Chemoselective lipase-catalyzed synthesis of amido derivatives from 5-hydroxymethylfurfurylamine

Chemoselec i e Lipase-Ca alyzed Syn hesis o Amido De i a i es om 5‑Hyd oxyme hyl u u ylamine An ía Pin o , I án La ande a, Alexey Volko ,*and Vicen e Go o -Fe nández* Ci e This: ACS Sus ainable Chem. Eng. 2023, 11, 10284−10292 Read Online ACCESS Me ics & Mo e A icle Recommenda ions * sı Suppo ing In o ma ion ABSTRACT: The acyla ions o u u ylamine and 5-hyd oxyme hyl u u ylamine (HMFA) ha e been s udied inding immobilized Candida an a c ica lipase B (CALB) as an ideal bioca alys . CALB was used immobilized on wo di e en suppo s (No ozyme 435 and EziG-CALB), wi h he polyme -coa ed con olled po osi y glass ca ie ma e ial om EnginZyme being an excellen ca ie o yield an ac i e and s able enzyma ic p epa a ion o he acyla ion o he p ima y amine g oup. The amoun o he acyl dono in he eac ion was a key ac o o achie e he mono- and chemoselec i e N-p o ec ion o HMFA wi h la ge excess o e hyl ace a e leading o he o ma ion o he N,O-diace yla ed p oduc . Thus, a se ies o 16 nonac i a ed es e s we e used o selec i ely modi y he amine g oup o HMFA, ob aining 9 hyd oxy amides unde mild eac ion condi ions and wi h quan i a i e yields h ough ch oma og aphy- ee ans o ma ions. The in luence o subs a e concen a ion was s udied, esul ing in comple e con e sions in all cases a e 22 h (100−1000 mM). Excellen esul s we e obse ed a 100 and 200 mM o HMFA, while highe concen a ions led o longe eac ion imes and, o some ex en , he o ma ion o he diace yla ed p oduc (up o 7% a e 22 h a 1 M). A e his op imiza ion, a me ic analysis was pe o med o con i m he high sus ainabili y o he p esen ed p ocess (E- ac o o 1.1 excluding sol en s) upon in ensi ica ion o he bio ans o ma ion o 1 g a 200 mM HMFA concen a ion. The possibili y o ob aining o hogonally p o ec ed HMFA-de i ed amido es e s has been achie ed h ough a clean and sequen ial one-po p ocess using EziG-CALB, which in ol ed he use o e hyl me hoxy ace a e as he nonac i a ed es e o N-acyla ion and he ac i a ed inyl ace a e o O-p o ec ion. KEYWORDS: acyla ion, chemoselec i e p ocess, enzyme immobiliza ion, 5-hyd oxyme hyl u u ylamine, lipases ■INTRODUCTION The sea ch o biobased chemicals and uels om aw biomass is cu en ly highly appealing o eplace adi ional ossil sou ces. Lignocellulosic biomass is a aluable sou ce o pla o m molecules such as u an de i a i es wi h mul iple applica ions in chemical indus y, o ins ance, in he manu ac u ing o adhesi es and polyme s. 1,2 In his con ex , 5-hyd oxyme hyl u u al (HMF, 1,Figu e 1) is conside ed a key molecule o biomass alo iza ion and also a e sa ile syn he ic building block, 3−5 wi h p ima y hyd oxy and o myl g oups being modula unc ionali ies o p oduce di e en amilies o aluable compounds. 6,7 Thus, chemical oxida ions o HMF p o ide access o 2,5-di o myl u an (DFF), 5- hyd oxyme hyl-2- u anca boxylic acid (HMFCA), 5- o myl-2- u anca boxylic acid (FFCA), and 2,5- u andica boxylic acid (FDCA); HMF educ ion leads o 2,5-bis(hyd oxyme hyl)- u an (BHMF, 2); while 5-hyd oxyme hyl-2- u u ylamine (HMFA, 3) can be ob ained h ough di ec HMF amina- ion. 8−11 Nowadays, he use o enzymes is pa icula ly a ac i e in o ganic syn hesis due o hei chemo-, egio-, and s e eo- selec i e eac i i y unde mild eac ion condi ions. Pa icula ly, s aigh o wa d and selec i e ans o ma ions o p oduce HMF de i a i es ha e been ex ensi ely s udied in he las decade. 12−18 Thus, a as numbe o bioca alys s ha e been iden i ied o he p oduc ion o a ious HMF de i a i es such Recei ed: Feb ua y 9, 2023 Re ised: June 15, 2023 Published: July 6, 2023 Resea ch A icle pubs.acs.o g/jou nal/ascecg © 2023 The Au ho s. Published by Ame ican Chemical Socie y 10284 h ps://doi.o g/10.1021/acssuschemeng.3c00775 ACS Sus ainable Chem. Eng. 2023, 11, 10284−10292 Downloaded ia CSIC on July 19, 2023 a 09:38:41 (UTC). See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles. as oxidases, 19 alcohol dehyd ogenases, 20 amine ansami- nases, 21−23 and educ i e aminases. 24 N-Subs i u ed u u yl amines a e o high in e es as p ecu so s o biologically ac i e compounds, 24−26 hei chemical p epa a ion p o iding excellen esul s ia educ i e amina ion o ob ain he co esponding N-alkyla ed de i a- i es, 27−29 while enzyma ic app oaches a e s ill in hei in ancy o his pu pose. The e o e, de eloping sus ainable and selec i e syn he ic ou es owa d N-p o ec ed u u yl amines unde mild eac ion condi ions would be o g ea in e es , en isaging an acyl g oup as an excellen choice as i can be de ached la e i equi ed. Lipases a e e sa ile enzymes able o ca alyze hyd oly ic and syn he ic ans o ma ions, cu en ly inding impo an applica ions in he indus ial sec o . 30−32 In ecen yea s, he use o lipases o he syn hesis o HMF de i a i es has been conside ably exploi ed by means o lipase- ca alyzed ( ans)es e i ica ion eac ions (Scheme 1). 33−42 The eac ion be ween HMF and di e en es e s o ca boxylic acids allows he selec i e unc ionaliza ion o he hyd oxyl g oup main aining unal e ed he aldehyde unc ionali y (Scheme 1, le ). 33−37 The ( ans)es e i ica ion o BHMF, howe e , usually p oceeds owa d he o ma ion o he co esponding die- s e s, 38−42 al hough depending on he eac ion condi ions, monoes e s can be selec i ely ob ained o a ce ain ex en (Scheme 1, igh ). Un o una ely, he wo k wi h he co esponding amino alcohol de i a i e (HMFA) emains unexplo ed, while he p esence o he amine and hyd oxyl g oups o e s a a ie y o syn he ic possibili ies o p oduce a wide ange o N- o /and O-p o ec ed compounds such as, e.g., he (hyd oxy) amides (Scheme 1, bo om). Based on he excellen ac i i y and selec i i y displayed by lipases, mainly Candida an a c ica lipase ype B (CALB), 43,44 owa d amide o ma ion unde mild condi ions, he ein, he chemoselec i e lipase-ca alyzed acyla ion o he HMFA p ima y amine g oup was ho oughly in es iga ed. ■EXPERIMENTAL SECTION Ma e ials and Equipmen . Chemical eagen s we e pu chased om Sigma-Ald ich, VWR In e na ional, and The mo Fishe Scien i ic and used as ecei ed. Pa icula ly, u u ylamine and HMFA ha we e used as subs a es o lipase-ca alyzed eac ions we e acqui ed om Sigma-Ald ich. Rega ding he enzyme a ailabili y, C. an a c ica ype B lipase (CALB) was used as wo di e en immobilized o ms: No ozyme 435 is suppo ed on he esin Lewa i VP OC 1600 and i was kindly dona ed by No ozymes, 45 while EziG-CALB is p oduced by EnginZyme and is suppo ed on a polyme -coa ed con olled po osi y glass ca ie EziG Ambe . 46 Rega ding o he enzymes employed in his con ibu ion, immobilized Candida ugosa lipase (CRL), immobilized Pseudomonas cepacia (PSL), and lyophilized lipase AK om Pseudomonas luo escens (AK) we e pu chased om Sigma-Ald ich; C. an a c ica ype A lipase (CALA) and The momyces lanuginosus lipase (TLL) we e ob ained om Immozymes and Mei o Sangyo, espec i ely, bo h used as immobilized p epa a ions; inally, immobilized Aspe gillus nige lipase (ANL) was ob ained om Bioca alys s L d. Thin-laye ch oma og aphy (TLC) analyses we e conduc ed using Me ck Silica Gel 60 F254 p ecoa ed pla es and isualized wi h a UV lamp and po assium pe mangana e o anillin s ains. Column ch oma og aphy pu i ica ions, when equi ed, we e pe o med using silica gel 60 (230−240 mesh). 1H-, 13C-, and DEPT NMR expe imen s we e eco ded on a B uke AV300 MHz spec ome e using CDCl3and MeOD as he sol en s. All chemical shi s (δ) a e gi en in pa s pe million (ppm) and e e enced o he esidual sol en signal as in e nal s anda d. IR spec a we e eco ded on a Jasco FT/IR-4700 spec opho ome e , and νmax alues a e gi en in cm−1 o he main abso p ion bands o he syn hesized compounds. High- esolu ion mass spec a (HRMS) expe imen s we e ca ied ou by elec osp ay ioniza ion in posi i e mode (ESI+) using a Mic o To Q spec ome e . Gas ch oma og aphy (GC) analyses we e pe o med on an Agilen HP6890 GC ch oma og aph equipped wi h an FID de ec o . A HP-1 column (30 m ×0.32 mm ×0.25 μm) was used o he de e mina ion o con e sion alues and p oduc pe cen ages (see addi ional in o ma ion in Sec ion 5 o he Suppo ing In o ma ion). Lipase-Ca alyzed Ace yla ion o Fu u ylamine (4) Using E OAc (5a) in an O ganic Sol en . Amine 4(20 mg, 0.2 mmol, 100 mM) was dissol ed in a hyd ophobic o ganic sol en (2 mL) such as e -bu yl me hyl e he (MTBE), die hyl e he (E 2O), e hyl ace a e (E OAc), o 2-me hyl e ahyd o u an (2-MeTHF) inside an E lenmeye lask. Then, No ozyme 435 o EziG-CALB (20 mg, 1:1 w/w enzyme:4 a io) and E OAc (59 μL, 0.6 mmol, 3 equi ) we e successi ely added ( he acyl dono was added only o he eac ions wi h MTBE, E 2O, and 2-MeTHF). The eac ion was shaken a 250 Figu e 1. HMF (1), BHMF (2), HMFA (3), and u u ylamine (4) chemical s uc u es. Scheme 1. Lipase-Ca alyzed T ans o ma ions Using HMF (1), BHMF (2), and HMFA (3) ACS Sus ainable Chemis y & Enginee ing pubs.acs.o g/jou nal/ascecg Resea ch A icle h ps://doi.o g/10.1021/acssuschemeng.3c00775 ACS Sus ainable Chem. Eng. 2023, 11, 10284−10292 10285 pm o 2 h a 30 °C, and a e his ime an aliquo was aken and analyzed by GC, obse ing he quan i a i e con e sion. The eac ion was il e ed, and he enzyme was washed wi h CH2Cl2(2 ×1 mL). The il a e was e apo a ed unde educed p essu e, a o ding N- ( u an-2-ylme hyl)ace amide (6) as a yellow oil. The spec oscopic da a ma ch wi h he ones ob ained ia chemical ace yla ion o 4using ace ic anhyd ide and ie hylamine (see he SI). R (E OAc): 0.48. IR (nea ): 3274, 3077, 1646, 1544, 734, and 599 cm−1.1H-NMR (300 MHz, CDCl3)δ7.35 (dd, J= 2.0, 0.9 Hz, 1H), 6.31 (dd, J= 3.2, 1.9 Hz, 1H), 6.22 (dd, J= 3.2, 0.9 Hz, 1H), 5.88 (b s, 1H), 4.42 (d, J= 5.5 Hz, 2H), and 2.00 (s, 3H) ppm. 13C-NMR (75 MHz, CDCl3)δ 169.9 (C), 151.4 (C), 142.3 (CH), 110.6 (CH), 107.6 (CH), 36.7 (CH2), and 23.3 (CH3) ppm. ESI-TOF-sHRMS: [M + Na]+ calcula ed o C7H9NNaO2: 162.0532; ound: 162.0525. Gene al P ocedu e o he EziG-CALB-Ca alyzed Selec i e N-Acyla ion o HMFA (3). The co esponding acyl dono 5a−p (0.16 mmol, 1.3 equi ) was added o a mix u e o HMFA (3, 15 mg, 0.12 mmol, 100 mM), EziG-CALB (1:1 w/w enzyme:3), and 2- MeTHF (1.2 mL). The mix u e was shaken o 2 h a 250 pm a 30 °C, and a e his ime, he eac ion c ude was analyzed by TLC and GC analyses. The eac ion was il e ed, and he enzyme was washed wi h CH2Cl2(2 ×1 mL). The il a e was concen a ed unde educed p essu e, a o ding he co esponding hyd oxy amides 7a−i wi h excellen pu i ies ha we e hen ully cha ac e ized. The only excep ions we e he eac ions wi h e hyl phenylace a e (5e), whe e he eac ion c ude was d ied on a eeze-d ye o e nigh and hose using benzyl ace a e (5j), 4-ni ophenyl ace a e (5k), e hyl cap a e (5o), and me hyl lau a e (5p) because he p oduc o ma ion was obse ed in comple e con e sion, bu he un eac ed acyl dono was no sepa a ed. Fo ins ance, he wash o he eac ion c udes con aining he hyd oxy amides 7h and 7i wi h cold E 2O (3 ×2 mL) allowed he isola ion o he p oduc wi h excellen pu i y. N-[(5-(Hyd oxyme hyl) u an-2-yl)me hyl]ace amide (7a). Yellow oil (19.8 mg, 99% isola ed yield). R (E OAc): 0.25. IR (nea ): 3390, 3280, 1623, 1545, 998, and 796 cm−1.1H-NMR (300 MHz, CDCl3)δ 6.44 (b s, 1H), 6.16 (d, J= 3.1 Hz, 1H), 6.12 (d, J= 3.2 Hz, 1H), 4.51 (s, 2H), 4.33 (d, J= 5.5 Hz, 2H), 3.15 (b s, 1H), and 1.94 (s, 3H) ppm. 13C-NMR (75 MHz, CDCl3)δ170.4 (C), 154.0 (C), 151.3 (C), 108.7 (CH), 108.4 (CH), 57.3 (CH2), 36.8 (CH2), and 23.2 (CH3) ppm. ESI-TOF-HRMS: [M + Na]+calcula ed o C8H11NNaO3: 192.0631; ound: 192.0634. N-[(5-(Hyd oxyme hyl) u an-2-yl)me hyl]-2-me hoxyace amide (7b). Yellow oil (23.3 mg, 99% isola ed yield). R (E OAc): 0.25. IR (nea ): 3298, 2918, 2849, 1649, 1195, and 1116 cm−1.1H-NMR (300 MHz, CDCl3)δ6.93 (b s, 1H), 6.18 (d, J= 3.1 Hz, 1H), 6.15 (d, J= 3.2 Hz, 1H), 4.52 (s, 2H), 4.41 (d, J= 5.9 Hz, 2H), 3.88 (s, 2H), 3.37 (s, 3H), and 2.97 (b s, 1H) ppm. 13C-NMR (75 MHz, CDCl3)δ 169.8 (C), 154.2 (C), 150.9 (C), 108.6 (CH), 108.4 (CH), 71.9 (CH2), 59.3 (CH3), 57.3 (CH2), and 35.9 (CH2) ppm. ESI-TOF- HRMS: [M + Na]+calcula ed o C9H13NNaO4: 222.0737; ound: 222.0739. N-[(5-(Hyd oxyme hyl) u an-2-yl)me hyl]p opionamide (7c). Yellow oil (21.4 mg, 99% isola ed yield). R (E OAc): 0.40. IR (nea ): 3350, 3173, 1645, 1539, 997, and 790 cm−1.1H-NMR (300 MHz, CDCl3)δ6.17 (d, J= 3.2 Hz, 1H), 6.13 (d, J= 3.2 Hz, 1H), 4.52 (s, 2H), 4.35 (d, J= 5.5 Hz, 2H), 2.95 (b s, 2H), 2.20 (q, J= 7.6 Hz, 2H), and 1.12 ( , J= 7.6 Hz, 3H) ppm. 13C-NMR (75 MHz, CDCl3)δ174.1 (C), 154.0 (C), 151.5 (C), 108.7 (CH), 108.2 (CH), 57.3 (CH2), 36.7 (CH2), 29.6 (CH2), and 9.8 (CH3) ppm. ESI-TOF- HRMS: [M + Na]+calcula ed o C9H13NNaO3: 206.0788; ound: 206.0790. 2-Chlo o-N-[(5-(hyd oxyme hyl) u an-2-yl)me hyl]ace amide (7d). Yellow oil (23.7 mg, 99% isola ed yield). R (E OAc): 0.56. IR (nea ): 3278, 3079, 1652, 1537, 1010, and 792 cm−1.1H-NMR (300 MHz, CDCl3)δ7.05 (b s, 1H), 6.19 (appa en q, J= 3.4 Hz, 2H), 4.54 (s, 2H), 4.43 (d, J= 5.7 Hz, 2H), 4.04 (s, 2H), and 3.59 (s, 1H) ppm. 13C-NMR (75 MHz, CDCl3)δ166.2 (C), 154.2 (C), 150.4 (C), 108.9 (CH), 108.8 (CH), 57.5 (CH2), 42.6 (CH2), and 37.0 (CH2) ppm. ESI-TOF-HRMS: [M + Na]+calcula ed o C8H10ClNNaO3: 226.0241; ound: 226.0245. N-[(5-(Hyd oxyme hyl) u an-2-yl)me hyl]-2-phenylace amide (7e). Whi e powde (28.6 mg, 99% isola ed yield). R (E OAc): 0.65. Mp: decomposi ion obse ed be ween 141 and 161 °C. IR (nea ): 3355, 3280, 1631, 1539, 1003, 999, and 691 cm−1.1H-NMR (300 MHz, CDCl3)δ7.40−7.19 (m, 5H), 6.16 (d, J= 3.2 Hz, 1H), 6.07 (d, J= 3.1 Hz, 1H), 5.98 (b s, 1H), 4.51 (s, 2H), 4.35 (d, J= 5.7 Hz, 2H), and 3.57 (s, 2H). 13C-NMR (75 MHz, CDCl3)δ171.2 (C), 153.9 (C), 151.3 (C), 134.7 (C), 129.6 (2CH), 129.1 (2CH), 127.5 (CH), 108.7 (CH), 108.2 (CH), 57.4 (CH2), 43.7 (CH2), and 36.9 (CH2) ppm. ESI-TOF-HRMS: [M + Na]+calcula ed o C14H15NNaO3: 268.0944; ound: 268.0947. N-[(5-(Hyd oxyme hyl) u an-2-yl)me hyl]bu y amide (7 ). B own powde (20.8 mg, 90% isola ed yield). R (E OAc): 0.50. Mp: 79−81 °C. IR (nea ): 3277, 2962, 2931, 2872, 1625, 1543, 1275, 996, and 758 cm−1.1H-NMR (300 MHz, MeOD-d4)δ6.22 (d, J= 3.2 Hz, 1H), 6.17 (d, J= 3.2 Hz, 1H), 4.46 (s, 2H), 4.33 (s, 2H), 2.18 ( , J= 7.4 Hz, 2H), 1.57−1.70 (sep , J= 7.4 Hz, 2H), 0.93 ( , J= 7.4 Hz, 3H). 13C-NMR (75 MHz, MeOD-d4)δ174.5 (C), 154.1 (C), 151.5 (C), 107.8 (CH), 107.4 (CH), 56.0 (CH2), 37.4 (CH2), 35.8 (CH2), 18.9 (CH2), 12.6 (CH3). ESI-TOF-HRMS: [M + Na]+calcula ed o C10H15NNaO3: 220.0944; ound: 220.0939. N-[(5-(Hyd oxyme hyl) u an-2-yl)me hyl]hexanamide (7g). O - ange powde (21.7 mg, 82% isola ed yield). R (E OAc): 0.63. Mp: 94−97 °C. IR (nea ): 3283, 2954, 2930, 2871, 2449, 1623, 1540, and 1022 cm−1.1H-NMR (300 MHz, MeOD-d4)δ6.22 (d, J= 3.2 Hz, 1H), 6.17 (d, J= 3.1 Hz, 1H), 4.46 (s, 2H), 4.33 (s, 2H), 2.20 ( , J= 7.5 Hz, 2H), 1.61 (quin , J= 7.5 Hz, 2H), 1.39−1.21 (m, 4H), 0.91 ( , J= 6.8 Hz, 3H). 13C-NMR (75 MHz, MeOD-d4)δ174.7 (C), 154.1 (C), 151.5 (C), 107.8 (CH), 107.4 (CH), 56.0 (CH2), 35.8 (CH2), 35.5 (CH2), 31.1 (CH2), 25.3 (CH2), 22.0 (CH2), 12.9 (CH3). ESI-TOF-HRMS: [M + Na]+calcula ed o C12H19NNaO3: 248.1257; ound: 248.1258. N-[(5-(Hyd oxyme hyl) u an-2-yl)me hyl]decanamide (7h). Yel- low powde (29.8 mg, 90% isola ed yield). R (50% E OAc/Hexane): 0.21. Mp: 115−118 °C. IR (nea ): 3283, 2918, 2849, 1626, 1542, 1002, and 808 cm−1.1H-NMR (300 MHz, MeOD-d4)δ6.22 (d, J= 3.2 Hz, 1H), 6.17 (d, J= 3.1 Hz, 1H), 4.46 (s, 2H), 4.33 (s, 2H), 2.20 ( , J= 7.5 Hz, 2H), 1.61 (quin , J= 6.8 Hz, 2H), 1.30 (d, J= 3.5 Hz, 12H), 0.90 ( , J= 6.8 Hz, 3H). 13C-NMR (75 MHz, MeOD-d4)δ 175.2 (C), 154.6 (C), 152.1 (C), 108.4 (CH), 107.9 (CH), 56.5 (CH2), 36.3 (CH2), 36.1 (CH2), 32.2 (CH2), 29.8 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 26.1 (CH2), 22.9 (CH2), 13.6 (CH3). ESI-TOF-HRMS: [M + Na]+calcula ed o C16H27NNaO3: 304.1883; ound: 304.1883. N-[(5-(Hyd oxyme hyl) u an-2-yl)me hyl]dodecanamide (7i). Pale yellow powde (33.1 mg, 91% isola ed yield). R (50% E OAc/ Hexane): 0.26. Mp: 119−121 °C. IR (nea ): 3282, 2918, 2849, 1624, 1464, 1199, and 1002 cm−1.1H-NMR (300 MHz, MeOD-d4)δ6.22 (d, J= 3.2 Hz, 1H), 6.17 (d, J= 3.2 Hz, 1H), 4.46 (s, 2H), 4.33 (s, 2H), 2.20 ( , J= 7.5 Hz, 2H), 1.60 (appa en , J= 7.3 Hz, 2H), 1.30 (d, J= 4.4 Hz, 16H), 0.90 ( , J= 6.8 Hz, 3H). 13C-NMR (75 MHz, MeOD-d4)δ175.2 (C), 154.6 (C), 152.1 (C), 108.4 (CH), 107.9 (CH), 56.5 (CH2), 36.3 (CH2), 36.1 (CH2), 32.2 (CH2), 29.9 (2CH2), 29.8 (CH2), 29.6 (CH2), 29.6 (CH2), 29.4 (CH2), 26.1 (CH2), 22.9 (CH2), 13.6 (CH3). ESI-TOF-HRMS: [M + Na]+ calcula ed o C18H31NNaO3: 332.2196; ound: 332.2196. S udy o he In luence o he Subs a e Concen a ion in he EziG-CALB-Ca alyzed N-Acyla ion o HMFA (3). E hyl ace a e (1.3 equi ) was added o a mix u e o HMFA (3, 30−150 mg, 0.24−1.2 mmol, 200−1000 mM), EziG-CALB (15 mg), and 2- MeTHF (1.2 mL). The co esponding mix u e was shaken be ween 2 and 22 h a 30 °C and 250 pm, aking aliquo s egula ly ha we e analyzed by GC (see he SI). The eac ions we e s opped once he comple e disappea ance o he s a ing ma e ial was achie ed. In all cases, hyd oxy amide 7a was ob ained as a majo componen (93− 99% yield). Highe concen a ions o he subs a e a o ed he o ma ion o he diace yla ed p oduc 8a o some ex en , pa icula ly unde p olonged eac ion imes (1−7%). Scale-Up o he EziG-CALB-Ca alyzed N-Acyla ion o HMFA (3). E hyl ace a e (1.0 mL, 10.22 mmol, 1.3 equi ) was added o a ACS Sus ainable Chemis y & Enginee ing pubs.acs.o g/jou nal/ascecg Resea ch A icle h ps://doi.o g/10.1021/acssuschemeng.3c00775 ACS Sus ainable Chem. Eng. 2023, 11, 10284−10292 10286 mix u e o HMFA (3, 1.00 g, 7.87 mmol, 200 mM), EziG-CALB (500 mg, 1:2 w/w enzyme:3), and 2-MeTHF (38.3 mL). The mix u e was shaken o 5 h a 250 pm a 30 °C, and a e his ime, he eac ion c ude was analyzed by GC analyses. The eac ion was il e ed, and he enzyme was washed wi h CH2Cl2(2 ×10 mL). The il a e was concen a ed unde educed p essu e, a o ding he hyd oxy amide 7a (1.13 g, 85% yield) and 99% pu i y h ough GC analysis. Gene al P ocedu e o he EziG-CALB-Ca alyzed Diace yla- ion o HMFA (3). A suspension o HMFA (3, 15 mg, 0.12 mmol, 100 mM) and EziG-CALB (1:1 w/w enzyme:3) in E OAc (1.2 mL) was shaken o 2 h a 250 pm a 30 °C, and a e his ime, he enzyme was il e ed and washed wi h CH2Cl2(2 ×1 mL). The il a e was concen a ed unde educed p essu e, a o ding amido es e 8a wi h excellen pu i y ha was ully cha ac e ized. 5-(Ace amidome hyl) u an-2-(yl)me hyl Ace a e (8a). Yellow oil (78 mg, 99% isola ed yield). R (E OAc): 0.65. IR (nea ): 2988, 2940, 1733, 1370, 1230, and 1048 cm−1.1H-NMR (300 MHz, CDCl3)δ 6.33 (d, J= 3.2 Hz, 1H), 6.19 (d, J= 3.1 Hz, 1H), 5.93 (b s, 1H), 4.99 (s, 2H), 4.40 (d, J= 5.5 Hz, 2H), 2.07 (s, 3H), and 2.00 (s, 3H) ppm. 13C-NMR (75 MHz, CDCl3)δ170.7 (C), 169.9 (C), 152.3 (C), 149.3 (C), 111.8 (CH), 108.6 (CH), 58.2 (CH2), 36.7 (CH2), 23.3 (CH3), and 21.0 (CH3) ppm. ESI-TOF-HRMS: [M + Na]+ calcula ed o C10H13NNaO4: 234.0739; ound: 234.0737. Gene al P ocedu e o he One-Po Sequen ial Double Acyla ion o HMFA Using EziG-CALB. EziG-CALB (20 mg) and e hyl me hoxy ace a e (5b, 24 μL, 0.21 mmol, 1.3 equi ) we e added o a solu ion o HMFA (3, 20 mg, 0.16 mmol, 100 mM) in 2-MeTHF (1.5 mL). The mix u e was shaken o 2 h a 30 °C and 250 pm, un il he comple e consump ion o he s a ing amine was obse ed by GC analysis owa d he o ma ion o me hoxyace amide 7b. The ea e , inyl ace a e (9, 44 μL, 0.47 mmol, 3 equi ) was added o he eac ion mix u e, and he eac ion was shaken o an addi ional 2 h a 30 °C and 250 pm obse ing he disappea ance o 7b and he o ma ion o p oduc 10 (GC analysis). The eac ion was il e ed, and he enzyme washed wi h CH2Cl2(2 ×1 mL). The il a e was concen a ed unde educed p essu e, eco e ing 10 wi h excellen pu i y (36.4 mg, 96% isola ed yield). {5-[(2-Me hoxyace amido)me hyl] u an-2-yl}me hyl Ace a e (10). Yellow oil. R (E OAc): 0.60. IR (nea ): 3343, 3135, 3112, 1739, 1658, 1262, and 751 cm−1.1H-NMR (300 MHz, CDCl3)δ6.85 (b s, 1H), 6.33 (d, J= 3.2 Hz, 1H), 6.21 (d, J= 3.2 Hz, 1H), 5.00 (s, 2H), 4.46 (d, J= 5.8 Hz, 2H), 3.92 (s, 2H), 3.41 (s, 3H), and 2.07 (s, 3H) ppm. 13C-NMR (75 MHz, CDCl3)δ170.7 (C), 169.6 (C), 152.0 (C), 149.3 (C), 111.7 (CH), 108.7 (CH), 72.0 (CH2), 59.3 (CH3), 58.2 (CH2), 35.9 (CH2), and 21.0 (CH3) ppm. ESI-TOF- HRMS: [M + Na]+calcula ed o C11H15NNaO5: 264.0844; ound: 264.0842. ■RESULTS AND DISCUSSION In a i s se o expe imen s, u u ylamine (4, 100 mM) was selec ed as a model subs a e due o i s comme cial a ailabili y a low p ice, while he p esence o a single eac i e g oup acili a ed he iden i ica ion o ac i e lipases o he acyla ion o he p ima y amino g oup. Due o he high eac i i y o amines, he use o nonac i a ed acyla ing agen s such as E OAc was ecommended o a oid he backg ound eac ion. 43 Thus, a lipase sc eening was pe o med unde s anda d eac ion condi ions (3 equi o E OAc, MTBE, 30 °C, 24 h and 250 pm) o p oduce N-( u an-2-ylme hyl)ace amide (6). The esul s a e depic ed in Figu e 2a, while a mo e comp ehensi e Figu e 2. Lipase-ca alyzed ace yla ion o 4: (a) unde s anda d condi ions (100 mM 4in MTBE, 3 equi o 5a, and lipase:4(1:1 w/w) a 30 °C and 250 pm o 24 h), and (b) sol en sc eening using 100 mM 4, 3 equi o 5a, and EziG-CALB (1:1 w/w enzyme:4) a 30 °C and 250 pm o 2 h. ACS Sus ainable Chemis y & Enginee ing pubs.acs.o g/jou nal/ascecg Resea ch A icle h ps://doi.o g/10.1021/acssuschemeng.3c00775 ACS Sus ainable Chem. Eng. 2023, 11, 10284−10292 10287 da a can be ound in Table S1. CALB was iden i ied as he mos e icien enzyme unde he chosen condi ions, which was in line wi h he excellen eac i i y ound in he li e a u e o he classical kine ic esolu ion o acemic u u yl amines using he comme cial p epa a ion No ozyme 435 as he bioca a- lys . 47−49 The eac ion wi h he No ozyme 435 p epa a ion led o quan i a i e con e sion in o ace amide 6unde non- op imized eac ion condi ions, mo i a ing us o use a ecen ly desc ibed immobilized CALB based on EnginZyme echnol- ogy. 46 Gladly, also quan i a i e con e sion was ob ained, being bo h supe io esul s han he ones ob ained wi h o he lipases (TLL, PSL, AKL, CRL, CALA, and ANL). In o de o ob ain u he in o ma ion abou he syn he ic bene i s o bo h CALB p epa a ions, a ime s udy was pe o med inding ha e y sho eac ion imes (30 min, Figu e S1) led o con e sions o e 90% o bo h ca alys s, equi ing only 2 h o a quan i a i e con e sion o he s a ing compound. A his poin , and inside a collabo a i e p ojec , we decided o explo e he syn he ic possibili ies o EziG-CALB mo e in dep h. Thus, he use o hese immobilized bioca alys s was p io i ized, and sol en sc eening was pe o med (Figu e 2b and Table S2). Comple e con e sions we e eached a e 2 h wi h a se ies o e he s such as MTBE, E 2O, and 2-MeTHF. Rema kably, his las sol en has been p e iously iden i ied as an ideal bio enewable medium o hyd olase-ca alyzed Figu e 3. Recycling s udies o he ace yla ion o u u ylamine (4, 20 mg, 100 mM) using immobilized EziG-CALB (20 mg), E OAc (3 equi ) in 2-MeTHF a 30 °C and 250 pm. Con e sion alues o he 10 eac ions we e de e mined by GC analyses o he eac ion c udes, eco e ing he enzyme a e each use by il a ion and wash wi h 2-MeTHF. Table 1. Sc eening o Lipases o he Enzyma ic Ace yla ion o HMFA (3) a en y enzyme 7a (%) b 1 <3 2 No ozyme 435 >99 3 EziG-CALB >99 4 TLL 83 5 PSL 69 6 AK 31 a Reac ion condi ions: 3(100 mM in 2-MeTHF), 1.3 equi o 5a, and 1:1 w/w enzyme:3 a io o 24 h a 30 °C and 250 pm. b Con e sion calcula ed by GC analyses o he eac ion c udes (see he SI o de ails). ACS Sus ainable Chemis y & Enginee ing pubs.acs.o g/jou nal/ascecg Resea ch A icle h ps://doi.o g/10.1021/acssuschemeng.3c00775 ACS Sus ainable Chem. Eng. 2023, 11, 10284−10292 10288 eac ions. 50 Al e na i ely, E OAc was also e alua ed, p esen - ing he ad an age o being u ilized as bo h he acyl dono and sol en ha would simpli y he eac ion p o ocol. Explo ing he po en ial o EziG-CALB p epa a ion, a ecyclabili y s udy was pe o med o e 10 cycles in he ace yla ion o u u ylamine (Figu e 3), g an ing o e 93% con e sion a e 1 h o eac ion in all o he expe imen s. Nex , he bes enzymes ound o he ace yla ion o amine 4 we e applied in he lipase-ca alyzed ace yla ion o 5- hyd oxyme hyl u u ylamine (3). Due o he exis ence o wo compe ing unc ional g oups in his molecule (alcohol s amine), and in an a emp o de elop a chemoselec i e p ocess, 51 a lowe amoun o he acyl dono , i.e., e hyl ace a e, was en isaged o a oid he occu ence o he N,O-diace yla ion eac ion (Table 1). In ac , he e a e examples in he li e a u e whe e he CALB-ca alyzed acyla ion o amino alcohols has led o a iable mix u es o N- and O-acyla ed compounds depending on he eac ion medium and subs a e em- ployed. 52,53 G a i yingly, in ou case, using 1.3 equi o E OAc and biobased 2-MeTHF as he sol en , bo h immobilized CALB p epa a ions p o ided comple e con e - sions (en ies 2 and 3) owa d N-ace yla ed p oduc 7a, while TLL, PSL, and lipase AK om P. luo escens led o mode a e o high con e sion alues (31−83%, en ies 4−6). In e es ingly, he eac ions s opped a he chemo- and monoselec i e N-p o ec ion o HMFA, allowing o ob ain he hyd oxy amide 7a wi h excellen yield. Impo an ly, when he eac ions we e ca ied ou in E OAc as he sol en , he o ma ion o he amido es e 8a was obse ed as he unique p oduc , which was clea ly obse ed due o he shi o he me hylene signal, p e iously assigned o he ee hyd oxyl g oup a 4.51 ppm, ha in he es e appea ed a 4.99 ppm (see Expe imen al Sec ion and NMR spec a in he SI). This ac highligh s he impo ance o he selec ion o adequa e eac ion condi ions o selec i e bio ans o ma ions. A his poin , he scope o he lipase-ca alyzed acyla ion o HMFA was in es iga ed wi h a a ie y o acyl dono s o syn hesize a amily o HMFA-de i ed amides (7a−i) and o compa e he eac i i y when p esen ing di e en s uc u al mo i s in he acyl dono (Figu e 4 and Table S3). All o he eac ions we e ca ied ou a 100 mM subs a e concen a ion, mild eac ion condi ions (30 °C), and sho eac ion imes (2 h) using EziG-CALB (1:1 weigh a io enzyme:subs a e) and 1.3 equi o acyl dono s 5a−pin bio enewable 2-MeTHF. In e es ingly, comple e con e sions we e achie ed in all cases o he selec i e N-p o ec ion o HMFA, leading o hyd oxy amides 7a−iwi h ull con e sion, quan i a i e yields, and in mos o he cases p oduc s we e eco e ed h ough simple enzyme il a ion and subsequen sol en e apo a ion. On one hand, he use o acyl dono s 5a−e,m−pbea ing di e en acyl g oups (Figu e 4; R1= Me, MeOCH2, E , ClCH2, Ph, nP , pen yl, nonyl, and undecyl) led o excellen esul s p o iding a s aigh o wa d app oach o a se ies o HMFA- de i ed amides 7a−i ha we e eco e ed wi h excellen pu i y a e a column ch oma og aphy- ee p o ocol. In e es ingly, halogena ed de i a i e 7d was syn hesized, ha can be easily en isaged as a compound o u he selec i e ans o ma ions. On he o he hand, he use o di e en alkoxy g oups in he nonac i a ed es e s o p oduce 7a wi h 5 −k(R2= Me, nP , nBu, iP , Bn, and 4-NO2C6H4), 7b wi h 5l (R2= Me), 7 wi h 5m (R2= Me), and 7i wi h 5p (R2= Me) was ound o be compa ible wi h EziG-CALB, a o ding in all cases he desi ed hyd oxy amides wi h excellen yields. Only a e y s e ically hinde ed es e such as e -bu yl ace a e did no lead o any con e sion owa d 7a. Be o e p oceeding wi h he scale-up o he eac ion, lipase- ca alyzed ace yla ion o HMFA was es ed a di e en subs a e Figu e 4. Scope o he EziG-CALB-ca alyzed chemoselec i e acyla ion o HMFA. ACS Sus ainable Chemis y & Enginee ing pubs.acs.o g/jou nal/ascecg Resea ch A icle h ps://doi.o g/10.1021/acssuschemeng.3c00775 ACS Sus ainable Chem. Eng. 2023, 11, 10284−10292 10289 concen a ions (200, 300, 400, 500, and 1000 mM). The empe a u e, E OAc a io, he amoun o EziG-CALB, and sol en (30 °C, 1.3 equi , 15 mg, and 1.2 mL, espec i ely) we e kep cons an . Moni o ing he eac ions using GC analyses showed comple e con e sions in all cases, al hough longe eac ion imes (up o 22 h) we e equi ed o mo e concen a ed ans o ma ions. Un o una ely, he use o highe HMFA concen a ions and longe eac ion imes led o he o ma ion o mode a e amoun s o he diace yla ed p oduc 8a (up o 7%, see Figu e S2). The ea e , he lipase-ca alyzed p ocess wi h 200 mM HMFA was conduc ed a 1 g scale (7.87 mmol), ob aining he desi ed hyd oxy amide 7a a e 5 h and a simple ee-column wo k-up consis ing o enzyme il a ion and sol en e apo a ion (85% isola ed yield). An en i onmen al impac analysis was pe o med, making use o he E- ac o concep , 54 o he bio ans o ma ion a 1 g scale (Figu es S3 and S4). Taking in o accoun he eagen s, ca alys , and sol en s employed in his p o ocol, we could con i m ha ou me hodology p esen ed a alue o 53.6, wi h he mos -con ibu ing o he ac o (97.9%) being he o ganic sol en s ha we e used in he eac ion medium and downs eam p ocess o wash he enzyme. In ac , excluding sol en s, ou enzyma ic me hod p esen ed an excellen alue o 1.1, demons a ing i s high po en ial, especially i he o ganic sol en s could be eu ilized. Las bu no he leas , we decided o explo e he possibili y o accessing he o hogonally p o ec ed amido es e using a ully enzyma ic app oach, ideally pe o med in one po (Scheme 2). Fo ha eason, we ook ad an age o he excellen chemoselec i i y displayed by EziG-CALB in he monoselec i e N-acyla ion o HMFA using e hyl me hoxy ace a e (5b) in 2-MeTHF. Once he eac ion eached comple e con e sion o hyd oxy amide 7b a e 2 h, an ac i a ed acyl dono such as inyl ace a e (9, 3 equi ) was added. Unde hese condi ions and a e 2 h o addi ional eac ion ime, amide 10 was isola ed (96%) possessing wo di e en acyl moie ies as O- and N-subs i u ions. The demons a ed s aigh o wa d eac ion sequence o such class o compounds opens he doo o easy access o, on one hand, di e en amido es e s by selec ing he p ope (non) ac i a ed es e s, and on he o he hand, O-p o ec ed HMFA de i a i es a e selec i e N-dep o ec ion, i equi ed. ■CONCLUSIONS Lipases a e sui able enzymes o he unc ionaliza ion o amines, which is highly a ac i e when aluable building blocks and pha macologically ac i e molecules a e syn hesized. This is he case o C. an a c ica lipase ype B (CALB), ha is usually he enzyme o choice o amide o ma ion, and he ein i was employed o p epa e a se ies o amides s a ing om u u ylamine and especially om he bi unc ional 5-hyd oxy- me hyl u u ylamine. Two immobilized p epa a ions o CALB, one on an ac ylic esin suppo and he o he on a glass po ous ma e ial ca ie , ha e yielded chemoselec i e unc ionaliza ion o he p ima y amino g oup o HMFA. Rema kably, EziG- CALB was shown o be an excellen bioca alys ha can be used o se e al cycles in a biobased o ganic sol en such as 2- MeTHF wi hou signi ican loss o he enzyme ac i i y. Con olling he amoun o he acyl dono was ound o be he key ac o o selec i e unc ionaliza ions, and he use o only 1.3 equi o E OAc led o N-[(5-(hyd oxyme hyl) u an-2- yl)me hyl]ace amide (7a). Highe acyl dono concen a ions p o ided s aigh o wa d access o he co esponding amido es e 8a. Rema kably, using he op imized eac ion condi ions, chemoselec i e N-acyla ion o HMFA was demons a ed in he syn hesis o nine hyd oxy amides wi h di e en ( unc ion- alized) acyl subs i uen s. This app oach was shown o be easily scalable o 1 g o subs a e, and easible a high subs a e concen a ions (up o 1 M). Mo eo e , i is cha ac e ized wi h a good en i onmen al impac as demons a ed by he E- ac o calcula ion o he ee-column scale-up p ocess (53.6 and 1.1, including and excluding sol en s, espec i ely). A las , he o ma ion o o hogonally N,O-dip o ec ed amido es e 10 was demons a ed h ough a one-po wo-s ep ans o ma ion, ia sequen ial addi ion o e hyl me hoxy ace a e and inyl ace a e as acyl dono s o N- and O-p o ec ion, espec i ely, unde e y mild eac ion condi ions. This me hodology can be en isaged as a p omising ool o he design o selec i ely modi ied HMFA de i a i es. ■ASSOCIATED CONTENT * sı Suppo ing In o ma ion The Suppo ing In o ma ion is a ailable ee o cha ge a h ps://pubs.acs.o g/doi/10.1021/acssuschemeng.3c00775. S uc u es o all chemical compounds s udied, op i- miza ion o he eac ion condi ions, de elopmen o GC analy ical me hods, en i onmen al assessmen calcula- ions, and copies o 1H-NMR, 13C-NMR, and DEPT NMR spec a o syn hesized de i a i es (PDF) ■AUTHOR INFORMATION Co esponding Au ho s Alexey Volko −EnginZyme AB, 171 65 Solna, Sweden; Email: [email p o ec ed] Vicen e Go o -Fe nández −O ganic and Ino ganic Chemis y Depa men , Uni e si y o O iedo, O iedo 33006, Spain; o cid.o g/0000-0002-9998-0656; Email: [email p o ec ed] Au ho s An ía Pin o −O ganic and Ino ganic Chemis y Depa men , Uni e si y o O iedo, O iedo 33006, Spain; EnginZyme AB, 171 65 Solna, Sweden I án La ande a −O ganic and Ino ganic Chemis y Depa men , Uni e si y o O iedo, O iedo 33006, Spain; o cid.o g/0000-0003-4857-4428 Comple e con ac in o ma ion is a ailable a : Scheme 2. One-Po Two-S ep Enzyma ic P ocess o Ob ain O hogonally P o ec ed HMFA Amido Es e 10 ACS Sus ainable Chemis y & Enginee ing pubs.acs.o g/jou nal/ascecg Resea ch A icle h ps://doi.o g/10.1021/acssuschemeng.3c00775 ACS Sus ainable Chem. Eng. 2023, 11, 10284−10292 10290 h ps://pubs.acs.o g/10.1021/acssuschemeng.3c00775 Au ho Con ibu ions The manusc ip was w i en h ough con ibu ions o all au ho s. All au ho s ha e gi en app o al o he inal e sion o he manusc ip . No es The au ho s decla e no compe ing inancial in e es . ■ACKNOWLEDGMENTS Financial suppo om he Eu opean Union’s Ho izon 2020 esea ch and inno a ion p og amme unde he Ma ie Skłodowska−Cu ie G an Ag eemen Numbe 860414 (IN- TER aces) is g a e ully acknowledged. ■ABBREVIATIONS CALB: Candida an a c ica lipase ype B; equi : equi alen s; EziG: EnginZyme; HMF: 5-hyd oxyme hyl u u al; HMFA: 5- hyd oxyme hyl u u ylamine ■REFERENCES (1) Hou, Q.; Qi, X.; Zhen, M.; Qian, H.; Nie, Y.; Bai, C.; Zhang, S.; Bai, X.; Ju, M. 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