Cyclic Dipeptide-Based Ambidextrous Supergelators: Minimalistic

Aug 31, 2017 - Computational Modeling. The molecular structure for the compounds CDP-1, CDP-2, CDP-3, and CDP-4 were built using molden software.(48) ...
3 downloads 12 Views 2MB Size
Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Cyclic Dipeptide-based Ambidextrous Supergelators: Minimalistic Rational Design, Structure-Gelation Studies and In Situ Hydrogelation Shivaprasad Manchineella, N. Arul Murugan, and Thimmaiah Govindaraju Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00924 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 2, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

&\FOLF 'LSHSWLGH EDVHG $PELGH[WURXV 6XSHUJHODWRUV 0LQLPDOLVWLF 5DWLRQDO 'HVLJQ 6WUXFWXUH *HODWLRQ 6WXGLHV DQG ,Q 6LWX +\GURJHODWLRQ

6KLYDSUDVDG 0DQFKLQHHOOD‚ 1 $UXO 0XUXJDQÁ DQG 7KLPPDLDK *RYLQGDUDMX ‚ %LRRUJDQLF



&KHPLVWU\ /DERUDWRU\ 1HZ &KHPLVWU\ 8QLW -DZDKDUODO 1HKUX &HQWUH IRU

$GYDQFHG 6FLHQWLILF 5HVHDUFK -1&$65 -DNNXU %HQJDOXUX

.DUQDWDND ,QGLD ( PDLO

WJUDMX#MQFDVU DF LQ Á 'LYLVLRQ

RI 7KHRUHWLFDO &KHPLVWU\ DQG %LRORJ\ 6FKRRO RI %LRWHFKQRORJ\ .7+ 5R\DO ,QVWLWXWH

RI 7HFKQRORJ\ 6

6WRFNKROP 6ZHGHQ

.(F\FOR / 3KH

/ *OX 2W%X @ ZHUH GHVLJQHG EDVHG RQ WKH LQIRUPDWLRQ WKDW DURPDWLF DPLQR DFLG GHULYDWLYHV IDFLOLWDWH WKH VHOI DVVHPEO\ WR IRUP LQMHFWDEOH RUJDQRJHOV )LJXUH >F\FOR / 3KH / $VS 2W%X @ DQG &'3

2Q WKH RWKHU KDQG &'3

>F\FOR / /HX / *OX 2W%X @ ZHUH FKRVHQ WR

XQGHUVWDQG WKH VLJQLILFDQFH RI DPLQR DFLG VLGH FKDLQ IXQFWLRQDOLW\ RQ WKH JHODWLRQ SURSHQVLW\ RI &'3V LQ RUJDQLF DQG DTXHRXV VROYHQWV DV ZHOO DV WKHLU LQ VLWX JHODWLRQ DELOLW\ XQGHU SK\VLRORJLFDO FRQGLWLRQV 7KH WHUWLDU\ EXW\OR[\FDUERQ\O %RF PRLHW\ ZDV FKRVHQ IRU VLGH FKDLQ SURWHFWLRQ RI / JOXWDPLF DFLG DQG / DVSDUWLF DFLG RZLQJ WR WKH DELOLW\ RI WHUWLDU\ EXW\O JURXS LQ GULYLQJ PROHFXODU JHODWLRQ RI VKRUW SHSWLGH GHULYDWLYHV V\QWKHVHV URXWHV 6FKHPH 6 6

$OO WKH &'3 ZHUH SUHSDUHG WKURXJK WKH UREXVW

7KH )PRF SURWHFWHG OLQHDU GLSHSWLGHV V\QWKHVL]HG ZHUH

VXEMHFWHG WR LQWUDPROHFXODU F\FOL]DWLRQ E\ WUHDWLQJ ZLWK SLSHULGLQH LQ GLFKORURPHWKDQH WR REWDLQ FRUUHVSRQGLQJ &'3V LQ JRRG \LHOG 7KH FKHPLFDO SXULW\ DQG LQWHJULW\ RI WKH LQWHUPHGLDWH GLSHSWLGHV DQG &'3V ZDV HYDOXDWHG XVLQJ 105 PDVV VSHFWURPHWU\ DQG HOHPHQWDO DQDO\VLV 6XSSRUWLQJ ,QIRUPDWLRQ *HODWLRQ 6WXGLHV 7KH JHODWLRQ DELOLW\ RI &'3V LQ RUJDQLF DQG DTXHRXV VROYHQWV ZDV VWXGLHG E\ KHDWLQJ DQG FRROLQJ F\FOH WKURXJK LQYHUVLRQ WXEH PHWKRG DOVR NQRZQ DV WDEOH WRS UKHRORJ\ DV

ACS Paragon Plus Environment

Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

H[SODLQHG LQ WKH H[SHULPHQWDO VHFWLRQ &'3

VKRZHG DPELGH[WURXV JHODWLRQ EHKDYLRU DQG

IRUPHG JHOV LQ ERWK RUJDQLF DQG DTXHRXV VROYHQWV DW OHVV WKDQ ZW

FRQFHQWUDWLRQ 7DEOH

7KH FRUUHVSRQGLQJ SKRWRJUDSKV RI JHOV LQYHUWHG YLDOV DUH VKRZQ LQ )LJXUH )LJXUH $ 7KXV &'3 PROHFXOH RI &'3

EHKDYHV DV VXSHUJHODWRU 0*&

HQWUDSV DSSUR[LPDWHO\

ZW

XSSHU SDQHO

ZKLFK LQIHUV WKDW RQH

ZDWHU PROHFXOHV DQG UHVXOWV LQ WKH IRUPDWLRQ RI

K\GURJHOV )XUWKHU WKH JHODWLRQ DELOLW\ RI &'3

&'3

DQG &'3

ZDV VWXGLHG WR XQGHUVWDQG

WKH VLJQLILFDQFH RI IXQFWLRQDO JURXSV LQ GULYLQJ WKH JHODWLRQ WKURXJK D VLPSOH VWUXFWXUH JHODWLRQ SURSHUW\ VWXG\ &'3

ZLWK / SKHQ\ODODQLQH DQG NHHSLQJ DOO RWKHU IXQFWLRQDOLWLHV VDPH DV &'3

ZDV H[SHFWHG WR SURYLGH LQVLJKW LQWR WKH VLJQLILFDQFH RI DURPDWLF IXQFWLRQDOLW\ SKHQROLF LQ W\URVLQH YHUVXV SKHQ\O RI SKHQ\ODODQLQH IRU JHODWLRQ &'3 VLPLODU WR &'3 WKDQ

ZW

DQG IRUPHG JHOV LQ RUJDQLF DQG DTXHRXV VROYHQWV DW JHODWRU FRQFHQWUDWLRQV OHVV

DQG KHQFH TXDOLILHG DV VXSHUJHODWRU 7DEOH

FRQWDLQLQJ RUJDQR DQG K\GURJHOV RI &'3 &'3

VKRZHG H[FHOOHQW JHODWLRQ SURSHUW\

DUH VKRZQ LQ )LJXUH

ZLWK SKHQ\ODODQLQH DJDLQVW W\URVLQH LQ &'3

LQGLFDWLQJ WKDW DURPDWLF ±2+ LQ &'3

7KH SKRWRJUDSKV RI LQYHUWHG YLDOV ORZHU SDQHO )LJXUH %

VKRZHG VLPLODU JHODWLRQ SURSHUW\ DQG KHQFH

LV QRW WKH PDMRU JHODWLRQ GULYLQJ IXQFWLRQDOLW\ 7KH

VLJQLILFDQFH RI HWK\OHQH OLQNHU ZKLFK FRQQHFWV WKH &'3 FRUH ZLWK %RF JURXS LQ &'3 DQG &'3 ZDV HYDOXDWHG E\ VWXG\LQJ WKH JHODWLRQ DELOLW\ RI &'3

FRQWDLQLQJ / DVSDUWLF DFLG PHWK\OHQH

OLQNHU LQ SODFH RI / JOXWDPLF DFLG HWK\OHQH OLQNHU &'3

ZDV IRXQG WR EH SRRU JHODWRU DV LW

IRUPHG ORRVH JHO RQO\ ZLWK DURPDWLF VROYHQW PJ P/

ZW

7DEOH

GLFKORUREHQ]HQH DW D YHU\ KLJK FRQFHQWUDWLRQ

6XEVHTXHQWO\ ZH VWXGLHG WKH JHODWLRQ SURSHQVLW\ RI &'3

/ OHXFLQH WR VWXG\ WKH UROH RI DOLSKDWLF YHUVXV DURPDWLF DV LQ &'3V

ZLWK

IXQFWLRQDOLW\ LQ GULYLQJ

JHODWLRQ 7KH RSWLPL]HG VHFRQG DPLQR DFLG / JOXWDPLF DFLG ZLWK %RF SURWHFWLRQ ZDV UHWDLQHG DV LQ &'3

DQG &'3

7KH VWXGLHV UHYHDOHG WKH IDLQW JHODWLRQ SURSHQVLW\ RI &'3

ACS Paragon Plus Environment

FRPSDUHG

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

WR &'3 &'3

DQG &'3

+RZHYHU &'3

Page 14 of 40

VKRZHG UHODWLYHO\ JRRG JHODWLRQ SURSHUW\ FRPSDUHG WR

LQ WHUPV RI D QXPEHU RI VROYHQWV WKDW FDQ EH JHOOHG &'3

JHODWHV VHOHFWLYHO\ DURPDWLF

VROYHQWV DW ORZHU FRQFHQWUDWLRQV DQG DTXHRXV VROYHQWV DW UHODWLYHO\ KLJKHU FRQFHQWUDWLRQV 7DEOH 1HYHUWKHOHVV WKH JHOV IRUPHG IURP &'3

ZHUH IRXQG WR EH UHODWLYHO\ OHVV VWDEOH DW DPELHQW

FRQGLWLRQV FRPSDUHG WR WKH ORQJ VKHOI OLIH RI JHOV IRUPHG IURP &'3 WKH K\GURJHOV RI &'3

DQG &'3

DQG &'3

SUHSDUHG DW WKHLU 0*& DQG DW ZW

6SHFLILFDOO\

UHPDLQHG VHOI

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

DQG &'3

FRPSDUHG WR &'3

ZLWK

7KH %RF GHSURWHFWHG &'3V ZLWK IUHH FDUER[\OLF DFLG IXQFWLRQDOLW\

&'3 $ &'3 $ &'3 $ DQG &'3 $ 6FKHPH 6 6

ZHUH IRXQG WR EH QRQ JHODWRUV LQ

ERWK RUJDQLF DQG DTXHRXV VROYHQWV XQGHU WKH VLPLODU JHODWLRQ FRQGLWLRQV VWXGLHG DW SK\VLRORJLFDO S+ DQG WHPSHUDWXUH ZKLFK LV LQ DJUHHPHQW ZLWK HDUOLHU UHSRUWV IURP WKH OLWHUDWXUH &'3

DQG &'3

7KXV

ZHUH FKRVHQ WR FDUU\ RXW IXUWKHU JHO FKDUDFWHUL]DWLRQ DQG LQ VLWX JHODWLRQ RU

SURSHQVLW\ IRU IRUPLQJ LQMHFWDEOH JHOV LQ SUHVHQFH RI GUXJ PROHFXOHV EHFDXVH RI WKHLU H[FHOOHQW JHODWLRQ SURSHUWLHV &KDUDFWHUL]DWLRQ RI &'3 *HOV

ACS Paragon Plus Environment

Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

)(6(0 $QDO\VLV 7KH VHOI DVVHPEOHG QDQRVWUXFWXUHV RI &'3V WKDW HQFDSVXODWH VROYHQW PROHFXOHV WR IRUP PROHFXODU JHOV ZHUH YLVXDOL]HG E\ )(6(0 )LJXUH VKRZV WKH )(6(0 PLFURJUDSKV RI &'3

DQG &'3

[HURJHOV IURP WROXHQH FKORURIRUP DQG ZDWHU 7KH &'3

[HURJHO LQ WROXHQH VKRZHG WKH SUHVHQFH RI EXQGOHG ILEHU DVVHPEOLHV ZLWK QDQRPHWHU ILEHU GLDPHWHU DQG VHYHUDO PLFURQV LQ OHQJWK )LJXUH $ 7KH PRUSKRORJLFDO DQDO\VLV RI &'3 [HURJHOV LQ FKORURIRUP DQG ZDWHU UHYHDOHG WKH SUHVHQFH RI LQWHUSHQHWUDWHG ' QHWZRUN RI QDQRILEHUV )LJXUH % & 6LPLODUO\ &'3

[HURJHOV IURP WROXHQH FKORURIRUP DQG ZDWHU

VKRZHG WKH SUHVHQFH RI HQWDQJOHG ' QDQRILEHU QHWZRUNV )LJXUH ' ) 7KH SUHVHQFH RI H[WHQGHG QDQRILEHU QHWZRUN LQ [HURJHO VDPSOHV FRQILUPHG WKH QRQFRYDOHQW LQWHUDFWLRQ GULYHQ VHOI DVVHPEO\ RI &'3V LQ RUJDQLF DQG DTXHRXV VROYHQWV 7KHUPDO 6WDELOLW\ RI &'3 *HOV 7KHUPDO VWDELOLW\ RI &'3 RUJDQRJHOV FKORURIRUP DQG K\GURJHOV ZDWHU ZDV HYDOXDWHG E\ PHDVXULQJ WKH FRUUHVSRQGLQJ PHOWLQJ WHPSHUDWXUH 7J RI JHOV SUHSDUHG DW YDULRXV JHODWLRQ FRQFHQWUDWLRQV )LJXUH

$ VWHDG\ ULVH LQ 7J YDOXH ZLWK

LQFUHDVH LQ &'3 FRQFHQWUDWLRQ ZDV REVHUYHG IRU ERWK RUJDQR DQG K\GURJHOV RI &'3

DQG &'3

LQGLFDWLQJ WKH SUHVHQFH RI VWURQJ LQWHUPROHFXODU LQWHUDFWLRQV 7KH REVHUYHG KLJK 7J YDOXHV RI &'3 JHOV DVFHUWDLQHG WKH SUHVHQFH RI VWURQJ DWWUDFWLYH LQWHUDFWLRQV RI JHODWRUV ZLWK VROYHQW PROHFXOHV 9LVFRHODVWLF 3URSHUWLHV RI &'3 *HOV 7KH YLVFR HODVWLF SURSHUWLHV DQG SK\VLFDO VWUHQJWK RI RUJDQR DQG K\GURJHOV RI &'3

DQG &'3

ZHUH HYDOXDWHG E\ PHDVXULQJ WKH VWRUDJH *¶ DQG

ORVV PRGXOXV *´ XVLQJ RVFLOODWRU\ UKHRORJ\ PHDVXUHPHQWV XQGHU DPELHQW FRQGLWLRQV 7KH &'3 K\GURJHOV ZHUH SUHSDUHG LQ ZDWHU DQG WKH RUJDQRJHOV LQ FKORURIRUP DW

ZW

)UHTXHQF\ VZHHS H[SHULPHQWV DW

&'3 JHOV DW URRP

VWUDLQ ZHUH FRQGXFWHG IRU ZW

WHPSHUDWXUH 7KH VWRUDJH PRGXOXV RI &'3

FRQFHQWUDWLRQ

RUJDQR DQG K\GURJHOV ZHUH IRXQG WR EH LQ WKH UDQJH

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

RI

Page 16 of 40

3D DQG ZHUH QHDUO\ RQH RUGHU JUHDWHU WKDQ WKH FRUUHVSRQGLQJ ORVV PRGXOXV )LJXUH $

% 6LPLODUO\ RUJDQR DQG K\GURJHOV RI &'3

H[KLELWHG RQH RUGHU KLJKHU *¶ WKDQ *¶¶ )LJXUH

& ' 2YHUDOO WKHVH YLVFRHODVWLF PHDVXUHPHQWV VKRZHG WKDW *¶ LV JUHDWHU WKDQ *´ ZKLFK FRQILUPHG WKH IRUPDWLRQ RI VWURQJ &'3 RUJDQR DQG K\GURJHOV +\GURJHQ %RQGLQJ ,QWHUDFWLRQV 7KH SUHVHQFH RI VWURQJ LQWHUPROHFXODU K\GURJHQ ERQGLQJ LQWHUDFWLRQV WKH PDMRU GULYLQJ IRUFH IRU WKH VHOI DVVHPEO\ RI &'3V ZDV HYDOXDWHG E\ WKH FRQFHQWUDWLRQ DQG WHPSHUDWXUH GHSHQGHQW + 105 VWXGLHV + 105 VSHFWUD RI &'3 LQ '062 G DW

. VKRZHG D PDUJLQDO GRZQILHOG VKLIW RI DPLGH SURWRQV DV D IXQFWLRQ RI

LQFUHDVHG FRQFHQWUDWLRQ &'3

WR

P0 7KH WHPSHUDWXUH GHSHQGHQW + 105 VSHFWUD RI

P0 UHFRUGHG DW

SURWRQV IURP

UHFRUGHG

SSP WR

. VKRZHG XSILHOG VKLIW RI DPLGH SSP 'G

SSP DQG

WR

SSP 'G

SSP ZLWK

LQFUHDVH LQ WHPSHUDWXUH FRQILUPLQJ WKH SUHVHQFH RI VWURQJ LQWHUPROHFXODU K\GURJHQ ERQGLQJ LQWHUDFWLRQV )LJXUH 6 XSILHOG VKLIWHG WR

6LPLODUO\ DPLGH SURWRQV RI &'3 'G

RI WHPSHUDWXUH IURP

. WR

P0 DW

DQG

SSP

SSP DQG

SSP 'G

SSP UHVSHFWLYHO\ DV D IXQFWLRQ

. )LJXUH 6

7KHUHIRUH FRQFHQWUDWLRQ DQG WHPSHUDWXUH

GHSHQGHQW + 105 VSHFWURVFRS\ DQDO\VLV FRQILUPHG WKH SUHVHQFH RI VWURQJ LQWHUPROHFXODU K\GURJHQ ERQGLQJ LQ &'3

DQG &'3

ZKLFK SOD\ D NH\ UROH LQ WKHLU VHOI DVVHPEO\ WR IRUP

QDQRILEHUV DQG LQ WXUQ WKH JHODWLRQ RI RUJDQLF DQG DTXHRXV VROYHQWV &'3 &\FORGH[WULQ +RVW *XHVW 6WXGLHV 7KH VLJQLILFDQW FRQWULEXWLRQ RI WHUWLDU\ EXW\OR[\FDUERQ\O %RF PRLHW\ LQ GULYLQJ WKH &'3 K\GURJHODWLRQ WKURXJK YDQ GHU :DDOV LQWHUDFWLRQV DORQJVLGH K\GURJHQ ERQGLQJ RI WKURXJK WKH FRUH ODFWDP ULQJ ZDV HYDOXDWHG E\ XVLQJ F\FORGH[WULQ

&' EDVHG KRVW JXHVW FKHPLVWU\ H[SHULPHQWV

ACS Paragon Plus Environment

&' ZDV FKRVHQ KRVW DV LW

Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

SUHIHUHQWLDOO\ HQFDSVXODWHV %RF PRLHW\ RYHU EHQ]\O JURXS DQG

DGDPDQWDQH FDUER[\OLF DFLG

$& ZDV XVHG DV FRPSHWLQJ JXHVW WR UHOHDVH WKH %RF PRLHW\ IURP WKH VKRZHG WKDW &'3

K\GURJHO

ZW

&' FDYLW\

WUDQVIRUP WR VRO XSRQ DGGLWLRQ RI

&'

JHO DQG DWWHPSWV WR FRQYHUW LW EDFN WR JHO LQFOXGLQJ LQFXEDWLRQ RI WKH VRO IRU

2XU VWXG\ PRO

RI

K XQGHU WKH

VWXGLHG H[SHULPHQWDO FRQGLWLRQV ZHUH XQVXFFHVVIXO )LJXUH $ % +RZHYHU WKH K\GURJHO ZDV HIIHFWLYHO\ UHVWRUHG E\ WKH DGGLWLRQ RI FRPSHWLQJ JXHVW $& ZLWK VWURQJ DIILQLW\ WRZDUGV WR

PRO

&' UHOHDVHV LW IURP ERXQG &'3

RI JHO ,Q WKLV FDVH $& E\ GLUHFWO\ FRPSHWLQJ ZLWK

%RF PRLHW\ HQFDSVXODWHG LQ WKH KRVW FDYLW\ DQG IDFLOLWDWHV WKH VHOI DVVHPEO\ RI &'3V UHVWRULQJ WKH JHODWLRQ RI WKH VROYHQW )LJXUH & 7KH VWXG\ FRQILUPHG WKH KRVW JXHVW FRPSOH[DWLRQ RI &' DQG &'3

WKURXJK %RF PRLHW\ HQFDSVXODWLRQ LQ

JXHVW FRPSOH[DWLRQ E &' DQG &'3

&' KRVW FDYLW\ )LJXUH ' ) 7KH KRVW

WKURXJK %RF PRLHW\ ZDV IXUWKHU DVFHUWDLQHG E\ WKH +

105 VWXG\ 7KH + 105 VSHFWUXP RI &'3

E &'

Y Y VKRZHG D EURDGHQLQJ RI E &' SURWRQV LQ WKH UHJLRQ RI XSILHOG VKLIW )LJXUH 6

7KH XSILHOG VKLIW RI

EURDGHQLQJ HVWDEOLVKHG WKH E &' &'3

P0 HDFK LQ '062 G ' 2 SSP WR

&' SURWRQV DFFRPSDQLHG E\ WKH VLJQDO

KRVW JXHVW FRPSOH[DWLRQ WKURXJK %RF PRLHW\

$GGLWLRQDOO\ KRVW JXHVW LQGXFHG UHYHUVLEOH K\GURJHODWLRQ RI &'3 DOWHUQDWLYH DGGLWLRQ RI

SSP ZLWK PDUJLQDO

ZDV SHUIRUPHG E\ WKH

&' DQG $& 5HYHUVLEOH FRQYHUVLRQ RI K\GURJHOV WR VROXWLRQ ZDV

REVHUYHG ZLWK WKH DOWHUQDWH DGGLWLRQ RI

&' DQG $& WR WKH &'3 K\GURJHOV ZKLFK IXUWKHU

DXJPHQWV WKH VLJQLILFDQFH RI W%RF JURXS LQ GULYLQJ WKH JHODWLRQ RI &'3V 7KH UHYHUVLEOH JHO VRO VWXGLHV ZHUH UHVWULFWHG WR WKUHH F\FOHV EHFDXVH RI WKH REVHUYHG WXUELGLW\ RI &'3 VROXWLRQV GXH WR UHSHDWHG DGGLWLRQ RI

&' DQG $& $OWRJHWKHU WKH &'3

E &' KRVW JXHVW VWXG\ FRQILUPHG WKH

FRQWULEXWLRQ RI %RF PRLHW\ LQ GULYLQJ WKH PROHFXODU JHODWLRQ RI &'3V &'3

ACS Paragon Plus Environment

DQG &'3

7KLV

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 40

VWXG\ IXUWKHU H[SODLQV WKH REVHUYHG LQDELOLW\ RI &'3V ZLWK IUHH FDUER[\OLF DFLG JURXSV &'3 $ DQG &'3 $ WR IRUP PROHFXODU JHOV ,Q VLWX +\GURJHODWLRQ 6WXGLHV 7KH DELOLW\ WR IRUP LQ VLWX JHOV ZDV HYDOXDWHG E\ VWXG\LQJ WKH JHODWLRQ RI &'3V XSRQ LQMHFWLQJ WKHLU 1 PHWK\O P0 S+

ž&

S\UUROLGRQH 103 VROXWLRQV LQWR 3%6

103 ZDV FKRVHQ DV WKH SUHIHUUHG VROYHQW WR GLVVROYH WKH &'3V DQG IRU LQ

VLWX JHODWLRQ VWXGLHV EHFDXVH RI LWV SURYHQ ELRFRPSDWLELOLW\ )'$ DSSURYHG DQG HVWDEOLVKHG XVH LQ VXEFXWDQHRXV LQMHFWLRQ IRUPXODWLRQV (/,*$5'Œ

&RQFHQWUDWLRQ RI &'3V LQ 103 ZDV

RSWLPL]HG WR DFKLHYH LQ VLWX JHODWLRQ XSRQ LQMHFWLRQ LQWR D YLDO FRQWDLQLQJ 3%6 DW

ž& &'3

IDLOHG WR IRUP LQ VLWX JHODWLRQ DW YHU\ KLJK FRQFHQWUDWLRQV VWXGLHG 2Q WKH RWKHU KDQG &'3 ZW

LQ 103 LQVWDQWDQHRXVO\ IRUPHG D JHO XSRQ LQMHFWLRQ LQWR 3%6 VROXWLRQ XVLQJ D

FRPPHUFLDOO\ DYDLODEOH V\ULQJH )LJXUH $ ' DQG WKH K\GURJHO IRUPHG LQ VROXWLRQ ZDV IRXQG WR EH VWDEOH XQGHU DPELHQW FRQGLWLRQV 7KH LQDELOLW\ RI &'3

WR IRUP K\GURJHOV LQ VLWX LV DWWULEXWHG

WR WKH GLIIHUHQWLDO VHOI DVVHPEO\ SDWWHUQV RI SHSWLGHV FRQWDLQLQJ SKHQ\ODODQLQH DQG W\URVLQH EHFDXVH RI WKH YDULDWLRQ LQ HOHFWURQ GHQVLW\ RQ WKH VLGH FKDLQ DURPDWLF JURXS + RQ 3KH 2+ RQ 7\U

7KH SRVVLEOH DSSOLFDWLRQ RI &'3

IRU WKH SDUHQWHUDO DGPLQLVWUDWLRQ RI ELRORJLFDOO\

LPSRUWDQW PROHFXOHV ZDV GHPRQVWUDWHG E\ ORDGLQJ K\GURSKRELF FXUFXPLQ DQG WKH SURSHQVLW\ IRU LQ VLWX JHODWLRQ ZDV VWXGLHG $V VKRZQ LQ )LJXUH ( + FXUFXPLQ

ZW

RI &'3 ORDGHG &'3

LQVWDQWDQHRXVO\ IRUPHG LQ VLWX JHOV LQ 3%6 VROXWLRQ XSRQ LQMHFWLRQ 7KH DELOLW\ RI &'3 LQ VLWX JHOV DORQH DQG ZLWK ORDGHG FDUJR )LJXUH

WR IRUP

KLJKOLJKWHG LWV SRWHQWLDO XVH IRU SDUHQWHUDO

DGPLQLVWUDWLRQ RI GUXJV DQG ELRPROHFXOHV DQG DOVR SRVVLEO\ PDQ\ RWKHU ELRPHGLFDO DSSOLFDWLRQV &\WRWR[LFLW\ 6WXG\ 7KH F\WRFRPSDWLELOLW\ RI DPELGH[WURXV VXSHUJHODWRU DQG LQ VLWX JHO IRUPLQJ &'3

ZDV HYDOXDWHG E\ PLWRFKRQGULDO DFWLYLW\

GLPHWK\OWKLD]RO

\O

GLSKHQ\OWHWUD]ROLXP EURPLGH 077 DVVD\ LQ PXULQH DGUHQDO PHGXOOD GHULYHG 3&

ACS Paragon Plus Environment

Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

SKHRFKURPRF\WRPD FHOOV 7KH PHWDEROLF DFWLYLW\ RI 3& FRQFHQWUDWLRQV RI &'3 FRQFHQWUDWLRQ RI &'3 ZLWK &'3

VKRZHG !

FHOOV LQ SUHVHQFH RI GLIIHUHQW

ZDV DVVHVVHG 7KH F\WRWR[LFLW\ GDWD RI 3& DIWHU

K LQ FXOWXUH LV VKRZQ LQ )LJXUH

FHOOV WUHDWHG ZLWK GLIIHUHQW

7KH YLDELOLW\ RI FHOOV WUHDWHG

YLDELOLW\ ZKLFK LV FRPSDUDEOH WR WKH XQWUHDWHG FRQWURO FHOOV DQG KHQFH

FRQILUPHG LWV F\WRFRPSDWLEOH QDWXUH 7KXV F\WRWR[LFLW\ VWXG\ FOHDUO\ GHPRQVWUDWHG WKDW &'3

LV

QRQ WR[LF DQG D SRWHQWLDO FDQGLGDWH IRU DSSOLFDWLRQV LQYROYLQJ OLYH FHOOV 6WUXFWXUH DQG (QHUJHWLFV RI 6HOI DVVHPEO\ IURP 0RGHOLQJ 2SWLPL]HG GLPHU VWUXFWXUHV DV REWDLQHG IURP 0

; OHYHO RI WKHRU\ IRU &'3V

DUH VKRZQ LQ )LJXUH $ ' 7KH GLPHULF

VWUXFWXUHV RI &'3V REWDLQHG IURP % /