Filtering the NMR Spectra of Mixtures by Coordination to Paramagnetic Cu2+

Fil e ing he NMR Spec a o Mix u es by Coo dina ion o Pa amagne ic Cu2+ Juan Co ea, Ana Ga cia-Ba andela, Llo encSocias-Pin o, and Edua do Fe nandez-Megia* Ci e This: Anal. Chem. 2022, 94, 10907−10911 Read Online ACCESS Me ics & Mo e A icle Recommenda ions * sı Suppo ing In o ma ion ABSTRACT: The pa amagne ic spin elaxa ion (PSR) il e allows he selec i e NMR signal supp ession o componen s in mix u es acco ding o hei complexa ion abili y o a pa amagne ic ion. I elies on he as e elaxa ion o nuclei in pa amagne ic en i onmen s and hus is complemen a y o classical di usion and elaxa ion il e s. So a , he PSR il e has es ablished Gd3+ as he sole PSR agen , es ic ing he pa amagne ic il e ing epe oi e. He ein, we p esen Cu2+ as a obus PSR agen wi h cha ac e is ic il e ing p ope ies. While Gd3+ depends on unspeci ic ion-pai in e ac ions wi h anionic componen s, Cu2+ s ands ou o il e ing species ia o de ed coo dina ion complexes. An e alua ion o he pa amagne ic e ec o Cu2+ o e mo e han 50 small molecules and polyme s has un eiled di e en sensi i i ies o Cu2+ (especially high o py idines, diamines, polyamines, and amino alcohols) and p ecise il e ing condi ions o mix u es (1H, COSY, and HMQC) ha we e challenged wi h a es bed o comme cial d ugs. The ad an age o in eg a ing Cu2+ and Gd3+ o he s epwise PSR il e ing o complex mix u es is also shown. As na u e seldom p o ides pu e compounds, chemis s ha e de o ed g ea e o s o he sepa a ion o complex mix u es. Fo una ely, he NMR analysis o mix u es sides eps he necessi y o physical sepa a ions unde ce ain condi ions. NMR il e s ake ad an age o di e ences in he di usion coe icien s and elaxa ion imes o he componen s o he selec i e signal supp ession o small molecules and mac o- molecules, espec i ely. 1,2 Wi h he aim o widening he NMR il e ing po olio beyond molecula weigh limi s, ou g oup has desc ibed he pa amagne ic spin elaxa ion (PSR) il e , 3,4 which elies on he as e elaxa ion o nuclei in pa amagne ic en i onmen s. 5,6 The addi ion o minu e concen a ions o Gd3+ (pa amagne ic) o mix u es allows he selec i e supp ession o pa icula componen s om he 1D and 2D spec a, acco ding o hei Gd3+ complexa ion abili y (Figu e 1). Since Gd3+ has he la ges spin momen (S= 7/2) and a high elec onic co ela ion ime (τsca. 10−8s), he Solomon− Bloembe gen−Mo gan equa ions p edic a selec i e dec ease o ans e se elaxa ion imes (T2) o species in as chemical exchange wi h Gd3+. 7−9 The in e se p opo ionali y be ween T2and he spec al line wid h 10 leads o hei selec i e signal supp ession, wi hou a ec ing he esolu ion and chemical shi o o he componen s in he mix u e. Since complexa ion o Gd3+ is mainly elec os a ic (ion-pai ), he PSR il e a ec s mo e anionic species han neu al and ca ionic species. Success ul applica ions o he PSR il e include he sequen ial NMR il e ing o mix u es o in e es in he pha maceu ical and ood indus ies 4,11 and he as sc eening o DNA ligands. 12 In e es ingly, he PSR il e bene i s om a a he low sensi i i y o size and molecula weigh ha makes i compa ible wi h classical elaxa ion and di usion il e s. The ad an age o his is aken o he il e ing o lowe PSR- sensi i e species, which leads o line-b oadening a he han a ull signal embedmen in he baseline, by implemen ing a complemen a y sho T2- il e , such as he Ca −Pu cell− Meiboom−Gill (CPMG). 13,14 Recei ed: May 6, 2022 Accep ed: July 25, 2022 Published: July 27, 2022 Figu e 1. PSR il e s based on Gd3+ (ion-pai ) and Cu2+ (coo dina ion) complexes. Le e pubs.acs.o g/ac © 2022 The Au ho s. Published by Ame ican Chemical Socie y 10907 h ps://doi.o g/10.1021/acs.analchem.2c01983 Anal. Chem. 2022, 94, 10907−10911 Downloaded ia UNIV DE SANTIAGO DE COMPOSTELA on Sep embe 1, 2022 a 07:15:24 (UTC). See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles. While he PSR il e based on ion-pai Gd3+-complexes is highly e icien in simpli ying he NMR analysis o complex mix u es, he de elopmen o PSR il e s wi h o he pa a- magne ic ions displaying al e na i e complexa ion modes would g ea ly ex end he u ili y o his echnology. He ein, we desc ibe ou e o s owa d a PSR il e based on he mo e coo dina ing Cu2+ (S= 1/2, τsca. 10−9s), a pa amagne ic ion aimed a selec i ely il e ing he NMR signals o species ia coo dina ion complexes (Figu e 1). The easibili y o a Cu2+ PSR il e was con i med by he selec i e supp ession o glucosamine in he p esence o glucose (Figu e 2C), a supp ession un easible o ep oduce in he p esence o Gd3+ (Figu e S4) because o he simila sensi i i y o bo h componen s o his ion. 4 Con e sely, he coo dina ing 1,2-amino alcohol moie y o glucosamine ensu es a selec i e il e ing in he p esence o Cu2+. The scope o Cu2+ as coo dina ing PSR agen was assessed by analyzing he pa amagne ic e ec on he 1H NMR spec a o a collec ion o mo e han 50 small molecules and polyme s o in e es in he pha maceu ical and ood indus ies, which display a la ge a ie y o unc ional g oups. Depending on he ex en o signal b oadening ( om no e ec o comple e supp ession), hese species we e assigned o se en g oups (Table 1), wi h he mo e sensi i e ones comp ising highly coo dina ing compounds. 1H NMR spec a o ep esen a i e molecules in he uppe , medium, and bo om pa s o Table 1, eco ded in he absence/p esence o Cu2+, a e shown in Figu es S1−S3: (1S,2S)-2-amino-1-phenyl-1,3-p opanediol, adenosine, suc ose. As a ule o humb, he b oadening e ec o Cu2+ on he 1H NMR spec a o py idines, diamines, polyamines, and amino alcohols was conside ably highe han wi h Gd3+, 4 in consis ency wi h a highe coo dina ion abili y. Nex , he possibili y o pe o ming selec i e supp essions by Cu2+ among he se en g oups in Table 1 was assessed in wo- componen mix u es (Figu e 2A). I was con i med ha he mo e dis an he g oups, he easie he selec i e supp essions. Also, he impossibili y o pe o ming supp essions wi hin a single g oup and be ween some neighbo ing g oups. As a esul , he ini ial g oups ( e lec ing he pa amagne ic b oad- ening e ec ) we e educed o jus h ee ca ego ies (designa ed as Blue, Yellow, and Red), acco ding o hei ease o supp ession by Cu2+. In addi ion, om da a in Figu e 2A, gene al condi ions o selec i e 1H, COSY, and HMQC supp essions be ween ca ego ies we e de e mined (concen- a ion o Cu2+/leng h o a complemen a y CMPG T2- il e ; Figu e 2B): Figu e 2. (A) Success ul and ailed PSR supp essions in wo-componen mix u es (D2O, 500 MHz). (B) PSR il e ing condi ions o selec i e 1H, COSY, and HMQC supp essions. Rep esen a i e examples o selec i e supp essions be ween Blue-Yellow-Red ca ego ies. 1H NMR spec a (D2O, 500 MHz, 300 K) o a mix u e o he ollowing: (C) glucosamine (2 mg/mL) and glucose (2 mg/mL) be o e (a) and a e (b) he addi ion o Cu2+ (2 mM), (D) 2-amino-1-phenyl-1,3-p opanediol (1 mg/mL) and adenosine (3 mg/mL) be o e (c) and a e (d) he addi ion o Cu2+ (0.16 mM) + T2- il e (CPMG, 30 ms), and (E) adenosine (1.2 mg/mL) and glucose (1.6 mg/mL) be o e (e) and a e ( ) he addi ion o Cu2+ (13 mM) + T2- il e (CPMG, 100 ms). Analy ical Chemis y pubs.acs.o g/ac Le e h ps://doi.o g/10.1021/acs.analchem.2c01983 Anal. Chem. 2022, 94, 10907−10911 10908 •Blue-Red: > 2.0 mM Cu2+ •Blue-Yellow: < 2.0 mM Cu2+, < 50 ms CPMG •Yellow-Red: > 2.0 mM Cu2+, > 50 ms CPMG Figu e 2 depic s ep esen a i e examples o success ul supp essions wi hin hese h ee ca ego ies. Fo ins ance, he a o emen ioned glucosamine/glucose supp ession can be easily a ionalized now, conside ing hei espec i e inclusion in o he Blue and Red ca ego ies (Figu e 2C). Simila ly, Figu e 2D,E show selec i e supp essions ha exploi he use o complemen a y T2- il e s be ween componen s o con iguous ca ego ies. Rema kably, none o hese supp essions could be ealized wi h Gd3+ as PSR ion (Figu es S4−S6), con i ming he coo dina ion abili y o Cu2+ as esponsible o he selec i i y achie ed. O he ep esen a i e spec a o success ul (di e en ca ego ies) and un ui ul (same ca ego y) supp essions in he wo-componen mix u es shown in Figu e 2A a e included in he SI (Figu es S7−S12). Addi ional ad an ages ha eme ged on he use o Cu2+ s Gd3+ as PSR agen include he possibili y o wo king in a wide pH- ange (Gd3+ ends o p ecipi a e a pH > 7.0) and a smalle b oadening e ec o e he esidual HOD signal. The eliabili y o Table 1 o p edic Cu2+ PSR il e s in complex mix u es was challenged wi h a es bed o comme cial d ugs, namely (i) an amoxicillin/cla ulanic acid an ibio ic (amoxicillin, cla ulanic acid, PEG, PVP); (ii) Ca iban, a d ug o ea nausea and omi ing in p egnancy (doxylamine, py idoxine, suc ose); (iii) he an ibio ic P ode ma (doxycy- cline, suc ose); and (i ) A epodin, a medicine o he ea men and diagnosis o sup a en icula achyca dia (adenosine iphospha e, glycine, benzyl alcohol). We s a ed analyzing Amoxicillin/Cla ulanic acid Cin amed, an an ibio ic ha con ains wo ac i e ing edien s, he penicillin-like an ibio ic amoxicillin (Blue) and he be a- lac amase inhibi o cla ulanic acid (no included in Table 1 bu expec ed o be Blue by simila i y). NMR- isible excipien s comp ise poly(e hylene glycol) (PEG) and poly inylpy oli- done (PVP), bo h Red species. A ending o he Blue and Red ca ego ies o he cons i uen s, a selec i e PSR supp ession was an icipa ed o amoxicillin and cla ulanic acid in he p esence o Cu2+.Figu e 3 shows hei selec i e il e ing, con i ming he p edic i e cha ac e o Table 1, and he h ee colo ca ego ies p oposed. When lowe concen a ions o Cu2+ we e assessed, i was e en possible o a ain a s epwise supp ession o he Blue componen s, il e ing i s he mo e coo dina ing amoxicillin. In e es ingly, he use o Gd3+ did no a o d a clean supp ession o any componen , con i ming Cu2+ as a PSR agen wi h cha ac e is ic il e ing p ope ies. Then, we p oceeded o analyze h ee comme cial d ugs (Ca iban, P ode ma, A epodin) con aining Yellow and Red componen s, whe e a complemen a y CPMG il e was expec ed o success ul supp essions. Ca iban is a medicine used o ea nausea and omi ing in p egnan women ha con ains doxylamine (an ihis amine) and py idoxine ( i amin B6) as ac i e ing edien s, bo h subs i u ed py idines ha belong o he Yellow ca ego y. The mix u e also includes suc ose (Red) as NMR- isible excipien . As p edic ed, he only addi ion o Cu2+ did no esul in a clean il e ing o doxylamine and py idoxine. Howe e , concomi an applica ion o a sho CPMG il e a o ded hei clean supp ession, lea ing una ec ed he esolu ion and chemical shi o he suc ose signals (Figu e 4). Rema kably, he ideli y o he PSR-CPMG il e was also demons a ed in 2D COSY and HMQC expe imen s, whe e he CPMG sequence was used as an exci a ion block eplacing he i s exci a ion pulse. 15 No unexpec edly, he use o Gd3+ as PSR agen was again unsuccess ul, ei he in he absence o p esence o CPMG il e s. The an ibio ic P ode ma con ains wo NMR- isible componen s, he ac i e ing edien doxycycline (Yellow) and suc ose (Red) as excipien . He e again, he di ec il e ing o he mos sensi i e Yellow componen was un easible by he Table 1. Pa amagne ic B oadening E ec (G oups 1−7) and Ease o Supp ession by Cu2+ (Blue, Yellow, Red Ca ego ies) in 1H NMR (4 mg/mL in D2O, 500 MHz, 300 K) a a G oup 7: supp ession o all signals a <1 mM Cu2+, G oup 6: supp ession o all signals a >1 mM Cu2+, G oup 5: b oadening o all signals a >1 mM Cu2+, G oup 4: supp ession o some signals a >1 mM Cu2+, G oup 3: b oadening o some signals a >1 mM Cu2+, G oup 2: educed signal esolu ion a >1 mM Cu2+, G oup 1: no e ec on signal esolu ion a >1 mM Cu2+. Figu e 3. 1H NMR spec a (D2O, 500 MHz, 300 K) o Amoxicillin/ Cla ulanic acid Cin amed (10 mg/mL) be o e (a) and a e he addi ion o 4 and 17 mM Cu2+ (b and c, espec i ely). Analy ical Chemis y pubs.acs.o g/ac Le e h ps://doi.o g/10.1021/acs.analchem.2c01983 Anal. Chem. 2022, 94, 10907−10911 10909 sole addi ion o Cu2+. Howe e , implemen a ion o a simul aneous sho CPMG il e allowed he clean supp ession o doxycycline (Figu e 5). Ve y simila il e ing condi ions also allowed he e icien il e ing o he Yellow componen s (adenosine iphospha e and glycine) o A epodin (Figu e S13). Ha ing es ablished he u ili y o Cu2+ as PSR agen wi h il e ing p ope ies dependen on he coo dina ion abili y o he componen s in a mix u e ( a he han ion-pai in e ac ions o Gd3+), we decided o assess he in eg a ion o bo h pa amagne ic ions in he il e ing o complex mix u es. To his end, we selec ed Ace ilcis eina Mylan, a comme cial mucoly ic d ug composed o ace ylcys eine (CuYellow/GdYellow) as ac i e ing edien , ci ic acid (CuBlue/GdBlue) as excipien , and wo swee ene s, D-manni ol (CuRed/GdYellow) and sodium sac- cha in (CuRed/GdRed). A ending o he Blue-Yellow-Red ca ego y o he componen s owa d Gd3+, we ha e p e iously epo ed he sequen ial supp ession o ci ic acid in a i s s ep, ollowed by he simul aneous supp ession o ace ylcys eine and D-manni ol (bo h GdYellow componen s). 4 He ein, he s onge Cu2+-coo dina ion o ace ylcys eine han D-manni ol (Table 1) has been exploi ed o he s epwise supp ession o he h ee componen s in a way una ainable wi h a single PSR agen . Thus, as shown in Figu e 6, a e an ini ial supp ession o ci ic acid wi h Gd3+, ace ylcys eine was il e ed wi h Cu2+, ollowed by a inal supp ession o D-manni ol wi h Gd3+. Applica ion o he PSR il e wi h Cu2+ s a s wi h he assignmen o he indi idual componen s in a mix u e o he Blue-Yellow-Red ca ego ies. As a i s app oach, use s a e Figu e 4. 1H, 1H−1H COSY, and 1H−13C HMQC spec a (D2O, 500 MHz, 300 K) o Ca iban (72 mg/mL) be o e (a, c, e) and a e (b, d, ) he addi ion o Cu2+ (13 mM) + T2- il e (CPMG, 75 ms). Figu e 5. 1H NMR spec a (D2O, 500 MHz, 300 K) o P ode ma (4 mg/mL) be o e (a) and a e (b) he addi ion o Cu2+ (4 mM) + T2- il e (CPMG, 75 ms). Figu e 6. NMR- isible componen s o Ace ilcis eina Mylan and hei espec i e Blue-Yellow-Red ca ego ies owa d Cu2+ and Gd3+.1H NMR spec a (D2O, 500 MHz, 300 K) o Ace ilcis eina Mylan (25 mg/mL) supplemen ed wi h saccha in (12.5 mg/mL) be o e (a) and a e he addi ion o (b) Gd3+ (25 μM) + T2- il e (CPMG, 25 ms), (c) Cu2+ (2.5 mM) + T2- il e (CPMG, 250 ms), and (d) Gd3+ (0.2 mM) + T2- il e (CPMG, 300 ms). Analy ical Chemis y pubs.acs.o g/ac Le e h ps://doi.o g/10.1021/acs.analchem.2c01983 Anal. Chem. 2022, 94, 10907−10911 10910 ad ised o ind s uc u al simila i ies be ween he species in a mix u e o in e es and hose in Table 1. Ne e heless, o a p ope inclusion o species in he Blue-Yellow-Red ca ego ies, he pa amagne ic e ec o Cu2+ on hei 1H NMR spec a should be de e mined as desc ibed in Table 1 (ex en o signal b oadening as a unc ion o he concen a ion o Cu2+). Once he species ha e been assigned o he h ee ca ego ies, selec i e supp essions could be expec ed by applica ion o he PSR condi ions shown in Figu e 2B. While clean supp essions ope a e o Blue species in he p esence o Red ones by he simple addi ion o mM concen a ions o Cu2+, he selec i e il e ing o species om con iguous ca ego ies (Blue-Yellow and Yellow-Red) a e un easible by he sole addi ion o Cu2+, being necessa y he implemen a ion o simul aneous CPMG il e s. Al hough he selec i e il e ing o species wi hin a ca ego y migh wo k in speci ic examples using inc easing concen a ions o Cu2+ o uning he leng h o CPMG il e s, his will no be o gene al applica ion because he 1HT2 alues o he species in a ca ego y will le el down in he p esence o a PSR agen , making unlikely hei selec i e supp ession. In conclusion, Cu2+ is p esen ed as a obus PSR agen wi h cha ac e is ic NMR il e ing p ope ies di e en han Gd3+, he a che ypal PSR agen so a . No only do he pa amagne ic p ope ies change be ween nuclei, bu also hei complexa ion modes di e , o e ing he oppo uni y o une he ou come o he PSR il e . While Gd3+ elies on he ion-pai complexa ion abili y o he componen s in a mix u e (mainly anionic species), Cu2+ s ands ou because o a g ea e capaci y o il e ing species ha pa icipa e in coo dina ion complexes, such as py idines, diamines, polyamines, and amino alcohols. An e alua ion o he pa amagne ic e ec o Cu2+ o e mo e han 50 small molecules and polyme s has un eiled h ee ca ego ies o compounds (Blue-Yellow-Red ca ego ies acco d- ing o hei ease o supp ession by Cu2+) and p ecise il e ing condi ions o 1H, COSY, and HMQC be ween hem. The in eg a ion o he speci ic il e ing p ope ies o Cu2+ and Gd3+ as PSR agen s o he analysis o complex mix u es has been also demons a ed, widening he ho izons o he PSR echnology o quali y con ol, na u al p oduc ex ac s, o he me abolic p o iling o biological samples. Finally, ha ing demons a ed he u ili y o Cu2+ as PSR agen , a mo e p ecise assignmen o species o he Blue-Yellow-Red ca ego ies is en isaged using he ans e se elaxa ion enhancemen (R2p), as p e iously done o Gd3+. 4 This app oach, which in ol es he analysis o he1HT2 alues o he species o in e es in he absence and p esence o Cu2+, will be he ocus o ou in es iga ions in he u u e and epo ed in due ime. ■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/acs.analchem.2c01983. Ma e ials, me hods, and NMR spec a (PDF) ■AUTHOR INFORMATION Co esponding Au ho Edua do Fe nandez-Megia −Cen o Singula de In es igación en Química Biolóxica e Ma e iais Molecula es (CIQUS), Depa amen o de Química O gánica, Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain; o cid.o g/0000-0002-0405-4933; Email: [email p o ec ed] Au ho s Juan Co ea −Cen o Singula de In es igación en Química Biolóxica e Ma e iais Molecula es (CIQUS), Depa amen o de Química O gánica, Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain Ana Ga cia-Ba andela −Cen o Singula de In es igación en Química Biolóxica e Ma e iais Molecula es (CIQUS), Depa amen o de Química O gánica, Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain Llo encSocias-Pin o −Cen o Singula de In es igación en Química Biolóxica e Ma e iais Molecula es (CIQUS), Depa amen o de Química O gánica, Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain Comple e con ac in o ma ion is a ailable a : h ps://pubs.acs.o g/10.1021/acs.analchem.2c01983 No es The au ho s decla e no compe ing inancial in e es . ■ACKNOWLEDGMENTS This wo k was suppo ed by he Spanish Minis y o Science and Inno a ion (RTI2018-102212-B-I00 and PID2021- 127684OB-I00), he Xun a de Galicia (ED431C 2018/30, and Cen o singula de in es igación de Galicia acc edi a ion 2019−2022, ED431G2019/03), Axencia Galega de Inno ación (IN845D 2020/09), and he Eu opean Union (Eu opean Regional De elopmen Fund-ERDF). ■REFERENCES (1) No oa-Ca ballal, R.; Fe nandez-Megia, E.; Jimenez, C.; Rigue a, R. 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