0
Po ada TFG_2023_24.pd
TFG
[Sub í ulo del documen o]
[FECHA]
[NOMBRE DE LA COMPAÑÍA]
[Di ección de la compañía]
Ximena Sánchez Iglesias
Gold-p omo ed cascade
eac ions in biological
media
José Luis Masca eñas Cid, i o e docen e do Depa amen o de Química O gánica, e Ma ía Tomás
Gamasa, co i o a e docen e do Depa amen o de Química O gánica , au o izan a p esen ación do
T aballo de Fin de G ao da alumna Ximena Sánchez Iglesias na con oca o ia de xullo do cu so
2023-2024, o cal oi ealizado baixo a súa di ección no Cen o Singula de In es igación en
Química Biolóxica e Ma e iales Molecula es da Uni e sidade de San iago de Compos ela
(CiQUS).
E pa a que así cons e asinamos o p esen e in o me en San iago de Compos ela o 30 de xuño de
2024.
TOMAS
GAMASA
MARIA -
09417809E
Fi mado
digi almen e po
TOMAS GAMASA
MARIA -
09417809E
Fecha: 2024.06.30
00:35:40 +02'00'
MASCAREÑAS
CID JOSE LUIS
- 34934771W
Fi mado digi almen e
po MASCAREÑAS CID
JOSE LUIS - 34934771W
Fecha: 2024.06.30
08:09:42 +02'00'
INDEX
ABBREVIATIONS .................................................................................................................................... 1
ABSTRACT ............................................................................................................................................... 2
1. INTRODUCTION ............................................................................................................................. 4
1.1 Bioo hogonal chemis y ......................................................................................................... 4
1.2 O ganome allic ca alysis in biological media ........................................................................ 6
1.2.1 Ru henium; oo s o bioo hogonal o ganome allic chemis y ................................................... 7
1.2.2 Palladium ................................................................................................................................... 8
1.3 Gold eac i i y in biological en i onmen s ............................................................................ 8
1.3.1 His o ical e iew.................................................................................................................... 8
1.3.2 Cycloisome iza ion o alkynes and cascade eac ions .......................................................... 11
1.3.3 Syn hesis and ele ance o ca bazoles ................................................................................. 12
2. OBJECTIVES AND WORK PLAN .............................................................................................. 14
3. RESULTS AND DISCUSSION ...................................................................................................... 15
3.1 P elimina y assays o a gold-cascade syn hesis o ca bazoles .......................................... 15
3.2 In i o op imiza ion ............................................................................................................... 16
3.2.1 Sc eening o o he eac ions condi ions ............................................................................... 16
3.2.2 Reac ion in bio ele an media ............................................................................................. 21
3.2.3 Compa ibili y wi h speci ic biomolecules ............................................................................ 22
3.3 Fluo escence s udies ............................................................................................................... 24
3.3.1 UV s udy............................................................................................................................... 24
3.3.2 Fluo escence s udy .............................................................................................................. 25
3.4 In cellulo expe imen s ............................................................................................................ 26
3.5 Expe imen al p ocedu es ...................................................................................................... 28
3.5.1 Gene al in o ma ion o in i o expe imen s ...................................................................... 28
3.5.2 Syn hesis o 1-iodo-2-(phenyle hynyl)benzene ..................................................................... 29
3.5.3 Syn hesis o 2-{[2-(phenyle hynyl)phenyl]e hynyl}aniline 1 ............................................... 29
3.5.4 Syn hesis o 6-phenyl-11H-benzo[a]ca bazole 2 ................................................................. 30
3.5.5 Rep esen a i e p ocedu e o eac ions a 200 mM scale using iP AuNT 2 as complex and
MeCN as sol en (sec ion 3.2.1) ........................................................................................................ 31
3.5.6 Rep esen a i e p ocedu e o he s udy o he dilu ion (sec ion 3.2.1)* .............................. 32
3.5.7 Rep esen a i e p ocedu e o eac ions a 1 mM scale using s ock solu ions in DMSO*
(sec ion 3.2.1) .................................................................................................................................... 32
3.5.8 In e nal s anda d me hod o yield quan i ica ion ............................................................... 33
3.5.9 Gene al in o ma ion o in cellulo expe imen s .................................................................. 34
3.5.10 Rep esen a i e p ocedu e o in cellulo eac ions ............................................................... 34
CONCLUSIONS ...................................................................................................................................... 35
REFERENCES ........................................................................................................................................ 38
ANNEX: CHARACTERIZATIONS...................................................................................................... 43
1
ABBREVIATIONS
allyl
p op-2-en-1-yl
NADH
nico inamide adenine dinucleo ide
( educed)
BSA
bo in se um albumin
NMR
nuclea magne ic esonance
13C-NMR
ca bon NMR
NTF2
bis( i luo ome hylsul onyl)amide
CuAAC
coppe -ca alyzed azide-alkyne
cycloaddi ion
OSCs
o ganic sola cells
d
double
OT
i luo ome hanesul ona e
dba
dibenzylideneace one
DEPT-135
dis o ionless enhancemen by
pola iza ion ans e
PBS
phospha e-bu e ed saline
δ
chemical shi
PhSH
phenyl hiol
DMEM
Dulbecco’s Modi ied Eagle
Medium
ppm
pa s pe million
DMSO
dime hyl sul oxide
PTA
1,3,5- iaza-7-phospaadaman ane
e.g.
exempli g a ia
. .
oom empe a u e
eq.
equi alen s
R
e en ion ac o
E 3N
ie hylamine
ROS
eac i e oxygen species
E OAc
e hylace a e
s
single
GSH
L-glu a hione
SM
s a ing ma e ial
1H-NMR
p o on NMR
SPAAC
s ain-p omo ed azide-alkyne
cycloaddi ion
HPLC-MS
high pe o mance liquid
ch oma og aphy – mass
spec ome y
TMB
1,3,5- ime hoxybenzene
HSA
human se um albumin
THF
e ahyd o u an
IED-DA
in e se elec on-demand
Diels-Alde eac ion
TLC
hin laye ch oma og aphy
J
coupling cons an
iple
JohnPhos
2-(di- e -
bu ylphosphino)biphenyl
e en ion ime
λ
wa eleng h
UV
ul a iole
m
mul iple
ol
olume
MeCN
ace oni ile
w/o
wi hou
2
ABSTRACT
In ecen yea s, he possibili y o in oducing abio ic eac ions inside li ing sys ems in a
biocompa ible way has a oused g ea in e es , hanks o he mul iple applica ions in chemical
biology and biomedicine ha could lead o he de elopmen o new he apies and ea men s.
Wi hin hese eac ions, he use o ansi ion me als as ca aly ic s uc u es can open he doo o new
eac i i ies o be ca ied ou wi hin biological sys ems. Among hem, gold, a p ecious me al long
conside ed highly ine , has begun o gain impo ance in he ield o Bioo hogonal Chemis y.
In his Final Deg ee P ojec , i is p oposed he de elopmen o a gold-p omo ed double
cycliza ion h ough a cascade mechanism wi hin biological media. Thus, he s udy in ol es he
op imiza ion o he ans o ma ion owa ds biological condi ions, ha is, high dilu ion, wa e and
ai compa ibili y, as well as he ole ance o he p esence o di e en biomolecules and complex
media. We ha e ob ained p omising esul s sugges ing he iabili y o hese cascade p ocesses in
biological en i onmen s and e en in li e mammalian cells (A549). This s a egy cons i u es an
impo an new app oach o build up high complexi y molecules om simple compounds in a
s aigh o wa d, elegan and bioo hogonal manne in li ing cells.
RESUMEN
En los úl imos años, la posibilidad de in oduci eacciones abió icas den o de sis emas i os
de mane a biocompa ible ha despe ado un g an in e és, g acias a las múl iples aplicaciones en
química biológica y biomedicina que pod ían conduci al desa ollo de nue as e apias y
a amien os. Den o de es as eacciones, el uso de me ales de ansición como es uc u as
ca alí icas pe mi e ab i la pue a a nue as eac i idades que lle a a cabo den o de sis emas
biológicos. En e ellos, el o o, un me al p ecioso que du an e mucho iempo ha sido conside ado
ine e, ha comenzado a gana ele ancia en el campo de la Química Bioo ogonal.
En es e T abajo de Fin de G ado se p opone el es udio de una eacción de doble ciclación
p omo ida po o o a a és de un mecanismo en cascada en el in e io de medios biológicos. Así,
se ha op imizado la ans o mación pa a alcanza condiciones biológicas; es o es, al as diluciones
y compa ibilidad con el ai e y el agua, así como ole ancia a la p esencia de di e en es
biomoléculas y medios complejos. Los esul ados ob enidos son p ome edo es, sugi iendo la
iabilidad de es os p ocesos en cascada, no solo en medios biológicos, sino ambién en el in e io
de células de mamí e o (A549). Es a es a egia cons i uye un impo an e nue o en oque pa a la
cons ucción de moléculas de al a complejidad a pa i de compues os sencillos de una mane a
simple, elegan e y bioo oogonal den o de células i as.

3
RESUMO
Nos úl imos anos, a posibilidade de in oduci eaccións abió icas den o de sis emas i os de
manei a biocompa ible despe ou un g an in e ese, g azas ás múl iples aplicacións en química
biolóxica e biomedicina que pode ían conduci ao desen ol emen o de no as e apias e
a amen os. Den o des as eaccións, o uso de me ais de ansición como es u u as ca alí icas
pe mi e desen ol e no as eac i idades den o de sis emas biolóxicos. En e eles, o ou o, un
me al p ecioso que du an e moi o empo oi conside ado ine e, comezou a gaña ele ancia no
campo da Química Bioo ogonal.
Nes e T aballo de Fin de G ao p oponse o es udo dunha eacción de dob e ciclación p omo ida
po ou o a a és dun mecanismo en cascada no in e io de medios biolóxicos. Así, op imizouse a
ans o mación pa a alcanza condicións biolóxicas; is o é, al as dilucións e compa ibilidade co
ai e e auga, así como ole ancia á p esencia de di e en es biomoléculas e medios complexos. Os
esul ado ob idos son p ome edo es, suxe indo a iabilidade des es p ocesos en cascada, non solo
en medios biolóxicos, senón amén no in e io de células de mamí e o (A549). Es a es a exia
cons i úe un impo an e no o en oque pa a á cons ución de moléculas de al a complexidade a
pa i de compos os sinxelos dunha manei a simple, elegan e e bioo oogonal den o de células
i as.
4
1. INTRODUCTION
Na u e has his o ically se ed as a sou ce o inspi a ion o science. The complex ne wo k o
chemical ans o ma ions ha makes li e possible cons i u es he basis o Chemical Biology,
which applies chemical knowledge o he explo a ion o li ing sys ems, he de elopmen o
echniques o he syn hesis and modi ica ion o biomolecules, as well as he design o new
chemical p ocesses based on biology.
1.1 Bioo hogonal chemis y
Bioo hogonal chemis y, de ined as eac ions ha do no dis up he in insic biochemical
p ocesses ha ake place inside li ing sys ems,1 was i s desc ibed by Ca olyn R. Be ozzi in
2003. These chemical eac ions, cha ac e ized by a good biocompa ibili y and high selec i i y,
mus ole a e physiological condi ions o pH and empe a u e.2 Ne e heless, de eloping new
bioo hogonal ans o ma ions in complex li ing sys ems is eally challenging, due o he
ne wo ks o in e connec ed me aboli es, ions and biopolyme s esponsible o he a ay o cellula
p ocesses ha happen wi hin cells, ha c ea e an in ica e media o conduc non-na u al eac ions.3
The ise o bioo hogonal ans o ma ions in ecen yea s has been possible due o hei impac
in mul iple ields. I can be implemen ed in medicinal chemis y (e.g. biomedical imaging o
bio he apeu ics), ma e ials science (e.g. polyme syn hesis o ene gy s o age ma e ials), basic
esea ch (e.g. p o ein modi ica ion and syn hesis) and bio echnology.4,5 This opens he possibili y
o e ol ing e icien me hods o pe o m gene ic code expansion, d ug labeling iden i ica ion and
deli e y, me abolic enginee ing and di e en ypes o bioconjuga ion.5 I was his signi ican
impac wha ea ned Be ozzi ( . Meldal and Sha pless) he Nobel P ize in 2022, “ o he
de elopmen o click chemis y and bioo hogonal chemis y”.
Howe e , compa ed o adi ional o ganic syn hesis, whe e concen a ion o eac an s,
sol en s and empe a u e can be modula ed, ai and mois u e can be elimina ed and ca alys can
be added, i necessa y, bioo hogonal eac ions ace an uncon ollable en i onmen .4
Thus, o success ully ca y ou a chemical eac ion unde physiological condi ions, some
equi emen s should be ollowed:
o Chemoselec i i y. Reac ions mus be selec i e o a ce ain unc ional g oup and ine
o he as compendium o chemical unc ionali ies ound in i o,1 a oiding compe ing
side eac ions engaging endogenous biological eac an s.4
o Biocompa ibili y. Li ing en i onmen s imply aqueous media and an ele a ed con ol
o pH and empe a u e, o a oid pe o ming oxic eac ions ha could in e e e in he
cu en unc ion o he medium.5
5
o Reac ion a e. I is impo an o op imize he kine ics o he eac ions wi h he objec i e
o educing he concen a ions used in i o, conside ing ha a g ea pa o hese
eac ions ollow second-o de a es, dependen on concen a ion o he wo eac an s.1
Analyzing hese cha ac e is ics, such as selec i i y and unc ional ole ance, i is no ed ha
bioo hogonal chemis y is, al hough concep ually di e en , qui e close o click chemis y in i s
a ibu es, being ai ly common o a bioo hogonal eac ion o mee he equi emen s o he la e .4
S udying i s his o y, he beginnings o bioo hogonal chemis y can be linked o p o ein
bioconjuga ion in he pu sue o a selec i e me hod o moni o ing biochemical p ocesses wi hin
cells.1 In his esea ch, he i s app oaches o bioo hogonal eac ions we e condensa ions
be ween aldehydes/ke ones and hyd azides, hyd azine o aminooxy compounds, whe e selec i i y
issues we e no able. Thus, he i s success in he sea ch o bioo hogonali y was achie ed wi h
azides, which demons a ed o be ine subs a es unde biological en i onmen s. They debu ed in
he S audinge liga ion,6 a ans o ma ion o azides in o amides by eac ion wi h a ylphosphine
de i a i es (Scheme 1).4
Scheme 1. S audinge liga ion.
F om hen on, an impo an se o bioo hogonal s a egies whe e de eloped, classi ied in wo
amilies o p ocesses: bioo hogonal bond- o ming eac ions and bioo hogonal bond-clea age
eac ions.
Scheme 2. Rep esen a i e: (a) bioo hogonal bond- o ming eac ion, (b) bioo hogonal bond-clea age eac ion.
Wi hin he i s ype o ans o ma ions, g aphically ep esen ed in Scheme 2a, i is wo h
highligh ing he well-known coppe -ca alyzed alkyne azide cycloaddi ion (CuAAC),7,8 epo ed
by Sha pless and Meldal (Scheme 3).
6
Scheme 3. Coppe -ca alyzed alkyne azide cycloaddi ion.
Essen ially, he eac ion consis s o a [3+2] addi ion be ween e minal alkynes and azides, o
egioselec i ely a o d 1,4-disubs i u ed-1,2,3- iazoles. I shows high selec i i y, g ea kine ics
and bioo hogonali y, hough i mus be said ha i s use, mos ly ela ed o he modi ica ion o cell
su ace glycans and p o eins,9 is limi ed by he oxici y o coppe and asco ba e (used o ob ain
CuI om a CuII sou ce), able o gene a e ROS ( eac i e oxygen species).10 Howe e , many
al e na i es ha e been desc ibed o a oid hese issues, bo h in he modi ica ion o azides11 and he
ligands o p o ec he coppe complex.12,13
In he ollowing yea s, mo e bio iendly al e na i es o he CuAAC eac ion ha e been
de eloped, such as he me al ee cycloaddi ion wi h s ained cycloalkynes (SPAAC),14 o he
in e se elec on-demand Diels-Alde 15 (IED-DA).2,4 The expansion o his ield has also allowed
o de elop new p ocedu es, such as palladium-ca alyzed c oss-couplings and u henium-ca alyzed
c oss-me a hesis eac ions, among o he examples.2
The second g oup o bioo hogonal ans o ma ions in ol es bond-b eaking eac ions (Scheme
2b). Typical syn he ic me hods o clea e chemical bonds use ha sh condi ions based on acidic-
basic, nucleophilic o edox eagen s no always compa ible wi h physiological condi ions. An
al e na i e app oach akes ad an age o he g ea eac i i y o he o ganome allic chemis y.
Towa ds his idea, many bond b eaking bioo hogonal eac ions p omo ed by ansi ion me al
ca alys s ha e al eady been desc ibed and inco po a ed o he a mo y o bioo hogonal chemis y.2
Along he yea s, he ield o bio hogonal o ganome allic chemis y has acqui ed a syn he ic
aim wi h nume ous examples o in acellula bond- o ming p ocesses in li e cells.
1.2 O ganome allic ca alysis in biological media
Me abolism is sus ained by my iads o enzymes ha wo k o conduc p ope ly each eac ion
ha akes place wi hin li ing sys ems. Many o hese bioca alys s ha e me al ions embedded in
hei ac i e si es, aking pa as edox cen e s o Lewis acids, such as Fe, Mn, Zn o Mg.16 I is
in e es ing o no ice ha na u al me alloenzymes do no use second o hi d ow ansi ion me al
ca alys s, being limi ed hei eac i i y mainly o edox p ocesses o acid-base ans o ma ions.
Whe eas he use o hese me alloenzymes in syn he ic chemis y oge he wi h he de elopmen
o a i icial ones has ecei ed a lo o a en ion, he explo a ion o a i icial ansi ion me al
ca alysis inside li e cells ha e been unconside ed o a long ime.
13
Scheme 12. Mechanism p oposed by Hi ano e al. o cascade cycliza ion wi h ca bazole o ma ion.
I can be obse ed ha his p ocedu e is a o ed by he o ho subs i u ion o he a ylalkyne
and he nucleophilic g oup, o cing p oximi y o bo h eac an s.47
The mechanism is suppo ed by checking he eac ion o a plausible in e media e (simila o
in e media e III lacking he gold a om), which when ea ed unde he s anda d eac ion condi ions
a o ds he same ca bazole.57

14
2. OBJECTIVES AND WORK PLAN
The p esen wo k, in he amewo k o Final Deg ee P ojec , aims o b oaden he scope o
in acellula bioo hogonal eac ions u ilizing gold ca alys s. We p oposed he de elopmen o
new biocompa ible gold-p omo ed cascade eac ions in biological o e en cellula en i onmen s.
Speci ically, we se ou hese goals:
(a) Syn hesis o bioac i e skele ons such as ca bazoles, by he in amolecula gold-p omo ed
double cycliza ion o designed subs a es h ough a cascade p ocess.
(b) Op imize he eac ion owa ds biocompa ible condi ions.
(c) Con i m he ole ance o he eac ion o di e en biological media and biomolecules.
(d) T ansla e he eac ion in o li e mammalian cells.
In he consecu ion o hese objec i es, he ollowing plan was p oposed:
MONTH
TASK
No embe
Bibliog aphical esea ch and amilia iza ion wi h he lab
Decembe
Syn hesis and cha ac e iza ion o eac an 1
Janua y
Syn hesis and cha ac e iza ion o p oduc 2
Feb ua y
Sc eening o gold ca alys s
Ma ch
Op imiza ion o in i o condi ions
Ap il
Re inemen o he inal condi ions
May
Biological media sc eening and biocompa ibili y s udy
wi h biomolecules
Memo y w i ing
June
Expe imen s in li e mammalian cells
Memo y w i ing
15
3. RESULTS AND DISCUSSION
In oducing a new chemical ans o ma ion in o a li ing sys em equi es o ollow ce ain s eps
o ensu e he iabili y o he p ocess, ha include he s udy o he eac ion and he op imiza ion
o he condi ions o gua an ee i s compa ibili y o aqueous and complex biological media. Finally,
he iabili y o he eac ion in li ing cells can be explo ed.
The p esen wo k was de eloped unde he di ec supe ision o las yea PhD s uden Cinzia
D’A ino.
3.1 P elimina y assays o a gold-cascade syn hesis o ca bazoles
Aiming o s udy new gold-p omo ed bioo hogonal cascade eac ions, we selec ed he one
desc ibed in o ganic sol en s by Hi ano e al (Scheme 11).48
The subs a e 1 was p epa ed h ough a wo-s ep syn hesis, s a ing om comme cial eagen s.
A Sonogashi a coupling be ween o-diiodobenzene and phenylace ylene using
bis( iphenylphospine)palladium(II) and coppe (I) iodide, in bu ylamine, p o ided 1-iodo-2-
(phenyle hynyl)benzene in 39% yield. This compound was subjec ed o a second Sonogashi a
coupling wi h 2-e hynylaniline, his ime in THF and E 3N, ob aining 2-((2-
phenyle hynyl)phenyl)e hynyl)aniline (1) in 75% yield. The o e all yield o his syn hesis was
29%.
Scheme 13. Syn he ic ou e o 2-((2-phenyle hynyl)phenyl)e hynyl)aniline 1.
Once ob ained compound 1, he gold-p omo ed cycliza ion was a emp ed ollowing Hi ano’s
o ganic eac ion condi ions. Thus, his aniline de i a i e was ea ed wi h
chlo o( iphenylphosphine)gold(I), using AgOT as chlo ide sca enge in MeCN and s i ed o
4.5 hou s. The p oduc 2 was o med in 50% yield, al hough in a lowe e iciency o he epo ed
by Hi ano (81%, in only 1.5 hou s).
16
Scheme 14. Syn hesis o 6-phenyl-11H-benzo[a]ca bazole 2 unde Hi ano’s condi ions.
To ansla e o ganic eac ions o biological media i is manda o y o pe o m he
ans o ma ions a physiological empe a u e, ha is, 37 ºC. Thus, we explo ed he eac ion a a
lowe empe a u e. When he eac ion was ca ied ou a oom empe a u e (25 ºC) and ime was
ise o 7 hou s, a 69% yield was ob ained, simila o he 70% epo ed in bibliog aphy.
In his case, he yield o 2 was de e mined by 1H-NMR using 1,3,5- ime hoxybenzene (TMB)
as in e nal s anda d.
Impo an ly, i was also con i med ha he eac ion is un iable wi hou he ca alys .
Scheme 15. Cycliza ion pe o med a . . wi h and wi hou ca alys . [Au] = Ph3PAuCl.
3.2 In i o op imiza ion
3.2.1 Sc eening o o he eac ions condi ions
The i s s udy unde aken was he selec ion o he gold ca alys using di e en gold
complexes.
17
0%
96%
0%
99%
AuCl
AuCl3
85%
75%
23%
55%
HAuCl4·3H2O
w/o
66%
0%
Scheme 16. Ca alys sc eening o he syn hesis o ca bazole 2.a
aCondi ions: 200 mM o 1 (0.12 mmol), 20 mol% Au ca alys , in MeCN (0.59 mL), o e nigh , unde N2 a mosphe e
a oom empe a u e. bYields we e de e mined by 1H-NMR using TMB as in e nal s anda d.
Impo an in o ma ion can be ex ac ed om his s udy. Fi s , he eac ion wi h PPh3AuCl
wi hou sil e sal led o he eco e y o compound 1, con i ming he need o he chlo ide
sca enge . This was also obse ed wi h iP AuNT 2 and iP AuCl (96% and 0%, espec i ely).
Mo eo e , gold(I) complexes showed a be e pe o mance compa ed o he gold(III) sal s.
In his sc eening, wo ca alys s, JohnPhos and iP AuNT 2, show excellen yields, abo e 90%.
Since he i s complex p esen s low cell-up ake and high cy o oxici y (as p e iously ound by he
g oup), he s udies con inued wi h iP AuNT 2.
Using his complex, i was ound ha high yields can be ob ained only a e 10 min o eac ion
(Table 1, en y 3).
18
Table 1. Time sc eening o he syn hesis o ca bazole 2.a
En y
Time
Yield (%)b
1
7 hou s
97
2
1 hou
98
3
10 min
99
aCondi ions: 200 mM o 1 (0.12 mmol), 20 mol% iP AuT 2, in MeCN (0.59 mL), unde N2 a mosphe e a oom
empe a u e. bYields we e de e mined by 1H-NMR using TMB as in e nal s anda d.
In he nex se o expe imen s, he pe o mance o he eac ion in di e en sol en s oge he
wi h he wa e compa ibili y was s udied. The esul s, shown in Table 2, demons a ed ha an
o ganic media was no necessa y, ob aining compa able yields when an aqueous milieu was used
(en y 3). In his case, he eac ions we e s i ed o 1 h o ensu e he o al consump ion o subs a e
unde he new eac ion media. Howe e , i should be poin ed ou he insolubili y o he subs a e
and p oduc in H2O, using he e m on wa e o e e o he he e ogeneous cha ac e o he eac ion.
Table 2. Sol en sc eening o he syn hesis o ca bazole 2.a
En y
Sol en
Yield (%)b
1
MeCN
98
2
DMSO
100
3
H2O
93
aCondi ions: 200 mM 1 (0.12 mmol), 20 mol% iP AuT 2, in di e en sol en s (0.59 mL), one hou and unde N2
a mosphe e a . . bYields we e de e mined by 1H-NMR using TMB as in e nal s anda d.
Once he wa e compa ibili y was con i med, he eac ion was es ed in an a mosphe e open
o ai , essen ial o he de elopmen o u u e in cellulo expe imen s. Simul aneously, ime was
o e looked again, due o he la e changes. The esul s a e summa ized in Table 3.

19
Table 3. Time and a mosphe e sc eening o he syn hesis o ca bazole 2.a
En y
Time
Yield (%)b
1
10 min
96
2
30 min
94
3
1 hou
89
4
24 hou s
89
aCondi ions: 200 mM 1 (0.12 mmol), 20 mol% iP AuT 2, in H2O (0.59 mL) unde open a mosphe e, . . bYields we e
de e mined by 1H-NMR using TMB as in e nal s anda d.
These esul s again e ealed he as kine ics o he eac ion, as well as he ole ance o oxygen
and humidi y. Consequen ly, om now on, all he expe imen s a e pe o med open o ai , unless
o he wise no ed.
The nex ques ion aised is whe he he ca alys loading and s a ing ma e ial concen a ion
could be educed, since in cellulo expe imen s a e highly sensi i e o ele a ed amoun s o
eagen s, p oducing s ess, beha io al e a ion o e en cy o oxici y.
Fi s ly, he eac ion pe o med unde op imized condi ions, ha is, a 200 mM scale o
subs a e, in wa e , . ., 10 min, bu dec easing he gold loading o 10 mol%, led o 91% yield
(Table 4, en y 1). Subsequen ly, he in luence o he concen a ion o subs a e was s udied. As
shown in he able, he mola i y could be educed up o 40 imes, o 5 mM o s a ing ma e ial,
keeping he yields abo e 80% (en ies 3, 5, 7 and 9). The eac ion also ook place a 1 mM, wi h
an accep able 65% (en y 11). Impo an ly, no p oduc was de ec ed in he absence o he gold
complex (en ies 2, 4, 6, 8 and 10).
Table 4. Sc eening o ca alys loading and concen a ion o 1 o he syn hesis o ca bazole 2.a
20
En y
[1] (mM)
mmol SM
Vol (mL)
Ca alys
Yield (%)b
1
200
0.12
0.59
Yes
91
2
200
0.12
0.59
-
0
3
100
0.034
0.34
Yes
88
4
100
0.034
0.34
-
0
5
50
0.034
0.68
Yes
83
6
50
0.034
0.68
-
0
7
10
0.034
3.4
Yes
87
8
10
0.034
3.4
-
0
9
5
0.034
6.8
Yes
87
10
5
0.034
6.8
-
0
11
1
0.034
34.1
Yes
65
12
1
0.034
34.1
-
0
aCondi ions: Di e en concen a ion o 1 (0.12 mmol a 200 mM and 0.034 mmol in he ollowing), 10 mol% iP AuT 2,
in H2O (0.59 mL in 200 mM o 34.1 mL in 1 mM) unde open a mosphe e, . . bYields we e de e mined by 1H-NMR
using TMB as in e nal s anda d.
Du ing his expe imen a ion, i became e iden he necessi y o educing he eac ion olume
o be able o inally ans e he eac ion in o li ing sys ems. Fo his, he ope a ional p o ocol was
changed, equi ing he p epa a ion o s ock solu ions om bo h he subs a e and he ca alys . To
achie e i conside ing he lack o solubili y o he eac an s in wa e , i was p oposed he addi ion
o DMSO. Thus, s ock solu ions o compound 1 and he gold ca alys in DMSO we e p epa ed,
and hen added o he wa e used as eac ion media, in a Schlenk ube, in a p opo ion 9 o 1
(H2O/DMSO).
Again, a ime sc eening was de eloped in he aim o a wide unde s anding o he
ans o ma ion aking place. As indica ed in Table 5, he yields a e 10 min we e ema kable.
Table 5. Time sc eening o he syn hesis o ca bazole 2 a 1 mM scale.a
21
En y
Time
Yield (%)b
1
10 min
74
2
30 min
71
3
1 hou
77
aCondi ions: 1 mM o 1 (0.0058 mmol), 10 mol% iP AuT 2, in 9/1 H2O/DMSO (5.8 mL) unde open a mosphe e, . .
bYields we e de e mined by 1H-NMR using TMB as in e nal s anda d.
Bea ing in mind all his, i was de e mined ha he inal op imized condi ions in aqueous
media a e hose indica ed in Scheme 17.
Scheme 17. Final op imized condi ions o he syn hesis o ca bazole 2 in aqueous media.
3.2.2 Reac ion in bio ele an media
When a bioo hogonal esea ch is being conduc ed, he analysis o he eac ion wi hin
biological milieu is an in e es ing in i o s udy, since he inc easing complexi y o he media can
enligh en abou he iabili y o he p ocess.
The ollowing mediums we e es ed:
• PBS (phospha e bu e ed saline solu ion, pH 7.4). Solu ion o ino ganic sal s (NaCl,
KCl, Na2HPO4 and KH2PO4). I is commonly used o washes, dilu ions and o
main ain he in eg i y o issues and cells in biological and biochemical
in es iga ions.
• DMEM (Dulbecco’s Modi ied Eagle Medium). Cell cul u e medium designed o
euka yo ic cells’ g ow h, con aining nu ien s and di e en g ow h ac o s. In i s
composi ion he e a e di e se amino acids, i amins, ino ganic sal s and o he
componen s like phenol ed, sodium py u a e and dex ose.
• Cellula lysa es. Ex ac ion o cellula con en esuspended in PBS. Concen a ion
gi en in mg p o ein/mL PBS.
22
Fi s ly, he eac ion was pe o med in he p esence o PBS, obse ing he o ma ion o he
p oduc in an excellen yield (99%). In DMEM he eac ion was also e ec i e, al hough he yield
dec eased o 69%. Rema kable, he eac ion is also possible in p esence o cellula lysa es, leading
o a 59% yield o he p oduc .
[Au]
w/o
[Au]
w/o
[Au]
w/o
PBS
DMEM
LYSATES
0
50
100
Yield (%)
Scheme 18. Ba g aphic showing he esul s o he eac ion in di e en biological media, wi h and wi hou [Au].
Condi ions: 1 mM o 1 (0.0034 mmol), 10 mol% iP AuT 2, in 9/1 media/DMSO (3.4 mL) unde open a mosphe e, . .
Yields we e de e mined by 1H-NMR using TMB as in e nal s anda d. PBS 1x: 136.9 mM o NaCl, 2.68 mM o KCl,
10.14 mM o Na2HPO4 and 1.76 mM o KH2PO4. Lysa es: 2.05 mg p o ein/mL o sample.
These esul s allow o conclude ha he eac ion is highly o hogonal, ole a ing he p esence
o di e en ype o sal s and molecules. Conside ing he ou come o he expe imen a ion, he nex
s udies we e ca ied ou in PBS, whe e he e ec o he s udied biomolecules can be easily spo ed.
3.2.3 Compa ibili y wi h speci ic biomolecules
As p e iously s a ed, he complex ne wo k o biomolecules ha exis wi hin cells is an aspec
o ake in o conside a ion when an abio ic eac ion is in ended o be in oduced in cellula
en i onmen s. The e o e, he pe o mance o he eac ion in he p esence o some ep esen a i e
biomolecules was nex in es iga ed.
29
They we e ollowed by a g adual change o 5 min o H2O/MeCN (5:95). The la es condi ions
we e kep 1 min. Then, se ings we e e u ned o he ini ial ones (45:55) o 2 min. The
ch oma og am was eco ded using a UV de ec ion a λ = 300 nm.
3.5.2 Syn hesis o 1-iodo-2-(phenyle hynyl)benzene
P ocedu e adap ed om Hi ano e al.48
Bis( iphenylphosphine)palladium(II) dichlo ide (182.5 mg, 0.26 mmol, 2.5 mol%) and
coppe (I) iodide (49.3 mg, 0.26 mmol) we e dissol ed in bu ylamine (20 mL) in a pu ged ound
lask unde ni ogen a mosphe e. Phenylace ylene (1.2 mL, 11.2 mmol, 1.06 eq) and 1,2-
diiodobenzene (1.4 mL, 10.6 mmol, 1 eq) we e added o he s i ed solu ion, again unde N2 low.
The mix u e was s i ed o 24 hou s a 60 ºC unde a gon, showing he TLC (silica gel, n-
Hexane/E OAc 20:1) comple e disappea ance o he s a ing ma e ial.
The mix u e was cooled o oom empe a u e, quenched by addi ion o sa u a ed aqueous
NH4Cl and washed h ee imes wi h b ine. The o ganic laye was d ied o e Na2SO4, il a ed and
concen a ed unde acuum. The esidue was pu i ied by column ch oma og aphy on silica gel
wi h n-hexane/e hyl ace a e 20:1 o a o d 1-iodo-2-(phenyle hynyl)benzene (1.15 mg, 36%) as a
pale yellow oil.
1H-NMR (300 MHz, d-chlo o o m): δ 7.88 (d, 3J = 8.0 Hz, 1H), 7.63-7.58 (m, 2H), 7.53 (d,
3J = 7.7 Hz, 1H), 7.40-7.29 (m, 4H), 7.01 ( , 3J = 7.7 Hz, 1H); 13C-NMR (75 MHz, d-chlo o o m):
δ 138.9 (CHA ), 132.6 (CHA ), 131.8 (2xCHA ), 130.0 (CA ), 129.5 (CHA ), 128.8 (CHA ), 128.5
(2xCHA ), 128.0 (CHA ), 123.1 (CA ), 101.3 (CA ), 93.2 (Calk), 91.8 (Calk).
R : 0.47 (hexane/e hyl ace a e 20:1).
Da a in acco dance wi h he li e a u e.58
3.5.3 Syn hesis o 2-{[2-(phenyle hynyl)phenyl]e hynyl}aniline 1
P ocedu e ex ac ed om Hi ano e al.48

30
A mix u e o 1-iodo-2-(phenyle hynyl)benzene (1.15 mg, 3.77 mmol, 1.0 eq), coppe (I) iodide
(35.7 mg, 0.050 eq) and bis( iphenylphosphine)palladium(II) dichlo ide (131.6 mg, 5 mol%)
we e pu ged in a ound lask and subjec ed o ni ogen a mosphe e. Once hey we e dissol ed
wi h E 3N (10.2 mL), a solu ion o e hynylaniline (470.0 mg, 4.012 mmol) in THF (3.9 mL) was
added o he mix u e, ha was s i ed a 80 ºC o 5.0 h unde a gon wi h e lux.
The mix u e was cooled o oom empe a u e, quenched by addi ion o sa u a ed aqueous
NH4Cl and ex ac ed h ee imes wi h E OAc. The combined o ganic laye s we e washed wi h
b ine, d ied o e Na2SO4, il a ed and concen a ed unde acuum. The p oduc 2-((2-
(phenyle hynyl)phenyl)e hynyl)aniline (1) was pu i ied by column ch oma og aphy on silica gel
wi h hexane/E OAc 4:1 as a pale yellow solid (843.5 mg, 76%).
1H-NMR (300 MHz, d-chlo o o m): δ 7.63-7.53 (m, 4H), 7.42-7.30 (m, 6H), 7.14 ( , 3J = 7.7
Hz, 1H), 6.70 ( , 3J =8.9 Hz, 2H), 4.43 (s, 2H); 13C-NMR (75 MHz, d-chlo o o m): δ 148.4 (CA ),
132.3 (CHA ), 132.1 (CHA ), 132.0 (2xCHA ), 131.6 (CHA ), 130.1 (CHA ), 128.7 (CHA ), 128.5
(2xCHA ), 128.3 (CHA ), 127.9 (CHA ), 126.0 (CA ), 125.2 (CA ), 123.2 (CA ), 117.8 (CHA ), 114.2
(CHA ), 107.8 (CA ), 93.9 (Calk), 93.2 (Calk), 90.6 (Calk), 89.0 (Calk).
R : 0.36 (hexane/AcOE 8:2); 0.37 (hexane/ oluene 2:1). C22H15N [M+]: 294.23. : 10.5 min.
Da a in acco dance wi h he li e a u e.48
3.5.4 Syn hesis o 6-phenyl-11H-benzo[a]ca bazole 2
P ocedu e adap ed om Hi ano e al.48
A mix u e o chlo o( iphenylphosphine)gold(I) (2.9 mg, 0.0059 mmol, 5 mol%) and sil e
i luo ome hanesul ona e (1.5 mg, 0.0059 mmol, 0.05 eq) we e dissol ed in ace oni ile (0.59
mL) in a pu ged Schlenk ube unde a gon a mosphe e. Once 2-((2-
(phenyle hynyl)e hynyl)aniline (35.2 mg, 0.12 mmol, 1.0 eq) was added, he mix u e was s i ed
a 80 ºC un il TLC (silica gel, n-hexane/E AOc 9:3) showed comple e con e sion o he s a ing
ma e ial (4.5 h).
A e cooling he Schlenk o oom empe a u e, he mix u e was dilu ed wi h e hyl ace a e,
washed h ee imes wi h sa u a ed aqueous NH4Cl and b ine, d ied o e Na2SO4, il e ed and
concen a ed unde acuum.
31
The p oduc 2 was pu i ied by column ch oma og aphy (silica gel) wi h n-hexane/ oluene
(2:1), ob aining 6-phenyl-11H-benzo[a]ca bazole as pale o ange solid (17.6 mg, 50%*).
1-H-NMR (300 MHz, d-chlo o o m): δ 8.91 (s, 1H), 8.17 (d, 3J= 7.8 Hz, 1H), 8.01 (d, 3J= 7.9
Hz), 1H), 7.70 (d, 3J = 7.4 Hz, 2H), 7.64-7.51 (m, 7H), 7.45 (d, 3J = 8.1 Hz, 1H), 7.37 ( , 3J = 7.6
Hz, 1H), 7.05 ( , 3J = 7.6 Hz, 1H). 13C-NMR (75 MHz, d-chlo o o m): δ 141.4 (CA ), 138.9 (CA ),
136.8 (CA ), 135.4 (CA ), 132.3 (CA ), 129.5 (2xCHA ), 129.1 (CHA ), 128.5 (2xCHA ), 127.7
(CHA ), 125.8 (CHA ), 125.6 (CHA ), 124.8 (CHA ), 124.1 (CA ), 122.3 (CHA ), 121.1 (CHA ), 120.5
(CHA ), 120.4 (CA ), 119.8 (CHA ), 117.0 (CA ), 111.0 (CHA ).
R : 0.42 (hexane/e hyl ace a e 9:3); 0.29 (hexane/ oluene 2:1). C22H15N [M+]: 294.24. : 9.9
min.
Da a in acco dance wi h he li e a u e.59
* Yield sligh ly o e a ed due o emaining oluene sol en . The u he pu i ica ion o he
isola ion o he p oduc was pe o med ia HPLC-p epa a i e (60-95% o MeCN in wa e o 32
min).
3.5.5 Rep esen a i e p ocedu e o eac ions a 200 mM scale using iP AuNT 2 as complex
and MeCN as sol en (sec ion 3.2.1)
Aniline 1 (35.2 mg, 0.12 mmol, 1.0 eq) and iP AuNT 2 (20.8 mg, 0.024 mmol, 20 mol%) we e
added o a Schlenk ube and subjec ed o a pu ge by h ee acuum-ni ogen cycles.* Then, MeCN
(0.59 mL) was added o he mix u e, which was magne ically s i ed a oom empe a u e
o e nigh . O e ime, he solu ion u ned yellow. A e app oxima ely 16 hou s, he mix u e was
dilu ed wi h e hyl ace a e, washed wi h sa u a ed aqueous NH4Cl and b ine, d ied o e Na2SO4,
il e ed and concen a ed unde acuum. A yield o 96% was de e mined by 1H-NMR wi h TMB
as in e nal s anda d. The s a ing ma e ial con e sa ion was comple e.
* When he eac ions a e pe o med open o ai , bo h compound 1 and ca alys a e added o a
Schlenk ube in open a mosphe e.
32
3.5.6 Rep esen a i e p ocedu e o he s udy o he dilu ion (sec ion 3.2.1)*
Aniline 1 (10.0 mg, 0.034 mmol, 1.0 eq) and iP AuNT 2 (3.0 mg, 0.0035 mmol, 10 mol%)
we e s i ed in a ound lask wi h wa e (0.34 mL) a oom empe a u e unde open a mosphe e
o 10 min.
The mix u e, a cloudy suspension ha u ns yellow o e ime, was dilu ed wi h e hyl ace a e,
washed wi h sa u a ed aqueous NH4Cl and b ine, d ied o e Na2SO4, il e ed and concen a ed
unde acuum. A yield o 88% was de e mined by 1H-NMR wi h TMB as in e nal s anda d. The
s a ing ma e ial con e sa ion was comple e.
* Al e a ions: concen a ion o 1 was g adually educed and, he e o e, olume o wa e was
inc eased.
3.5.7 Rep esen a i e p ocedu e o eac ions a 1 mM scale using s ock solu ions in DMSO*
(sec ion 3.2.1)
A s ock solu ion o iP AuNT 2 (2.0 mM) in DMSO and ano he one o SM 1 (20 mM), again
in DMSO, we e p e iously p epa ed o add o wa e (5.2 mL) in a Schlenk ube. Bo h solu ions
we e pipe ed wi h a olume o 290 μL, i s he subs a e ollowed by he ca alys . Due o he
insolubili y o he s a ing ma e ial, a suspension was o med. I was s i ed a oom empe a u e
o 10 minu es.
The mix u e was ex ac ed wi h e hyl ace a e h ee imes, d ied o e Na2SO4 and hen il e ed
wi h Flo isil® (60-100 mesh) o elimina e he gold ca alys . Then, he solu ion was concen a ed
unde acuum. A yield o 74% was de e mined by 1H-NMR wi h TMB as in e nal s anda d. The
s a ing ma e ial con e sa ion was comple e.
* Times we e sc eened.
Fo he s udy o he biocompa ibili y, a hi d solu ion o 1.0 eq o a biomolecule was p epa ed
in PBS (0.1 mL) and added be o e compound 1 and ca alys o he eac ion media (3.0 mL).
33
3.5.8 In e nal s anda d me hod o yield quan i ica ion
Nuclea magne ic esonance (NMR) spec oscopy allows elucida ion o small and mac o
molecules, as well as quan i ica ion o signals hanks o he p opo ionali y be ween he esonance
line and he numbe o esonan nuclei.60
This enables he u iliza ion o an in e nal s anda d o analyze eac ion c udes, allowing he
easy ob en ion o he ans o ma ion yield by compa ing a well-known peak o he in e nal
s anda d wi h one o he p oduc whose in eg a ion ha e been p e iously iden i ied. This way, an
ideal in e nal s anda d would be one highly pu i ied, s able, chemically ine and soluble in he
NMR sol en used.61
In his esea ch i was used 1,3,5- ime hoxybenzene (TMB), whe e i s a oma ic peak (δ= 6.09
ppm) can be used o de e mine he yield o he eac ion. The compa a ion was made based on he
mean o h ee signals o he p oduc : wo a oma ic H om he ca bazole moie y (δ = 8.17 ppm, d,
1H; δ = 8.01 ppm, d, 1H) and he H om he NH g oup (δ = 8.91 ppm, s, 1H).
The known amoun o TMB added was calcula ed in base o a 1:1 ela ion be ween he
in es iga ed signals, assuming a100% yield. To simpli y he calcula ions, a ac o o 1/3 was
employed. This adjus men accoun s o he ac ha he u ilized peak o TMB in eg a es o h ee
p o ons (3H), while he co esponding peaks om he p oduc in eg a e o one p o on (1H). Thus,
o he expe imen s pe o med in Table 5 (En y 1):
𝑀𝑎𝑠𝑠𝑇𝑀𝐵 = 0.006 𝑚𝑚𝑜𝑙 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 · 1 𝑚𝑚𝑜𝑙 𝑇𝑀𝐵
1 𝑚𝑚𝑜𝑙 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 · 1 𝐻
3 𝐻 · 168.19 𝑚𝑔 𝑇𝑀𝐵
1 𝑚𝑚𝑜𝑙 𝑇𝑀𝐵 = 0.34 𝑚𝑔 𝑇𝑀𝐵
Scheme 25. Example o yield de e mina ion by in e nal s anda d o 1,3,5- ime hoxybenzene. Spec um
co esponding o p ocedu e desc ibed in Table 5.
The de e mined amoun o in e nal s anda d would be added o he c ude wi h he p e ious
p epa a ion o s ock solu ions.
34
3.5.9 Gene al in o ma ion o in cellulo expe imen s
All s eps we e pe o med on a s e ile clean bench Tesl a AV-100 a oom empe a u e.
Solu ions s o ed in a idge we e p e-wa med in a wa e ba h (37ºC) be o e use.
A549 cell cul u es we e incuba ed in DMEM (Dulbecco’s modi ied Eagle’s medium) acqui ed
om Sigma-Ald ich, which was en iched wi h penicillin (100 uni s/mL), s ep omycin (100
uni s/mL) and glu amine 5 mM, all om In i ogen. Cells we e seeded in well pla es a he
speci ied concen a ion wo days p io o ea men . P oli e a ing cul u es we e main ained in an
incuba o a 37 ºC, 5% CO2 and 95% humidi y.
3.5.10 Rep esen a i e p ocedu e o in cellulo eac ions
To pe o m he di e en expe imen s, 1.5 million cells pe pla e (100 nm) we e seeded wo
days be o e ea men . In he beginning o he expe imen a ion, he g owing media was e i ed
p io o he addi ion o he solu ions p e iously p epa ed in DMEM. T ea men o he cells was
ini ia ed wi h he i s one con aining he aniline 1 (100 o 50 μM, inal olume o 3 mL), o 30
minu es, using eshly p epa ed s ock solu ion (10 mM in DMSO). Once he ime is o e , he
media was aspi ed and cells we e washed wice wi h PBS (3 mL). Then, he second DMEM
solu ion was added, con aining iP AuNT 2 (50 and 25 μM, inal olume o 3 mL), om again
eshly p epa ed s ock solu ion (10 mM in DMSO).
A e 3 hou s, he media was collec ed and kep o pos e io analysis, as well as he 2 new
washes wi h PBS (3 mL). All hese ac ions we e lyophilized and il e ed by HPLC il e be o e
being subjec ed o HPLC-MS. Finally, cells we e ex ac ed wi h MeCN (1 mL) h ee imes. The
po ions we e concen a ed unde acuum, agi a ed by o ex and subjec ed o sonica ion o 5
min. Then, hey we e il e ed wi h HPLC il e s and analyzed by HPLC-MS o de e mine he
p oduc 2 o ma ion.

35
CONCLUSIONS
In he p esen Final Deg ee P ojec , a new bioo hogonal eac ion has been desc ibed based
on he in amolecula double cycling p omo ed by gold o an aniline by means o a cascade
mechanism. I s o iginal pa ame e s, based on o ganic syn hesis, ha e been op imized, modi ying
ca alys s and hei loads, imes, concen a ions, sol en s and a mosphe e, inally de e mining ha
he eac ion can be ca ied ou in mild condi ions. The pe ec ed pa ame e s allow he chemical
ans o ma ion o be ca ied ou in 9/1 H2O/DMSO, in 10 minu es, wi h a load o 10 mol% o
iP AuNT 2 as a ca alys , wi h a subs a e concen a ion o 1 mM, a oom empe a u e and in an
open a mosphe e.
I has also been shown ha he eac ion is pe ec ly compa ible wi h mo e complex media,
ob aining good esul s in bo h PBS, DMEM and cell lysa es. A he same ime, he o ma ion o
ca bazole om aniline has been ound o be pe ec ly ole an o he p esence o biomolecules
such as NADH, GSH, L-Cys eine o sodium asco ba e, which ac wi h e y li le inhibi ion in in
i o es s. Finally, p elimina y s udies ha e been de eloped on he adap a ion o he eac ion o
he in e io o cellula media, using he A549 line o hese in cellulo analyses, demons a ing i s
co ec unc ioning and bioo hogonali y.
These esul s could open he way o he de elopmen o bioo hogonal eac ions o he
o ma ion o s uc u es o g ea molecula complexi y h ough cascade mechanisms, ac ing in a
simple and sus ainable way. This could lead o he in si u o ma ion o cyclic and he e ocyclic
s uc u es o in e es o Medicinal Chemis y, highligh ing o example he an imic obial and
an i umo ac ion o annula ed de i a i es o ca bazoles.
In his way, i is in e es ing o con empla e he u u e op imiza ion o p elimina y assays in
cells, imp o ing he condi ions and ca ying ou quan i a i e s udies ha allow a be e
unde s anding o he eac ion, as well as ca ying i ou in di e en cell lines o obse e i s e ec s.
On he o he hand, he u u e modi ica ion o he inal s uc u e o he cycliza ion is p oposed wi h
he aim o making i luo escen , acili a ing i s in i o s udy h ough luo escence mic oscopy.
CONCLUSIONES
En el p esen e T abajo de Fin de G ado, se ha desc i o una nue a eacción bioo ogonal basada
en la doble ciclación in amolecula p omo ida po o o de una anilina median e un mecanismo en
cascada. Se han op imizado sus pa áme os o iginales, undamen ados en la sín esis o gánica,
modi icando ca alizado es y sus ca gas, iempos, concen aciones, disol en es y a mós e a,
de e minando inalmen e que la eacción se puede ealiza en condiciones sua es.
36
Los pa áme os pe eccionados pe mi en lle a a cabo la ans o mación química en 9/1
H2O/DMSO, en 10 minu os, con una ca ga de 10 mol% de iP AuNT 2 como ca alizado , con una
concen ación de sus a o de 1 mM, a empe a u a ambien e y en a mós e a abie a.
También se ha demos ado que la eacción es pe ec amen e compa ible con medios más
complejos, ob eniendo buenos esul ados an o en PBS, DMEM como en lisados celula es. Al
mismo iempo, la o mación del ca bazol a pa i de la anilina ha esul ado se pe ec amen e
ole an e a la p esencia de biomoléculas como NADH, GSH, L-Cis eína o asco ba o sódico, las
cuales ac úan con una muy pequeña inhibición en ensayos in i o. Po úl imo, se han desa ollado
es udios p elimina es sob e la adap ación de la eacción al in e io de medios celula es, u ilizando
la línea A549 pa a es os análisis in cellulo, demos ando su co ec o uncionamien o y
bioo ogonalidad.
Es os esul ados pod ían ab i el paso al desa ollo de eacciones bioo ogonales de o mación
de es uc u as de g an complejidad molecula a a és de mecanismos en cascada, ac uando de
mane a sencilla y sos enible. Es o pod ía da luga a la o mación in si u de es uc u as cíclicas y
he e ocíclicas de in e és pa a la Química Médica, des acando po ejemplo la acción
an imic obiana y an i umo al de los de i ados anillados de los ca bazoles.
De es a mane a, es in e esan e con empla la op imización u u a de los ensayos p elimina es
en células, mejo ando las condiciones y ealizando es udios cuan i a i os que pe mi an
comp ende mejo la eacción, así como lle a la a cabo en di e en es líneas celula es pa a obse a
sus e ec os. Po o o lado, se p opone la u u a modi icación de la es uc u a inal de la ciclación
con el obje i o de hace la luo escen e, acili ando su es udio in i o a a és de mic oscopía de
luo escencia.
CONCLUSIÓNS
No p esen e T aballo de Fin de G ado, desc ibiuse unha no a eacción bioo ogonal baseada
na dob e ciclación in amolecula p omo ida po ou o dunha anilina median e un mecanismo en
cascada. Op imizá onse os seus pa áme os o ixinais, undamen ados na sín ese o gánica,
modi icando ca alizado es e as súas ca gas, empos, concen ación, disol en es e a mos e a,
de e minando inalmen e que a eacción pódese ealiza en condicións sua es. Os pa áme os
pe eccionados pe mi en le a a cabo a ans o mación química en 9/1 H2O/DMSO, en 10
minu os, cunha ca ga de 10 mol% de iP AuNTF2 como ca alizado , cunha concen ación de
subs a o de 1 mM, a empe a u a ambien e e a mos e a abe a.
37
Tamén demos ouse que a eacción é pe ec amen e compa ible con medios máis complexos,
ob endo bos esul ados an o en PBS, DMEM como en lisados celula es. Ao mesmo empo, a
o mación do ca bazol a pa i da anilina esul ou se pe ec amen e ole an e á p esencia de
biomoléculas como NADH, GSH, L-Cis eína ou asco ba o sódico, os cales ac úan cunha moi
pequena inhibición en ensaios in i o. Po úl imo, desen ol é onse es udos p elimina es sob e a
adap ación da eacción ao in e io de medios celula es, u ilizando a liña A549 pa a es es análises
in cellulo, demos ando o seu co ec o uncionamen o e bioo ogonalidade.
Es es esul ados pode ían ab i o paso ao desen ol emen o de eaccións bioo ogonais de
o mación de es u u as de g an complexidade molecula a a és de mecanismos en cascada,
ac uando de manei a sinxela e sos ible. Is o pode ía da luga á o mación in si u de es u u as
cíclicas e he e ocíclicas de in e ese pa a a Química Médica, des acando po exemplo a acción
an imic obiana e an i umo al dos de i ados anulados dos ca bazoles.
Des a manei a, é in e esan e con empla a op imización u u a dos ensaios p elimina es en
células, mello ando as condicións e ealizando es udos cuan i a i os que pe mi an comp ende
mello a eacción, así como le ala a cabo en di e en es liñas celula es pa a obse a os seus
e ec os. Po ou o lado, p oponse a u u a modi icación da es u u a inal da ciclación co obxec i o
de acela luo escen e, acili ando o seu es udo in i o a a és de mic oscopio.
38
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45
HPLC-MS

46
6-phenyl-11H-benzo[a]ca bazole 2
1H-NMR
13C-NMR
DEPT-135
47
HPLC-MS