + (c') to (e') are 8.00:3.88,indicating the formation of the TEG-dithiol.The 13 C-NMR spectrum in Figure S2B shows only the signals corresponding to the thiol end groups, with only traces of signals corresponding to the carbons next to the -OH (E and F) at δ = 72.38 ppm and δ = 61.16ppm, possibly from traces of residual TEG-monothiol, indicating 100% conversion of the -OH groups to the thiols.

145g
/mol) are liquid at the reaction temperature and are miscible with MP-SH, no solvent was necessary 146 as a medium for the reaction.The main chain protons (g) and the methylene protons next to the -OH 147 (e and f) and the thioester (f') appear at δ = 3.61 ppm.For PEG1000-monothiol, the new methylene 148 protons next to the carbonyl group (e') appear at δ = 4.23 ppm, which makes the integral value of 149 internal protons of PEG1000 (g, e, f, and f'): 88 − 2 = 86.Therefore, the integral value of the internal 150

Catalysts 2019, 9 ,
x FOR PEER REVIEW 7 of 13 corresponding to the carbons next to the thioester (E' and F') and -OH end groups (E and F) appear simultaneously, indicating the formation of monothiol.
192 PEG1000-dithiol was obtained by reacting PEG1000-monothiol with fresh MP-SH and CALB for 24 193 hours under solvent-less conditions.Figure 8 shows the 13 CNMR spectrum of the PEG1000-dithiol.The 194 disappearance of the signals (F and E, Figure 6) at δ = 72.66ppm and δ = 61.72 ppm, corresponding 195 to the methylene protons next to the hydroxyl end-groups from the PEG1000-monothiol indicates full 196 conversion to PEG1000-dithiol in 24 hours with 85% reaction yield.

200 1 H
-NMR spectra shown in FigureS3verified the structure of the PEG2050 mono-and di-thiols that 201 were obtained with 100% and 94% reaction yield.