Recent Developments and Challenges in the Enzymatic Formation of Nitrogen–Nitrogen Bonds

Recen De elopmen s and Challenges in he Enzyma ic Fo ma ion
o Ni ogen−Ni ogen Bonds
Cha i omeni Angeli,
‡
Sa a A ienza-Sanz,
‡
Simon Sch öde , Annika Hein, Yongxin Li, Alexande A gy ou,
Angelina Osipyan, Hen ik Te holsen, and Sandy Schmid *
Ci e This: ACS Ca al. 2025, 15, 310−342
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ABSTRACT: The biological o ma ion o ni ogen−ni ogen
(N−N) bonds ep esen s in iguing eac ions ha ha e a ac ed
much a en ion in he pas decade. This in e es has led o an
inc easing numbe o N−N bond-con aining na u al p oduc s
(NPs) and ela ed enzymes ha ca alyze hei o ma ion ( e e ed
o in his e iew as NNzymes) being elucida ed and s udied in
g ea e de ail. While mo e de ailed in o ma ion on he biosyn hesis
o N−N bond-con aining NPs, which has only become a ailable in
ecen yea s, p o ides an unp eceden ed sou ce o biosyn he ic
enzymes, hei po en ial o bioca aly ic applica ions has been
minimally explo ed. Wi h his e iew, we aim no only o p o ide a
comp ehensi e o e iew o bo h cha ac e ized NNzymes and
hypo he ical bioca alys s wi h pu a i e N−N bond o ming ac i i y,
bu also o highligh he po en ial o NNzymes om a bioca aly ic pe spec i e. We also p esen and compa e con en ional syn he ic
app oaches o linea and cyclic hyd azines, hyd azides, diazo- and ni oso-g oups, iazenes, and iazoles o allow compa ison wi h
enzyma ic ou es ia NNzymes o hese N−N bond-con aining unc ional g oups. Mo eo e , he biosyn he ic pa hways as well as he
di e si y and eac ion mechanisms o NNzymes a e p esen ed acco ding o he di ec unc ional g oups cu en ly accessible o hese
enzymes.
KEYWORDS: Ni ogen−Ni ogen Bond, NNzymes, Na u al P oduc s, Bioca alysis, Heme Enzymes, Cupin Domain
1. INTRODUCTION
Since he isola ion o he i s ni ogen−ni ogen (N−N)
bond-con aining na u al p oduc (NP) mac ozamin in 1940
om an Aus alian cycad plan ,
1
hund eds o NPs con aining
N−N bonds ha e been epo ed om a ple ho a o
o ganisms.
2,3
These NPs a e pa icula ly no ewo hy due o
hei po en ial as he apeu ic agen s and p ecu so s o
syn hesizing biologically ac i e molecules. In addi ion o
NPs, N−N bond con aining g oups ha e al eady been used
as key-s uc u al mo i s in di e se compounds applied o dyes,
ag ochemicals, syn he ic ma e ials and cosme ics.
3−7
Cu en ly,
he biological ac i i ies o N−N bond-con aining compounds
including py azomycin (1), alanimycin (2), c emeomycin (3),
de i a i es o pipe aza e (L-4) and o he ela ed me aboli es
a e being in es iga ed (Figu e 1).
3,8−11
One such compound,
he N-ni oso-con aining NP s ep ozocin (5) is bo h a NP and
an FDA app o ed d ug. Ma ke ed unde he b and name
Zanosa , i is an an ineoplas ic agen used o ea panc ea ic
cance .
12−15
Pha maceu ical inno a ions pa icula ly bene i
om syn he ic and na u al compounds con aining N−N
bonds, mainly a oma ic o nona oma ic he e ocycles, because
o hei di e se ange o an i i al, an ibac e ial, an imala ial and
an icance ac i i ies.
3,8,16
As a ma e o ac , each yea , he
FDA’s CDER (The Uni ed S a es Food and D ug Admin-
is a ion’s Cen e o D ug E alua ion and Resea ch) app o es
an inc easing numbe o new d ugs con aining his in iguing
s uc u al mo i .
17
Fo ins ance, celecoxib (6), a syn he ic
py azole compound employed o pain elie in a h i is.
18,19
Daca bazine (7), a chemo he apy d ug p esen s he iazine
unc ionali y,
20
while hyd alazine (8), which is p esc ibed o
high blood p essu e,
21
con ains dual hyd azine g oups. The
angio ensin con e ing enzyme (ACE) inhibi o cilazap il (9)
(EMA app o ed) includes he common hyd azide unc ion-
ali y.
22
In addi ion, he chemo he apy agen s p oca bazine
(10), ca mus ine (11), and lomus ine (12) con ain N−N
unc ionali ies. The o me as a nonde i a ized hyd azine
Recei ed: Augus 30, 2024
Re ised: Decembe 4, 2024
Accep ed: Decembe 6, 2024
Published: Decembe 17, 2024
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g oup and he la e wo include he N-ni osamine
unc ionali y (Figu e 1).
15,23,24
Toge he , hese FDA app o ed d ugs and NPs con ain a
numbe o di e en N−N unc ionali ies, including: hyd azines
(R2N-NR2), hyd azides (R2N−N(R)C(�O)R), hyd azones
(R2C�N-NR2), azines (R2C�N−N�CR2), diazo- (R2C�
N+�N−), azo- (RN�NR), azoxy- (RN�N+(O−)R), ni os-
amines (R2N−N�O), iazenes (R2N−N�N-R) and a o-
ma ic he e ocycles. Howe e , di ec access o some unc ion-
ali ies ha e no been associa ed wi h an NNzyme (Figu e
2).
3,25
The o ma ion and clea age o N−N bonds ep esen a
undamen al biogeochemical p ocess.
26
Ca an o e al.
p oposed ha N−N bond- o ming enzymes (NNzymes),
which a e in ol ed in he biosyn hesis o seconda y
me aboli es, including cha ac e ized NPs, may ha e e ol ed
om enzymes ha pa icipa e in he ni ogen cycle.
27
Fo an
in-dep h analysis o enzymes in ol ed in he biosyn hesis o
p ima y ni ogen cycle me aboli es, including ni a e, ni i e,
ni ic oxide and ni ous oxide, eade s a e encou aged o
consul ecen e iews.
28−30
The g owing in e es in compounds con aining he N−N
mo i has led o a dedica ed and ongoing in es iga ion in o he
syn hesis o hese molecules.
31
Al hough accessible o o ganic
chemis s h ough he use o hyd azine and i s de i a i es, he
haza dous and explosi e na u e o hese subs ances makes
hem challenging o use in indus ial applica ions and
nega i ely impac s he en i onmen .
32
Ins ead, he di ec
coupling o ni ogen a oms, a he han hyd azine inco po-
a ion in o a molecule, ep esen s a highly e icien me hod
ha gi es g ea e eedom when cons uc ing molecules wi h
N−N bonds. Howe e , he syn he ic challenge associa ed wi h
he coupling oge he o wo ni ogens o o m an N−N bond
can be a ibu ed o he high nucleophilici y o he a oms
in ol ed. The e o e, he majo i y o he coupling me hods ely
on ni ogen ac i a ion o inc ease he elec ophilici y o one
ni ogen making coupling easible.
33
Examples o me hods ha
achie e his ni ogen ac i a ion a e elec ochemis y,
34
me al
ca alysis
35
and oxida i e o educ i e ans o ma ions
36−41
ha
ha e been de eloped o access a ich a ie y o molecules wi h
N−N bonds. Despi e he e o s made o imp o e classic
o ganic syn hesis me hods, se e al challenges mus be
o e come, including p oduc loss, low yields, poo accessibili y
o in e molecula eac ions, low enan io- and dias e eoselec-
i i y, he e o- and homocoupling selec i i y and he use o
haza dous chemicals.
42,43
A sui able solu ion o hese challenges is o employ he use
o enzymes as bioca alys s. Na u al NNzymes ha e adop ed
me hods o o e come his he modynamic obs acle, esul ing
in he occu ence o N−N bond-con aining unc ional g oups
in nume ous seconda y me aboli es. The ad en o genome
mining echniques has g ea ly acili a ed he explo a ion o he
biosyn he ic gene clus e s (BGCs) esponsible o he
p oduc ion o hese me aboli es. The enzyma ic machine y
esponsible o N−N bond o ma ion is g adually being
elucida ed wi hin he clus e s, leading o he disco e y o an
Figu e 1. Chemical s uc u es o N−N bond-con aining na u al p oduc s and FDA-app o ed d ugs. Comme cial names a e men ioned in b acke s.
Figu e 2. N−N bond-con aining unc ional g oups ound in na u al p oduc s (NPs). The blue backg ound ep esen s he unc ional g oups ha
can be o a e specula ed o be cons uc ed by ni ogen−ni ogen bond- o ming enzymes (NNzymes). The o ange backg ound highligh s emaining
N−N bond-con aining unc ional g oups ha can be ound in NPs, bu o which no co esponding NNzymes ha e ye been iden i ied.
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inc easing numbe o NNzymes. Thus, a deepe unde s anding
o he enzyma ic machine y esponsible o N−N bond
o ma ion in biosyn he ic pa hways and he de elopmen o
NNzymes in o b oadly applicable bioca alys s could acili a e
he c ea ion o e icien , cos -e ec i e, and mo e sus ainable
me hods o he syn hesis o N−N bond-con aining com-
pounds. I is he e o e necessa y o elucida e he unc ional and
s uc u al p ope ies o hese enzymes o expand hei
bioca aly ic capabili ies.
In ecen yea s, a numbe o e iews ha e p o ided an
o e iew o N−N bond o ma ion in NP biosyn hesis and
summa ized eme ging NNzymes and hei (p oposed)
enzyma ic mechanisms.
3,11,25,44−48
Howe e , since he las
e iew on (bio)syn he ic ou es o N−N bonds by he Ryan
g oup was published in 2022,
25
a ple ho a o no el N−N
bond-con aining NPs and hei biosyn he ic machine y ha e
been elucida ed. Impo an ly, mo e de ailed in o ma ion on he
unde lying enzyma ic mechanisms and s uc u al ea u es o
se e al NNzymes has also been un a eled since hen.
The e o e, his e iew aims o summa ize he mos ecen
unde s anding o he enzyma ic and chemical mechanisms
unde lying N−N bond o ma ion in NP biosyn hesis and o
p o ide a comp ehensi e o e iew o bo h cha ac e ized
NNzymes and hypo he ical bioca alys s wi h pu a i e N−N
bond o ming ac i i y. The enzymes a e classi ied acco ding o
he unc ional g oups hey gene a e wi hin he biosyn he ic
pa hways o di e en NPs. Fu he mo e, a special emphasis is
placed on NNzymes om a bioca aly ic pe spec i e, aiming o
de elop mo e sus ainable me hods o N−N bond o ma ion.
The classical syn he ic pa hways owa d hese unc ional
g oups can be ound in Sec ion 2.Sec ion 3 co e s he gene al
eac ion mechanisms and di e si y o NNzymes, and Sec ion 4
ocuses on he biosyn hesis o speci ic unc ional g oups.
Fu he mo e, Sec ion 5 add esses he majo challenges
cu en ly aced by classical syn he ic app oaches, wi h a
pa icula emphasis on he po en ial o NNzymes o excel in
a eas whe e cu en limi a ions could be o e come.
2. OVERVIEW ON CONVENTIONAL STRATEGIES TO
ACCESS N−N BOND-CONTAINING FUNCTIONAL
GROUPS
As highligh ed in his e iew, he cons uc ion o N−N bond-
con aining molecules in na u e is a ascina ing opic, especially
in e ms o how biological sys ems ingeniously o e come he
high elec onega i i y o ni ogen a oms o c ea e a di e se
a ay o N−N bond-con aining unc ional g oups. While
se e al e ol ed s a egies o o ming N−N bonds in na u e
appea o mi o known syn he ic me hodologies, o he s seem
o di e ge om hese con en ional ou es. Thus, a ho ough
unde s anding o he biosyn he ic p ocesses is undamen al o
ad ancing ou knowledge o chemical bonding and eac i i y,
as he N−N bond displays p ope ies ha a e dis inc om
hose o he C−C and C−N bonds,
49
and may also pa e he
way o he de elopmen o no el selec i e ou es o N−N
bond-con aining molecules. This sec ion o he e iew p o ides
a gene al o e iew o he di e se N−N bond-con aining
unc ional g oups and he con en ional me hods o hei
syn hesis. Gi en he impo ance o biosyn he ic N−N bond
o ma ion ia enzyma ic ca alysis, we ocus p ima ily on he
syn hesis o N−N bonds, which can also be accessed by
NNzymes. Fo a mo e de ailed discussion o he syn he ic
app oaches, eade s a e di ec ed o ecen e-
iews.
11,25,28,31,33,44,50−52
The e a e a numbe o po en ial e osyn he ic app oaches
ha can be employed o access molecules con aining N−N
bonds (Scheme 1). The mos equen ly used me hod in he
syn hesis o N−N bond-con aining molecules occu s om he
unc ionaliza ion and subsequen inco po a ion o he
Scheme 1. Ca aly ic App oaches o C ea ing an Elec ophilic Pa ne o Fo ming Ni ogen−Ni ogen (N−N) Bonds
a
a
A. The Raschig p ocess B. O e iew and classi ica ion o he me hods used o o m N−N bonds ia ac i a ion o an elec ophilic pa ne . C.
Re iew o gene al ypes o N−N bond o ming eac ions om Jiang-Lin Hu e al.
33
No ed eac ions: C1 ni ene ans e ,
56
C2 oxida i e
dehyd ogena ion,
57
C3 educ i e coupling,
58
and C4 adical coupling.
59
Unless o he wise indica ed, R ep esen s an alkyl o a yl (A ) g oup.
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hyd azine syn hon (Scheme 1A).
50
Fo med h ough he
Raschig p ocess, which has been modi ied o e he pas 100
yea s, hese syn hons p o ide access o a wide a ie y o N−N
unc ional g oups bu a e associa ed wi h signi ican oxico-
logical conce ns.
53
The implemen a ion o a hyd azine syn hon
as an indi ec solu ion will no be discussed he ein. Ins ead, he
p eac i a ion o an amine ia an elec ophilic pa ne , oge he
known as an amina ing eagen , allows o e icien linkage o
wo ni ogen a oms.
25
Typical s ong amina ing agen s such as
O-diphenylphosphinyl-hyd oxlamine (13)
54
and hyd oxyl-
amine O-sul onic acid (14)
55
and ha e been shown o amina e
a wide a ie y o amines (Scheme 1B).
A ecen e iew by Jiang-Lin Hu e al.
33
highligh ed a a ie y
o o he ca aly ic app oaches o c ea ing an elec ophilic
pa ne o o ming ni ogen−ni ogen (N−N) bonds
(Scheme 1C). This included: ni ene ans e , oxida i e
dehyd ogena ion, educ i e coupling, adical coupling and
cycloaddi ions.
33
The la e will no be u he discussed he ein
due o he lack o enzyme-media ed ou es.
Ni ene- ans e eac ions wo k by gene a ing highly eac i e
elec ophilic me al−ni ene species om me al ca alys s and
ni ene p ecu so s.
60
These eac ions p o ide access o a
di e se ange o N-a yl and alkyl hyd azides h ough he use o
a a ie y o ansi ion me al (TM)-based o ganome allic and
ino ganic complexes, including hose con aining Rh, I , Ni, Fe,
Cu, and Ag.
61
This me hodology is applicable no only o
ela i ely simple amines bu also o complex na u al molecules
such as b ucine o quinine.
62
Oxida i e dehyd ogena ion is he mos di ec and e icien
s a egy o pe o m a homo o he e o N−N bond o ma ion.
63
This p ocess can be conduc ed wi h he use o ansi ion me al
ca alys s, including complexes o Fe, Cu, and Co in
conjunc ion wi h oxidan s (e.g., H2O2), o adical ini ia o s,
such as azobis(isobu y oni ile) (AIBN). Coppe ca alysis has
been pa icula ly use ul due o i s e sa ili y in ae obic
oxida ion eac ions. An al e na i e app oach o his eac ion
employs elec ochemical me hods, pho o edox ca alysis, and a
hype alen halide-media ed unc ionaliza ion.
33
This ype o
eac ion allows o he o ma ion o a wide ange o p oduc s,
including azo dyes, azines, ca bazoles and he e ocyclic
compounds (py azoles, iazoles), while main aining good
unc ional g oup compa ibili y.
64
Reduc i e coupling elies on ni oa enes, which a e eadily
accessible compounds ha ac as di ec amina ing eagen s and
educing agen s. Al hough educ i e coupling has mo e
limi a ions han he i s wo ypes o eac ions, due o he
o hogonal o complemen a y eac i i y o he ca aly ic
complexes used, i can be combined wi h o he ypes o
ca alysis (e.g., ansi ion me als o bioca alys s) o achie e
mo e sophis ica ed ans o ma ions. I has been demons a ed
ha educ i e cycliza ion (he e ocycliza ion) o benzamidines
o 2-ni obenzaldehydes, whe he in amolecula o in e mo-
lecula , espec i ely, esul s in he e ec i e con e sion o bo h
in o 2H-indazoles.
52
This ype o eac ion equi es he use o
edox couples o Bi
65
o P,
58
o , al e na i ely, Cu-based
ca alys s, which can also be used o condensa ion/cyclo-
addi ion cascades.
33
Radical coupling me hods a e less
common bu in ol e he gene a ion o ni ogen-cen e ed
adicals, which can combine o o m N−N bonds. This
app oach is o en ini ia ed pho ochemically o by using adical
ini ia o s. This me al ee me hod allows use o a a ie y o
bi unc ionalized oxime es e eagen s.
The inc easing numbe o me hods o N−N bond
o ma ion highligh s he g owing in e es in his a ea o
chemis y. Howe e , many o hese app oaches ace challenges
ela ed o a om economy, cos e iciency, and sus ainabili y,
wi h limi ed conside a ion o achie ing high le els o s e eo-,
chemo- and egioselec i i y. Fu he mo e, many exis ing
me hods ely hea ily on ansi ion me al ca alysis and ha sh
eac ion condi ions. This sec ion will explo e he chemical
syn hesis o speci ic N−N unc ionali ies, emphasizing hei
signi icance and iden i ying po en ial a eas whe e NNzymes
could o e imp o emen s o e con en ional, mo e demanding
syn he ic N−N bond o ming ou es.
2.1. Hyd azines, Hyd azides, and Hyd azones. Hyd a-
zines (R2N−NR2) cons i u e a class o compounds cha ac-
e ized by he p esence o wo ni ogen a oms linked ia a
co alen bond, wi h one o ou alkyl o a yl subs i uen s.
Linea and cyclic hyd azine de i a i es, such as hyd azides
R2N−N(R)C(�O)R and hyd azones (R2C�N−NR2), a e
ex ensi ely employed in he pha maceu ical, ag ochemical,
polyme and dye indus ies, acili a ing a ange o chemical
p ocesses. While nonde i a ized hyd azines a e ypically no
ound in he inal s uc u e o na u al p oduc s due o hei
high eac i i y, hey a e widely u ilized in biosyn he ic
pa hways o o m an a ay o N−N bond-con aining na u al
p oduc s. Examples include, py azomycin (1),
8
s56-p1 (15),
66
o mycin (16)
67
and he iacsins (17).
68
As p e iously men ioned, he Raschig p ocess has been used
since he s a o he 20 h cen u y o syn hesize hyd azine
al hough he oxic na u e o he p ocess makes i un a o able.
Due o i s nucleophilici y, hyd azine eac s wi h aldehydes and
ke ones in o ganic sol en s, such as e hanol, me hanol, and
bu anol o o m hyd azones,
69
key in e media es in many well-
known syn he ic eac ions including he Wol −Kishne
educ ion,
70
he Wha on eac ion
71
and he Shapi o
eac ion.
72
I can easily decompose in o N2gas and has been
a pa o he o al syn hesis o many molecules including
scopadulcic acid B
73
and dysidiolide.
74
Hyd azine i sel , along wi h i s syn hesis, is limi ed by issues
o sa e y, sus ainabili y and selec i i y.
32,75
Bioca alysis p esen s
a p omising al e na i e, o e ing he po en ial o o e come
hese limi a ions. Se e al N−N bond o ming enzymes ha e
al eady been iden i ied, such as he cyclic hyd azine o ming
pipe aza e syn hase (PZS) K zT
76
and i s homologues
77−80
being among he mos ex ensi ely s udied. Fo linea
hyd azines, he hyd azine syn he ase (HS), Py N
81
and i s
homologues
82
ha e gained signi ican a en ion o hei ole in
N−N bond o ma ion a he ini ial s ages o NP biosyn hesis.
The nonde i a ized N−N bond is subsequen ly subjec ed o
u he ans o ma ions by o he enzymes wi hin he BGC.
These enzyma ic pa hways will be discussed in Sec ions 4.1.2
and 4.4.
2.2. Azoxy and Azo G oups. Azoxy compounds ep esen
a ela i ely mino ca ego y o na u ally occu ing molecules,
sha ing a common unc ional g oup and he gene al s uc u al
o mula RN�N+(O−)R ha ep esen s he o mally oxidized
coun e pa o he azo g oup (RN�NR).
83
The azoxy moie y
o seconda y me aboli es endows hem wi h he capaci y o
damage DNA, a p ope y ha is pe cei ed as bo h haza dous
and ca cinogenic. To da e, azoxy compounds ha e been
iden i ied in a numbe o na u ally occu ing sou ces, including
bac e ia, ungi and plan s.
84
In he biosyn hesis o 2, an azoxy
g oup was epo ed o be o med ia an azo compound ha is
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no p esen in he s uc u es o he inal seconda y
me aboli es.
67
The azo unc ional g oup is u ilized ubiqui ously in chemical
syn hesis such as in he Mi sunobu eac ion whe e i o ms pa
o he die hyl azodica boxyla e (DEAD) eagen , also as pa o
he adical ini ia o compound AIBN. Addi ionally, azo dyes,
o med by azo coupling eac ions, cons i u e o 60−70% o all
dyes used in he ood and ex ile indus ies.
85
The mos common me hod o he p epa a ion o a yl azo
compounds employs he use o highly ene ge ic diazonium
sal s.
86
The azoxy and azo g oups can also be accessed by
oxida i e c oss-coupling o anilines, o by educ i e coupling o
ni obenzene compounds.
83,87
T adi ional oxida i e coupling
me hods equen ly employ en i onmen ally un iendly
oxidan s, including pe oxides, coppe o sil e -based sal s,
and ansi ion me al-based ca alys s, such as u henium
complexes.
88
Gi en he signi ican impo ance o hese unc ionali ies, i is
necessa y o de elop al e na i e sus ainable me hods o hei
syn hesis. I has ecen ly been epo ed, ha oxida i e coupling
can be pe o med enzyma ically using AzoC,
89
which is no an
NNzyme bu allows he o ma ion o an N−N bond ia a
spon aneous adical mechanism. Addi ionally, p og ess was
made in he educ i e coupling o ni obenzene compounds
using a pho oenzyma ic app oach, which highligh s he
impo ance o ansi ioning o bio echnological me hods.
90
2.3. Diazo G oup. Diazo compounds (R2C�N+�N−),
hough ela i ely a e in na u e, play a signi ican ole in
seconda y me aboli e biosyn hesis due o hei unique
chemical p ope ies. In he 1950s, he i s diazo compounds
disco e ed included modi ied α-amino acids like azase ine
(18), 6-diazo-5-oxo-L-no leucine (DON, 19) and azo omycin,
which we e ob ained om di e en S ep omyces s ains.
91−93
Since hen, esea ch on diazo compounds has con inued,
esul ing in he disco e y o in iguing bioac i e molecules like
kinamycin D (20) and lomai i icin A (21). S udies on hese
diazo luo ene-based an i umo an ibio ics ha e e ealed he
c ucial ole o diazo g oups in hei biological ac i i y.
94
These
compounds a e bes known o hei in ol emen as e sa ile
in e media es in mode n syn he ic o ganic chemis y, such as
in he Bam o d−S e ens,
95
Doyle−Ki mse
96
and he Buch-
ne −Cu ius−Schlo e beck
97
eac ions.
98
They can also be
gene a ed in si u om p ecu so s such as hyd azones.
99
Due o hei ylide na u e, he he mal s abili y, eac i i y and
liabili y owa d s ong acids is an impo an conce n when
choosing o use diazo compounds in syn he ic ou es. Thei
eac i i y can be uned howe e by changing he subs i uen on
he diazo ca bon. Fo simple alipha ic and elec on dona ing
g oups, he unc ional g oup is uns able bu wi h a oma ic and
subs i u ed elec on wi hd awing g oups, i becomes mo e
s able. Al hough hey a e inc edibly use ul in e media es, hei
sa e y isk means ha ew a e made comme cially a ailable.
Especially o he highly haza dous and explosi e diazo-
me hane which when used in indus y equi es con inuous
p oduc ion and consump ion.
100
The e o e, in si u consump-
ion in a con inuous p ocess is key o main aining sa e y and
also u ilizing he unc ionali y. The bioca aly ic pa hways
p esen he e-in allow o sa e con ol o hese in e media es
while accessing a di e se ange o chemis ies.
101
2.4. N-Ni oso G oup. N-ni o g oups can be classi ied
in o se e al ypes based on he a oms o which he ni oso
g oup (NO) is di ec ly a ached.
102
The p esen e iew will
ocus on N-ni osamine compounds, in which he N−NO
bond o ma ion is ca alyzed by an NNzyme. N-ni oso
compounds can be u he classi ied as N-ni osamines
(R1N(−R2)−N�O), N-ni osohyd oxylamines (R1N-
(−OH)−N�O), and N-ni osamides (R1C(�X)N(−R2)−
N�O) wi h he de i a i es, N-ni osoca bama es, N-ni o-
sou eas and N-ni osoguanidine. Among he a ious bio-
logically ac i e NPs, he N-ni oso compounds ep esen he
la ges g oup, including L-alanosine (22), g amibac in (23),
(−)- agin (24), and chalkophomycin (25) due o hei abili y
o coo dina e me als and ac as me allopho es.
103
N-ni oso compounds ha e been demons a ed o possess
mu agenic and ca cinogenic p ope ies, whe eby hey can be
bioac i a ed, o ming a ca bonium ion ha acili a es alkyla ion
o di e se cellula mac omolecules.
104
Consequen ly, hey
ep esen a p omising class o compounds o po en ial
exploi a ion in chemo he apy.
105
One p ominen example is
s ep ozocin (5), which belongs o he N-ni osamides g oup
and exe s i s ac i i y by gene a ing elec ophilic DNA
alkyla ing agen and ni ic oxide. The con en ional syn he ic
me hodologies o accessing N-ni oso compounds ypically
employ a ange o ni osa ing eagen s, including ni osyl
halides (e.g., NOCl, NOB ), ni ogen oxides (e.g., NO, N2O3,
N2O4), ni o compounds (e.g.,CH3NO2), and ni i e sal s (e.g.,
alkyl ni i es, NaNO2); howe e , a majo i y o hem a e
oxic.
102,106
The disco e y o N-ni osamine impu i ies in ac i e
pha maceu ical ing edien s (APIs) has become a global
conce n. In 2021, he Eu opean Medicines Regula o y
Ne wo k es ablished he Ni osamines Implemen a ion O e -
sigh G oup (NIOG) o o e see isk and implemen mi iga ion
s a egies. One key app oach in ol es elimina ing hese oxic
ni osa ing agen s by modi ying sol en s o syn he ic ou es.
Bioca alysis p esen s a p omising al e na i e, o e ing a mo e
sus ainable syn hesis pa hway ha a oids he use o ni osa ing
agen s al oge he , hus p o iding enhanced con ol and
educing he isk o ca cinogenic impu i ies in pha maceu ical
p oduc s.
107
2.5. T iazenes and T iazoles. The iazenes a e linea
molecules comp ising h ee adjacen ni ogen a oms (RN�
N−NR2). A oma ic compounds con aining iazenyl g oups,
such as 1,2,3- iazole, ha e a p o ound impac on he ields o
syn he ic o ganic and medicinal chemis y.
108
In con as , he
chemis y o inyl and alkynyl iazenes was p e iously an a ea
o limi ed in es iga ion. Nowadays, i has become e iden ha
inyl and alkynyl iazenes a e highly in iguing compounds
wi h dis inc i e eac i i y.
109
T iazenes ha e been in es iga ed
o hei po en ial an icance p ope ies, employed as
p o ec ing g oups in NPs syn hesis and combina o ial
chemis y, and u ilized o gene a e no el he e ocycles. Thei
biological ac i i y is de i ed om hei capaci y o o m
diazonium sal s, which a e capable o alkyla ing DNA.
Addi ionally, iazenes can be ans o med in o a ange o
eac i e g oups ollowing he applica ion o sui able
eagen s.
110
T iazenes a e easily syn hesized om eadily
a ailable anilines o alkyl azides. Dialkyl iazenes can be
ob ained om he eac ion o an alkyl azide wi h he
app op ia e G igna d o alkyl li hium eagen .
111
I is
no ewo hy ha mos iazene syn heses we e op imized
be o e he 1930s and ha some o he mos use ul p epa a i e
ou es ha e ha dly changed in he las 100 yea s since hei
ini ial disco e y. Howe e , his longe i y has also mean ha
he sus ainabili y o he p ocess has no been eassessed.
112
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Gi en he signi icance o iazoles, he syn hesis o hese
compounds has been he subjec o conside able esea ch.
Following he ad en o he 1,3-dipola cycloaddi ion be ween
azide and e minal alkyne, also known as he Wa son
cycloaddi ion,
113
he Cu-ca alyzed azide−alkyne cycloaddi ion
eac ion (CuAAC), as p oposed by Sha ples, became he
p edominan me hod o syn hesizing 1,2,3- iazoles, mo e
commonly e e ed o as “click chemis y”.
114
Al hough
enzyma ic me hods a e unde ep esen ed in he e ocyclic
chemis y, he e a e a ew examples o newly disco e ed
BGCs ha ha e he po en ial o be explo ed in his ield, which
a e u he discussed in Sec ions 4.4 and 4.5.
T adi ional syn he ic app oaches o N−N bond o ma ion o
hese unc ional g oups o en in ol e ha sh condi ions, oxic
eagen s, and a endency owa d undesi able selec i i y. This
p esen s signi ican obs acles o achie ing e icien , con olled
N−N coupling eac ions. NNzymes, howe e , o e a
p omising al e na i e by ci cum en ing many o he challenges
while opening new a enues o he p oduc ion o complex N−
N unc ional g oups. Gi en his, a ho ough unde s anding o
he biosyn he ic p ocesses is undamen al o ad ancing ou
knowledge o chemical bonding and eac i i y. The in eg a ion
o bioca alysis in o syn he ic me hodologies holds he po en ial
o e olu ionize N−N bond cons uc ion, p o iding a mo e
sus ainable and selec i e app oach o accessing hese unique
unc ional g oups.
3. BIOSYNTHESIS OF N−N BOND-CONTAINING
FUNCTIONAL GROUPS CATALYZED BY NNZYMES:
GENERAL REACTION MECHANISMS AND
DIVERSITY
A di e se a ay o N−N bond-con aining unc ional g oups a e
biosyn hesized by known o hypo he ical NNzymes, o which
a comp ehensi e o e iew is gi en in Table 1, ca ego ized
acco ding o he unc ional g oup hey cons uc . In gene al,
NNzymes can be di ided in o heme-dependen , heme-
oxygenase−like dii on oxidase and oxygenase (HDO), and
cupin-dependen bioca alys s, bu also bioca alys s ha belong
o uncha ac e ized s uc u al g oups. Among he ela i ely
small bu well-es ablished g oup o heme-dependen enzymes,
he mos well-known ep esen a i e is he PZS K zT.
76
In
con as , he heme-like p o eins lack heme occupancy bu
coo dina e i on in he icini y o hei ac i e si e. The cupin
old is ound in one o he mos e sa ile p o ein amilies and
has been linked o he o ma ion o N−N bonds in nume ous
examples. The majo i y o he cupin-dependen bioca alys s
con ain i on in hei ac i e si e. Howe e , he cupin domain
can also coo dina e me al ions such as zinc, coppe , cobal o
manganese.
115
One pa icula ly illus a i e example is he
p o ein SznF/S zF om S ep omyces ach omogenes subsp.
s ep ozo icus, which possesses one HDO cen al domain and
a C- e minal monoi on cupin domain.
116
Mo eo e , o he
majo i y o NNzymes o hypo he ical enzymes in ol ed in N−
N bond o ma ion, no s uc u al in o ma ion is cu en ly
a ailable. Ne e heless, hese enzymes a e in ol ed in highly
in iguing biosyn he ic p ocesses ha make hem a subjec o
conside able in e es . Despi e he ema kable di e si y o N−N
bond-con aining NPs, NNzymes ha e been iden i ied o he
cons uc ion o speci ic unc ionali ies, including hyd azines,
diazo- and ni oso- g oups, iazenes and iazoles (Figu e 2).
This e iew will p o ide a de ailed desc ip ion o hese
bioca alys s.
The biosyn hesis o hese unc ional g oups can be achie ed
by ei he an in a- o in e molecula mechanism. The na u e o
his mechanism can be de e mined by he p esence o absence
o an ex e nal ni ogen dono . In addi ion, nume ous
mechanisms o NNzymes in ol e he o ma ion o an N−N
bond h ough he addi ion o ni ogen oxide species, including
ni ic oxide (NO), ni ous acid (HNO2), ni i e (NO2−),
ni a e (NO3−2), and he hyd oxylamine g oup (−NHOH).
These elec ophilic moie ies can be eadily a acked by he
second nucleophilic ni ogen a om o an amine g oup (R−
NH2), in a mo e gene al class o comp opo iona ion
(synp opo iona ion) eac ions,
117
he eby o ming he N−N
bond. No able examples o NNzymes ha exhibi his
mechanism a e K zT, C eM, T i28, T i17, among o he s,
which a e discussed in he ollowing sec ions. An addi ional
co e s a egy ha esul s in he o ma ion o N−N bonds
in ol es he ea angemen o he a ge molecule in he
icini y o he ac i e si e in he cupin domain, ul ima ely
leading o he o ma ion o an N−N bond. The mos
ex ensi ely s udied ep esen a i e ca alyzing a ea angemen
eac ion is SznF/S zF, while Spb40 can be included in bo h o
hese wo g oups acco ding o he p oposed eac ion
mechanism (Scheme 5). Ano he app oach o he biosyn hesis
o N−N bond-con aining NPs in ol es he spon aneous
ecombina ion o ansien ni ogen adicals wi hou he
in ol emen o NNzymes, as epo ed in he biosyn hesis o
azoxymycins,
118
in which he nonheme dii on oxygenase AzoC
(UniP o ID: A0A0K0PIV3) gene a es he ni oso p ecu so s
equi ed o N−N bond o ma ion (Figu e 3).
119
Howe e , he
o ma ion o N−N bonds ia a adical-media ed p ocess will
no be u he discussed he ein.
4. FUNCTIONAL GROUP DIVERSITY ACCESSIBLE TO
NNZYMES
4.1. Hyd azine Func ional G oup. 4.1.1. In amolecula
Hyd azine Fo ma ion ia Heme-Dependen Pipe aza e
Syn hases. Pipe azic acid (sys ema ic name: (S)-hexahyd o-
py idazine-3-ca boxylic acid; L-Piz, L-4) is a noncanonical,
seconda y α-hyd azino acid con aining a hyd azine bond in i s
1,2-diazinane he e ocyclic side chain. A la ge numbe o
bioac i e molecules a e de i ed om he s uc u al sca old o
Piz, which a e ound in hund eds o non ibosomal pep ide
syn he ase (NRPS)-de i ed seconda y me aboli es,
11
many o
which exhibi po en biological ac i i ies, such as he an icance
agen s himas a in,
149
luzopep ins
150
and depsidomycins,
151
he
an ibio ics monamycin
152
and ma lys a in,
153
o he an i ungal
ku zne ides (26).
154,155
Ano he example o Piz-con aining
biologically ac i e compounds is he KRAS G12D inhibi o
RMC-9805, which has ecen ly been in oduced in o Phase 1
clinical ials.
156
In 2012, he BGC o ku zne ides was
iden i ied
157
and i was ound by Neumann e al. ha an
FAD-dependen N-hyd oxylase K zI (UniP o ID: A8CF85) is
c i ical o he biosyn hesis o L-4 (Scheme 2). K zI is ac i e
owa d L-o ni hine (L-27) o p oduce a hyd oxylamine
in e media e, N5-hyd oxy-L-o ni hine (L-28).
158,159
The o -
ma ion o he N−N bond emained elusi e un il Du e al.
iden i ied a heme b-dependen syn hase K zT (o iginally
named o 4 in he ku zne ide BGC) ha cons uc s he
hyd azine bond o L-Piz in Ku zne ia sp. 744 (Scheme 2).
76
Mo e speci ically, he K zI-ca alyzed “ac i a ion” o L-27
yields L-28, which is he subs a e o a ing-closing
condensa ion ca alyzed by K zT, yielding he hyd azine bond
in L-4 (Scheme 2). This enzyma ic pai o N-hyd oxyla ing
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Table 1. Lis o Pu a i e and Con i med NNzymes Co e ed in This Re iew
P o ein name Accession numbe Na u al p oduc O ganism Func ional g oup MiBig En y
Pu a i e o
Con i med
NNzyme
a
e
In amolecula hyd azine g oup o ma ion
K zT UniP o ID: A8CF72 ku zne ides (26)Ku zne ia sp. 744 Hyd azine BGC0000378 Con i med
(in i o)
76
S aC UniP o ID: D3U9Y3 sangli eh in A S ep omyces la eolus Hyd azine BGC0001042 Con i med
(in i o)
120
Hm C UniP o ID: D9WMP1 himas a in S ep omyces himas a inicus
ATCC 53653
Hyd azine BGC0001117 Con i med
(in i o)
80
PadO UniP o ID: U5YN79 padanamides S ep omyces sp. RJA2928 Hyd azine - Con i med
(in i o)
80
Luz13 NCBI ID: UKU09931.1 luzopep ins Ac inomadu a luzonensis Hyd azine - Con i med
(in i o)
121
Ko 13 - ko komicins Mic omonospo a
sp. ATCC 55011
Hyd azine - Pu a i e 121
M PizS NCBI ID: WP_230415462 unknown Mic omonospo a a apacensis Hyd azine - Con i med
(in i o)
77
SpPizS NCBI ID: WP_111501290 unknown S ep acidiphilus pinicola Hyd azine - Con i med
(in i o)
77
PipS NCBI ID: WP_030722408.1 unknown S ep omyces g iseus subsp. g iseus Hyd azine - Con i med
(in i o)
79
PAI2 UniP o ID: A0A5B7 V5A7 unknown S ep omyces sp. YIM 121038 Hyd azine - Con i med
(in i o)
78
Ma F NCBI ID: WP_240489931 ma lys a ins Ac inomadu a a amen a ia DSM
43919
Hyd azine BGC0001443 Con i med
(in i o)
80
XF36 UniP o ID: A0A0M4QM67 ge umycins Pseudonoca dia
sp. HH130629-09
Hyd azine - Con i med
(in i o)
80
In e molecula ATP-dependen hyd azine g oup o ma ion
Spb40 UniP o ID: A0A1L7NQI6 s56-p1 (15)S ep omyces
sp. SoC090715LN-17
Hyd azine BGC0001764 Con i med
(in i o)
66
T i28 Unip o ID: I2MZC8,
UniP o ID: A0A4P8XS63
iacsins (17)S ep omyces sukubensis Ki asa o-
spo a au eo aciens
Hyd azine BGC0001983 (Ki asa ospo a au eo aciens) Con i med
(in i o)
68
Aza12/AzaE UniP o ID: A0A1G6ZJC9 azase ine (18)Glycomyces ha binensis
ATCC 43155
Hyd azine - Con i med
(in i o)
122−124
SFaza12/AzsN NCBI ID: WP_108953673.1 azase ine (18)S ep omyces agilis Hyd azine - Con i med
(in i o)
Py N/P J UniP o ID: A0A516ELE7 py azomycin (1)S ep omyces candidus Hyd azine - Con i med
(in i o)
8
Fo J Unip o ID: QTK22492,
NCBI ID:
WP_051869537.1 and
WP_012180808.1
o mycin (16)S ep omyces kaniha aensis,S ep-
omyces esis omyci icus,Salinis-
po a a enicola CNS-205
Hyd azine - Con i med
(in i o)
67
Apy9 NCBI ID: BDC79915 ac inopy idazinone A, B (55,54)S ep omyces
sp. MSD090630SC-05
Hyd azine - Con i med
(in i o)
125
Po 11 NCBI ID: WP_057724816.1 unknown Pseudomonas o ien alis Hyd azine - Con i med
(in i o)
125
In e molecula ni i e-dependen hyd azine g oup o ma ion
FzmP UniP o ID: U5YN85 os azinomycin (65)S ep omyces sp. Hyd azine BGC0000937 Pu a i e 126
KinJ UniP o ID: A0A385LMJ2 kinamycins (20)S ep omyces mu ayamaensis Hyd azine BGC0000236 Pu a i e 45
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Table 1. con inued
P o ein name Accession numbe Na u al p oduc O ganism Func ional g oup MiBig En y
Pu a i e o
Con i med
NNzyme
a
e
In e molecula ni i e-dependen hyd azine g oup o ma ion
Alp1J UniP o ID: Q1RQT7 p ekinamycin S ep omyces ambo aciens s ain
ATCC 23877
Hyd azine - Pu a i e 182
Lom29 UniP o ID: A0A059UDU0 lomai i icins (21)Salinispo a opica CNB-440 Sal-
inispo a paci ica DPJ-0016 and
DPJ-0019
Hyd azine BGC0000241 (Salinispo a opica)
BGC0000240 (Salinispo a paci ica)
Pu a i e 128,129
ORF38 Unip o ID: F6K0Z1 luos a ins (71) uncul u ed bac e ium
BAC AB649/1850
Hyd azine BGC0001596 (S ep omyces albus)
BGC0000223 (uncul u ed bac e ium)
BGC0001904 (Mic omonospo a osa ia)
Pu a i e 129
Azoxy unc ional g oup ia hyd azine in e media es
VlmO UniP o ID: Q84F35,
Unip o ID: E4N6B1
alanimycin (2)S ep omyces i idi aciens Ki asa-
ospo a se ae
(s ain ATCC 33774)
Hyd azine - Con i med
(in i o)
130
KaO NCBI ID: BBC93011.1 KA57-A S ep omyces ochei 7434AN4 Hyd azine - Pu a i e 130
ElaO NCBI ID: WP_189274298.1 elaiomycins S ep omyces a a us
NRRL-16927
Hyd azine - Pu a i e 130
AzdO BGC ID: BGC0002805 azody ecins (78)S.mi abilis P8-A2 Hyd azine - Con i med
(in i o)
131
Ady6 NCBI ID: BDI55413.1 and
BDI55430.1
azody ecins (78)S ep omyces sp. A1C6 and
S ep omyces sp. RM72
Azoxy - Pu a i e 132
Diazo unc ional g oup
C eM UniP o ID: A0A0K2JLU1 c emeomycin (3)S ep omyces c emeus Diazo BGC0001295 Con i med
(in i o and
in i o)
133
AzpL NCBI ID: WP_157538045.1 alazopep in (86)Ki asa ospo a aza ica S ep acidi-
philus g iseoplanus
Diazo BGC0002536 (Ki asa ospo a aza ica)
BGC0002457
(S ep acidiphilus g iseoplanus)
Con i med
(in i o)
134
Aha11 NCBI ID: UMM61389.1 asikamides A-C (88−90)S ep omyces asikensis Diazo BGC0002661 Con i med
(in i o)
135
SpiA7 NCBI ID: WP_189300626.1 spinamycin (93)S ep omyces albospinus Diazo - Con i med
(in i o)
136
Pzm18 NCBI ID: UUJ74630.1 penzoemycins A and B (98,99)S ep omyces sp. SCSIO 40020 Diazo - Pu a i e 137
A aA6 NCBI ID: BDI54813.1 a enalumic acid (95)S ep omyces sp. RI-77 Diazo - Con i med
(in i o)
138
CmaA6 Unip o ID: W5W4E6 p-couma ic acid (104)Ku zne ia albida DSM 43870 Diazo - Con i med
(in i o)
139
NapB4 UniP o ID: A7KGZ4,
Unip o ID: A7KH22
azame one (84)S ep omyces aculeola us S ep o-
myces sp. CNQ-525
Diazo BGC0001079 (S ep omyces aculeola us)
BGC0000652
(S ep omyces sp. CNQ-525)
Pu a i e 10
Ni osamide (N-ni osou ea) unc ional g oup
SznF/S zF UniP o ID: A0A411MR89 s ep ozocin (5)S ep omyces.ach omogenes
subsp. s ep ozo icus
N-Ni oso-amides BGC0002313 (SznF) BGC0002294 (S zF) Con i med
(in i o)
116,140
Ni osohyd oxylamine unc ional g oup
Aln AUnip o ID: A0A6B9JBV1 L-alanosine (22)S ep omyces alanosinicus N-Ni oso-hyd oxylamines - Pu a i e 141,142
G bD UniP o ID: B1G5G9 g amibac in (23)Pa abu kholde ia g aminis Pa a-
bu kholde ia caledonica
N-Ni oso-hyd oxylamines BGC0001999 (Pa abu kholde ia g aminis)
BGC0002563
(Pa abu kholde ia caledonica)
Pu a i e 143−145
HamA and/o HamE UniP o ID: A0A144VC93,
UniP o ID: A0A1 V2W1F5
(−)- agin (24) aldiazen (116)Bu kholde ia cenocepacia H111 N-Ni oso-hyd oxylamines BGC0001599 Pu a i e 146
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monooxygenase (NMO) and PZS is also widely dis ibu ed in
o he BGCs esponsible o he biosyn hesis o a ious Piz-
con aining molecules, wi h some no able K zI-K zT homolo-
gous pai s being S aB (UniP o ID: D3U9Y2)-S aC in he
biosyn hesis o sangli eh in A,
160
PadN (UniP o ID:
U5YL02)-PadO in he biosyn hesis o padanamides,
161
Hm M (UniP o ID: D9WMQ4)-Hm C in he biosyn hesis
o himas a in,
162
Ko 17-Ko 13 in he biosyn hesis o
ko komicins,
121
and Luz17 (NCBI ID: UKU09924.1)-Luz13
in he biosyn hesis o he luzopep ins.
121
This enzyma ic pai
does no always appea as wo dis inc genes, as Hu e al.
showed ha na u ally occu ing didomain NMO-PZS chime ic
enzymes exis which can p oduce L-4 in ac inobac e ia and
also in i o om L-27.
120
The disco e y o he K zI-K zT pai
in Piz monome p oduc ion has enabled he usage o a ge ed
me abolomics,
163
and genomic signa u e-based sc eening
me hods
164
o iden i y no el Piz-con aining na u al p oduc s.
Combined wi h speci ic p oduc iden i ica ion echniques like
15N NMR-based sc eening o Piz,
164,165
hese app oaches ha e
g ea ly simpli ied he iden i ica ion o Piz-p oducing o ganisms
and Piz-con aining NPs, and al eady led o he disco e y o
p e iously un epo ed an icance agen s such as inca na apep-
in B
165
and pe icho ins.
163
Despi e hese ad ances, s uc u al and mechanis ic knowl-
edge on how N−N bond o ma ion is achie ed by PZSs is s ill
e y limi ed. Size-exclusion ch oma og aphy expe imen s
e ealed ha K zT o ms dime s in solu ion, and s uc u al
p edic ions sugges homodime s wi h wo symme ical ac i e
si es (Scheme 3A).
76
F om he analysis o he PZS consensus
sequence and subsequen mu agenesis s udies, Du e al. ound
he conse ed esidue H65 in K zT o be c ucial o heme
binding and i s ca aly ic ac i i y (Scheme 3A).
76
Mo e
speci ically, hey p edic ed he heme i on o ac as a Lewis
acid, inducing pola iza ion o he hyd oxylamine bond o he
ac i a ed subs a e L-28 o allow a nucleophilic a ack by he
α-Non he δ-N o occu , elimina ing wa e in he p ocess
(Scheme 3B). Ou own s udies iden i ied he esidue C197 in
K zT o o m a dime -linking disul ide bond, being a leas
pa ly esponsible o dime iza ion o ha speci ic PZS.
77
Howe e , he C197S mu a ion does no nega i ely in luence
he ca aly ic ac i i y o K zT, and no ably, ce ain homologues,
such as S aC, na u ally lack a cys eine esidue in hei C-
e minal egion.
120
Thus, nei he a cys eine a ha posi ion no
a disul ide bond a e essen ial o unc ional PZS. Apa om
he na i e subs a e, K zT has been shown o exhibi some
p omiscui y, being sligh ly ac i e on D-28 and N4-hyd oxy-L-
diaminobu anoic acid (L-29) as well, yielding D-4 and 5-aza-L-
p oline (L-30), espec i ely.
76
Enzyme p o iling expe imen s
conduc ed on K zT e ealed p e e ed op imal eac ion
condi ions a 30 °C and low sal concen a ions, and a pH
op imum a ound 9, which po en ially bene i s dep o ona ion o
he α-amine o he subs a e, inc easing nucleophilici y.
77
Homologous PZS om Mic omonospo a a apacensis, M PizS
(52% sequence iden i y o K zT), S ep acidiphilus pinicola,
SpPizS (59% sequence iden i y o K zT), Ac inomadu a
a amen a ia DSM 9954, Ma F and Pseudonoca dia sp.
HH130629-09, XF36, we e s udied in i o as well and we e
shown o exhibi PZS ac i i y on L-28.
77,80
Recen ly, a mo e
de ailed p edic ion o he eac ion mechanism eme ged
(Scheme 3C) when Higgins e al. sol ed he c ys al s uc u e
o a PZS PipS om S ep omyces g iseus subsp. g iseus NRRL F-
5144 (54% sequence simila i y o K zT).
79
Table 1. con inued
P o ein name Accession numbe Na u al p oduc O ganism Func ional g oup MiBig En y
Pu a i e o
Con i med
NNzyme
a
e
Ni osohyd oxylamine unc ional g oup
ChmM UniP o ID: QNQ35080 CuII-chalkophomycin (25)S ep omyces sp. CB00271 N-Ni oso-hyd oxylamines - Pu a i e 103
T iazene unc ional g oup
T i17 UniP o ID: A0A7G3URI3,
UniP o ID: A0A4P8XUW1
iacsins (17)S ep omyces sukubensis
NRRL 18488 Ki asa ospo a
au eo aciens ATCC 10762
T iazene BGC0001983 (Ki asa ospo a au eo aciens) Con i med
(in i o)
68
T iazole unc ional g oup
P nB/8-AzgE UniP o ID: A0A6G9KGS5/
A0A7G3ZQC3
8-azaguanine (126)S ep omyces pa hocidini
ATCC 14510
1,2,3-T iazole BGC0002508 Pu a i e 147,148
a
Con i med ac i i y e e s o N−N bond o ma ion ca alyzed by he indica ed enzyme, bu has no necessa ily been con i med o he na i e subs a e wi hin he biosyn hesis o he espec i e NP.
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Hyd azinosuccinic acid (70) no only plays a ole in he
biosyn hesis o 65 bu has been p oposed o also se e as key
in e media e in he NPs kinamycins (20), lomai i icins (21),
luos a in C (71) and nenes a in C (72). Following
compa ison s udies in he whole genome o he p oduce
o ganisms o hose NPs, i became appa en ha despi e hei
s uc u al di e ences wi h 65, hey sha e a se o homologous
genes. Speci ically, in he genome o S ep omyces mu aya-
maensis ATCC 21414, p oduce o 20, a homologue o FzmP
called KinJ is p oposed o acili a e he N−N bond
o ma ion.
45,126
A e y ecen s udy on he biosyn hesis o
he same amily o NPs showed ha he inal diazo g oup is
ins alled a e he backbone o he NP has been cons uc ed by
a p o ein called AlpH (PDB ID: 8H3T).
182
AlpH is an O-
me hyl ans e ase-like enzyme ha in oduces he en i e L-
glu amylhyd azine in e media e (gluN2H3) in o he backbone
o 20. The o igin o he N−N bond in he in e media e
gluN2H3is p oposed o be gene a ed by he pu a i e FzmP
homologue, Alp1J. I is no ewo hy ha in he same alp BGC,
he p o eins Alp2F and Alp2G, also homologues o FzmM and
FzmL, we e iden i ied.
127
In he biosyn hesis o lomai i icin
(21), ano he hypo he ical NNzyme was assigned due o i s
sha ed homology wi h FzmP, called Lom29.
128,129,182
Al hough a diazo N−N bond is no always ound in he inal
NPs as in he case o 71 and 72, hei biosyn he ic pa hway will
include a s ep o diazo inco po a ion.
129,183,184
In he
biosyn hesis o 71, an uncha ac e ized enzyme has been
p oposed o acili a e his s ep, iden i ied as ORF38 (Scheme
8).
129
Despi e he e o s o explo e he BGCs o he
a o emen ioned NPs, all pu a i e enzymes ca alyzing 70
o ma ion emain o be biochemically cha ac e ized and
u he in es iga ion is equi ed o con i m hei na i e
unc ion in hese di e en biosyn he ic pa hways. Ne e heless,
he bioca aly ic po en ial o such NNzymes would be e y
use ul, as i would allow he in oduc ion o a e minal
hyd azine om an amine and ni i e.
4.1.2.3. Azoxy-Con aining NP Biosyn hesis ia Hyd azine
In e media es. VlmO is a unique example o memb ane-
bound NNzyme ha is esponsible o he syn hesis o he
hyd azine in e media e (52) in he alanimycin (2) bio-
syn he ic pa hway. The o ma ion o an N−N bond ia VlmO
is chemically analogous o he eac ions ca alyzed by zinc-
dependen hyd azine syn he ases om he cy osol cupin amily
(e.g., Fo J), ye exhibi s no homology wi h hem.
81,130,185,186
Despi e he ac ha he enzyma ic basis esponsible o azoxy
bond o ma ion has emained la gely enigma ic, ea ly esea ch
on he biosyn hesis o 2indica ed ha L- aline (73) and L-
se ine (41) unde go a hyd azine-azo-azoxy pa hway ia a N-
isobu ylhyd oxylamine in e media e (74).
186
This hypo hesis
was con i med when i was ound ha he hyd oxyla ion s ep
can be ca alyzed by a wo-componen , la in-dependen
monooxygenase (VlmH,VlmR).
9,187−190
In he BGC o 2,
VlmA (NCBI WP_014134059.1) ca alyzes he condensa ion
o 74 wi h se yl- RNA o o m an uns able es e in e media e,
O-se yl-isobu ylhyd oxylamine (75).
67,131
Addi ionally, he
heme-like dii on-dependen oxygenase VlmB (UniP o ID:
E4N6B3) was shown o be an essen ial in he biosyn hesis o 2,
accep ing 52 o med by VlmO and con e ing i o he inal
azoxy-con aining NP, ia an azo- (76) and an azoxy-
con aining in e media e (77) (Scheme 9A).
130
I on binding in VlmO is likely media ed by ou essen ial
esidues (D51, H82, H110, and D114) as si e-di ec ed
mu agenesis expe imen s e ealed.
130
These esidues a e
loca ed in a po en ial sol en -accessible ca i y ound in a
s uc u e model o VlmO ha was p edic ed by AlphaFold.
O he ecen ly ound homologues o he NNzyme VlmO and
he oxygenase VlmB ha e been iden i ied in he biosyn hesis o
many azoxy-con aining NPs, such as KA57-A (KaO-KaB
(NCBI ID: BBC93013.1)), elaiomycins (ElaO-ElaB (NCBI
ID: WP_114244573.1)) and azody ecins (78) (AzdO-AzdB
(BGC ID: BGC0002805)).
130
In i o s udies o he cha ac e iza ion o he enzyma ic pai s
ElaO/ElaB and KaO/KaB e ealed ha conse ed s eps mus
be in ol ed in he biosyn hesis o alipha ic azoxy me aboli es.
In addi ion o ha , VlmO/VlmB and hei homologues sha e
lexible subs a e speci ici y, while i was p o en ha hey can
accep subs a es wi h a ious alipha ic chains. In azody ecin
(78) biosyn hesis, he VlmO homologue AzdO ca alyzes an
in amolecula N−N bond o ma ion p oducing a hyd azine
p oduc (79) ha is u he ans o med in o he azoxy-
con aining p ecu so (80) o he inal NP (Scheme 8B).
131
In
addi ion, he memb ane p o eins Ady6 and Ady8 (GenBank
ID: BDI55415.1/BDI55432.1) o he e i in-like supe amily
om S ep omyces sp. RM72 (LC712332) and S ep omyces sp.
Scheme 9. Biosyn hesis o Azoxy-Con aining NPs ia Hyd azine In e media es
a
a
A, Biosyn hesis o alanimycin (2).
130
B, Biosyn hesis o azody ecin A (78).
132
The N−N unc ional g oup di ec ly o med by he NNzyme o
in e es is highligh ed in blue, while o he N−N bonds a e highligh ed in pu ple.
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A1C6 (LC712331) we e p oposed as he enzyme pai
ca alyzing he es e (81) ea angemen o he hyd azine
in e media e (79) and subsequen ly o he azoxy-con aining
molecule (80) (Scheme 9B). The biochemical cha ac e iza ion
o hese enzymes is s ill pending, bu he homology wi h VlmO
suppo s he hypo hesis ha Ady6 may ac as a hyd azine
syn hase.
132
The bioca aly ic ac i i y o VlmO/VlmB and hei
homologues owa d alipha ic azoxy compounds is p omising,
as he hyd oxylamine subs a e is no limi ed o amino acids.
Howe e , as wi h Me RS/cupin hyd azine syn he ases, he
nonhyd oxyla ed amine subs a e is mechanis ically es ic ed
o amino acids. To da e, se e al biologically ac i e NPs ha e
been iden i ied, including cal a inin, azoxybacillin, and
elaiomycin, which possess an i ungal o an ibac e ial ac i -
i ies.
83,191
The in iguing chemical s uc u es o azoxy
compounds and hei di e se biological ac i i ies ha e
p omp ed esea ch in he ield o na u al p oduc chemis y,
o al syn hesis, and biochemis y o iden i y new ou es owa d
no el azoxy compounds.
4.2. Diazo Func ional G oup. One o he main ques ions
a ising in he enzyma ic syn hesis o diazo moie ies in NPs is
he o igin o he ni ogen dono . Explo ing he biosyn hesis o
c emeomycin (3), Sugai e al. iden i ied ni ous acid (69) as
he sou ce o he dis al ni ogen in he diazo g oup in 3.
Ni ous acid is syn hesized ia an enzyma ic pa hway, la e
named as he aspa a e-ni osuccina e (ANS) pa hway, ha
in ol es wo key enzymes: he FAD-dependen monooxyge-
nase C eE (UniP o ID: A0A0K2JL70), which ca alyzes he
i e a i e oxida ion o 67 o ni osuccinic acid (82), and C eD
(UniP o ID: A0A0K2JL82), which con e s 82 o ni ous acid
(69), eleasing uma ic acid (83) (Scheme 10).
192
Based on
he obse a ions made by Win e e al., who es ablished ni i e
(68) as he ni ogen sou ce o he N−N bond in azame one
(84) biosyn hesis,
193
Sugai e al. in es iga ed he genome o
he azame one p oduce S ep omyces sp. CNQ-525. No ably,
hey ound c eE and c eD homologues o ming an ope on a a
di e en locus om he pu a i e azame one BGC. Gi en he
high po en ial o ac inobac e ia o p oduce seconda y
me aboli es, hey analyzed addi ional ac inobac e ial genomes
and ound he ANS pa hway widely dis ibu ed, o en nea
genes o seconda y me aboli e biosyn hesis. They ocused
hei esea ch on examining known BGCs o NPs con aining
N−N bonds, such as he hyd azine-con aining 65, and
iden i ied C eE and C eD homologues, named FzmM and
FzmL, espec i ely, as desc ibed in Sec ion 4.1.2.2. This
sugges s ha ni ous acid (69) could se e as he ni ogen
dono no only in diazo-con aining compounds like 3bu also
in o he N−N bond-con aining NPs, such as 65 and 84.
192
Fu he esea ch con i med he p esence o C eE and C eD
homologues in mul iple BGCs as desc ibed in Table 3 ha also
con ains pu a i e o con i med diazo- o ming enzymes.
136,138
Once he o igin o he ni ogen was es ablished, he nex
ques ion o add ess was whe he hese diazo moie ies we e
o med enzyma ically and which enzymes we e esponsible o
his p ocess. The i s diazo g oup- o ming enzyme disco e ed
was C eM, an enzyme pa o he c emeomycin BGC (c e)
ound by Waldman e al. in he genome o S ep omyces c emeus
NRRL 3241.
10
C emeomycin (3) is a pho osensi i e o-
diazoquinone wi h an ibac e ial and an ip oli e a i e ac i i y
ha was isola ed o he i s ime in 1967.
194,195
C eM,
p edic ed o be a a y acid-CoA ligase o he acyl-CoA ligases,
non ibosomal pep ide syn he ases and luci e ases (ANL)
supe amily, ca alyzes he diazo iza ion o 3-amino-2-hyd oxy-
4-me hoxybenzoic acid (3,2,4-AHMBA) (85) wi h 68 o
p oduce 3bo h in i o and in i o (Scheme 11A). Ini ial
cha ac e iza ion o N−N bond- o ming ac i i y o C eM was
challenging due o he spon aneous o ma ion o he diazo
g oup in 3unde ce ain cul u e condi ions, as obse ed by he
au ho s when es ing di e en cul u e media, some o which
p omo ed his unin ended eac ion.
133
To add ess his issue,
he c eM gene was exp essed and he biosyn hesis o 3
econs i u ed in Esche ichia coli, whose media did no ca alyze
nonenzyma ic diazo iza ion. Howe e , he low p oduc ion
le els and chemical ins abili y o 3hinde ed i s de ec ion in
i o. To u he con i m he ca aly ic ac i i y o C eM, he
au ho s in oduced a mu a ion in o a highly conse ed esidue,
E352, which is known o coo dina e an essen ial Mg2+ ion
equi ed o ATP binding in membe s o he a y acid-CoA
ligase amily. This mu a ion esul ed in he comple e
abolishmen o 3p oduc ion.
133
Ano he diazo-g oup con aining molecule is alazopep in
(86), a NP syn hesized by S ep acidiphilus g iseoplanus and
Ki asa ospo a aza ica.
134,196,197
86 is a ipep ide comp ising
wo molecules o he diazo-con aining amino acid DON (19)
Scheme 10. Fo ma ion o Ni i e (68) h ough he ANS Pa hway
192
Table 3. C eE and C eD Homologs Found in BGCs Rela ed o Diazo-G oup Fo ma ion
Na u al P oduc BGC C eE Homologue (Accession Numbe ) C eD Homologue (Accession Numbe ) e
alazopep in (86) azp AzpE (NCBI ID: WP_035850953.1) AzpD (NCBI ID: WP_035850955.1) 134
asikamides A−C (88−90) aha Aha2 (NCBI ID: UMM61380.1) Aha1 (NCBI ID: UMM61379.1) 135
spinamycin (93) spi SpiED (NCBI ID: WP_189300634.1) 136
penzoemycins A and B (98,99) pzm Pzm12 (NCBI ID: UUJ74624.1) Pzm11 (NCBI ID: UUJ74623.1) 137
a enalumic acid (95) a a A aE (NCBI ID: BDI54816.1) A aD (NCBI ID: BDI54817.1) 138
p-couma ic acid (104) cma CmaE (whole genome GenBank ID: CP007155.1) CmaD (whole genome GenBank ID: CP007155.1) 139
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326
and one molecule o L-alanine (40).
198,199
The an ibio ic and
an i umo p ope ies o 86 a e o in e es om a
pha maceu ical pe spec i e.
197
In 2021, Kawai e al. e ealed
he comple e biosyn he ic pa hway o his compound (azp
BGC) and iden i ied he NNzyme o his pa hway as he
ansmemb ane p o ein AzpL, which uses 68 and 5-oxo-L-
lysine (87) owa d he o ma ion o he diazo in e media e 19,
which is inco po a ed wice in o he inal p oduc (Scheme
11B). The ollowing s ep in his pa hway is he p oduc ion o
N-Ac-DON, om a N-ace yl ans e ase p o ein AzpI (NCBI
ID: WP_035850924.1). Kawai e al. conduc ed a compa a i e
analysis o po en ial AzpL homologues and iden i ied a numbe
o conse ed y osine, se ine and glu ama e esidues ha a e
likely o be in ol ed in he ca aly ic mechanism.
134
Fu he mo e, an alanine sc eening was conduc ed, which
e ealed ha he mu a ion Y93A comple ely abolished he
p oduc ion o N-Ac-DON. Con e sely, he subs i u ion o he
same esidue wi h phenylalanine esul ed in a educ ion in he
o ma ion o N-Ac-DON. These indings indica e ha Y93
plays a pi o al ole in he ca aly ic mechanism o AzpL.
134
Fu he e o s ha e been made o cha ac e ize o he enzymes
o he biosyn he ic pa hway.
200
Diazo g oups a e no always ound in he inal NP, ins ead,
hey equen ly occu in in e media es ha a e subsequen ly
ans o med in o o he N−N bond-con aining unc ional
g oups, such as hyd azones. This is he case wi h asikamides
A-C (88−90), compounds ha ha e a hyd azone g oup linking
he cyclic pep ide backbone o an alkyl 5-hyd oxylan h anila e
Scheme 11. P oposed Biosyn he ic Pa hways In ol ing Diazo-Bond-Fo ming NNzymes
a
a
A, Biosyn hesis o c emeomycin (3).
133
B, Biosyn hesis o alazopep in (86).
134
C, Biosyn hesis o asikamides A-C (88−90).
135
D, Biosyn hesis o
spinamycin (93).
136
E, Biosyn hesis o penzonemycins A−B (98,99).
137
F, Biosyn hesis o a enalumic acid (95).
138
G, Biosyn hesis o p-couma ic
acid (104).
139
The N−N unc ional g oup di ec ly o med by he NNzyme o in e es is highligh ed in blue, while o he N−N bonds a e
highligh ed in pu ple.
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(AHA, 91) moie y. They we e isola ed om S ep omyces
asikensis P46 by Ma e al o he i s ime in 2022.
135
They
iden i ied wo di e en BGCs esponsible o he biosyn hesis
o his molecule. The i s , sk BGC, encodes a non ibosomal
pep ide syn he ase (NRPS) pa hway o assembling he cyclic
pen apep ide sca old and he second BGC, aha, encodes he
genes in cha ge o syn hesizing he alkyl AHA moie y (91).
This clus e encodes genes ha sha e sequence simila i y o
genes om he biosyn he ic pa hway o 3. Aha11 (C eM
homologue) is an ATP-dependen a ylamine-diazo izing
(AAD) enzyme ha pe o ms he diazo iza ion eac ion o
91, o ming he in e media e diazo-AHA (92). Then, he diazo
compound unde goes a nonenzyma ic Japp−Klingemann
eac ion ha couples i wi h he cyclic pep ide gene a ing he
hyd azone-con aining asikamides (88−90) (Scheme 11C).
135
The same esea ch g oup dele ed he aha11 gene and ob ained
h ee new asikamides (I, J and K) ha show di e en s uc u e
o 88-90, in which he 2 subuni s a e connec ed by an
enaminone b idge ins ead o he hyd azone moie y. This
demons a es he implica ion o Aha11 in he enzyma ic
assembly o his junc ion, con i ming i s ole as an NNzyme.
201
I is plausible ha he same biosyn he ic logic employed o
he cons uc ion o he N−N bond o spinamycin (93), an
an i ungal an ibio ic disco e ed in 1966 ha con ains a
hyd azide moie y.
202,203
Kawai e al. isola ed his NP om
S ep omyces albospinus JCM3399.
136
I was demons a ed ha
a diazo in e media e (94) plays a pi o al ole in he
inco po a ion o he hyd azine bond in o he inal p oduc
(Scheme 11D). By que ying ANS pa hway genes (Scheme 10),
he spinamycin biosyn he ic gene (spi) clus e was unco e ed,
which su p isingly con ained C eE/C eD homologues in he
o m o a na u al used p o ein called SpiED (NCBI ID:
WP_189300634.1).
136
In e ms o i s s uc u al composi ion,
93 exhibi s an a yl polyene moie y, which is simila o ha
obse ed in a enalumic acid (95) (Scheme 10F). The la e
was he subjec o a p e ious s udy by he same esea ch g oup,
and ano he NNzyme called A aA6 was iden i ied in he
co esponding BGC.
138
While bo h BGCs display simila i ies,
hey also exhibi di e ences ha e lec he s uc u al a ia ions
obse ed in he inal NPs. An ATP-dependen homologue o
he diazo ase A aA6 was iden i ied wi hin he spi clus e and
named SpiA7. I is hypo hesized ha his enzyme pe o ms
diazo iza ion o he a oma ic amine 6-(3-aminophenyl)-2,4,6-
hep a ienoic acid 96, u ilizing ni i e 68 p oduced by SpiED as
a ni ogen sou ce. This esul s in he o ma ion o uns able
diazonium in e media e 94, ha apidly decomposes in o
cinnamic acid, due o he lack o s abilizing hyd oxy g oup in
he o ho posi ion. The ac i i y o SpiA7 owa d his subs a e
was pos ula ed on he basis o in i o e idence being
inconclusi e. Howe e , i s ac i i y owa d 3-aminocinnamic
acid (97) p o ided a con i ma ion ha SpiA7 is a diazonium-
o ming NNzyme. I is no ewo hy ha SpiA7 is ac i e owa d
anilines lacking a hyd oxyl g oup. This is pa icula ly signi ican
gi en ha o he diazo NNzymes equi e he p esence o a
hyd oxyl g oup o diazo iza ion. Fo example, in he
biosyn hesis o a enalumic acid (95), 88−90 and 3, he
a oma ic subs a es o diazo iza ion enzymes possess a
hyd oxyl g oup a he pa a o o ho posi ion o he amino
g oup o be diazo ized. Following he diazo iza ion, he
au ome iza ion o he hyd oxyl g oup con ibu es o he
s abiliza ion o he diazo in e media e. Howe e , his
s abilizing mechanism is absen in he syn hesis o 93, which
esul s in he low s abili y o he diazo in e media e and i s
subsequen apid ans o ma ion wi hin he biosyn he ic
pa hway. This ans o ma ion may occu spon aneously ia
he Japp-Klingemann eac ion, o i could be enzyme-
media ed, al hough he esponsible enzyme has no ye been
iden i ied.
136
The au ho s employed Japp-Klingemann chemi-
cally o be able o de ec he p oduc o med in he in i o
assays.
136
A simila mechanism o hyd azone moie y cons uc ion was
obse ed in he biosyn hesis o 98 and 99. Recen ly isola ed
om he ma ine o ganism S ep omyces sp. SCSIO 40020,
hese no el molecules possess a hyd azone moie y and a 3-
hyd oxyan h anilic acid (3-HAA) co e. A e isola ion, Liu e
al. iden i ied he pu a i e gene clus e encoding he enzyma ic
machine y o i s biosyn hesis (pzm BGC). The 3-HAA co e
was p oposed o be de i ed om a cho isma e pa hway
in ol ing he genes pzm6 o pzm9 (NCBI BGC:
ON345781.1). This clus e also con ains a C eM homologue
iden i ied as he AMP-binding p o ein Pzm18, ha is p oposed
o inco po a e ni a e 68 wi h a pu a i e subs i u ed benzoic
acid in e media e (100), leading o he o ma ion o he diazo
moie y in 101. Finally, he nonenzyma ic Japp-Klingemann
coupling eac ion is p edic ed o cons uc he hyd azone
moie y (Scheme 11E). Fu he expe imen al da a a e equi ed
o con i m he ac i i y o all he enzymes in ol ed in his
biosyn he ic pa hway.
137
Un il now, he diazo NNzymes desc ibed in he p eceding
pa ag aphs ha e been shown o c ea e an N−N bond ha
ei he emains in he inal molecule as a diazo moie y o
unde goes u he ans o ma ion o e.g. hyd azone o
py idazine. Howe e , his p inciple is no uni e sally
applicable. In ce ain NPs, diazo in e media es a e o med
ini ially, bu hen unde go a deamina ion p ocess, esul ing in
he elimina ion o he N−N bond as ni ogen gas (N2). This is
he case o a enalumic acid (95), a phenolic acid o iginally
ound in oa plan s, whe e i occu s as a s uc u al mo i in
a enan h amide compounds.
204
This NP was la e isola ed
om he bac e ium Rhodococcus sp. RV157.
205
In hei sea ch
o no el enzyme chemis ies ha exploi 69 de i ed om he
ANS pa hway simila ly o C eM, Kawai and colleagues mined
he genomes and iden i ied he a a clus e in S ep omyces sp.
RI-77.
138
Despi e hei e o s, hey we e unable o isola e 95
om his o ganism, likely because i is a do man seconda y
me aboli e BGC unde he gi en cul u e condi ions. The e o e,
hey exp essed his clus e he e ologously in S ep omyces
albido la us, success ully demons a ing he p oduc ion o he
compound. Du ing he biosyn hesis o 95, an ATP-dependen
diazo ase homologue o C eM, A aA6, pe o ms he
diazo iza ion o an a oma ic amino g oup. Speci ically, he
enzyme pe o ms diazo iza ion o 3-aminoa enalumic acid (3-
AAA) (102) in o 3-diazoa enalumic acid (3-DAA) (103).
This is ollowed by he educ i e enzyma ic subs i u ion o he
diazo g oup wi h a hyd ide, libe a ing N2, ca ied ou by he
enzyme A aA7 (NCBI ID: BDI54815.1) (Scheme 11F). The
au ho s u he pe o med a genome mining which e ealed
ha mo e han 100 ac inobac e ia ca y BGCs simila o he
a a clus e , indica ing ha his NP o i s de i a i es may be
p oduced by a wide a ie y o ac inobac e ia.
138
Ano he NP wi hou an N−N bond in i s s uc u e bu wi h
a diazo in e media e is p-couma ic acid (104). I s s uc u e is
simila o 95, di e ing only in he leng h o he ca bon chain. I
is a p ecu so in he la onoid biosyn he ic pa hway, no mally
de i ed om phenylalanine o y osine (43).
206
Kawai e al.
employed he a a BGC as a que y o iden i y he cma BGC in
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he genome o he a e ac inomyce e Ku zne ia albida JCM
3240.
139
He e ologous exp ession o he clus e and in i o
enzyme assays demons a ed i s ole in he biosyn hesis o 104.
In his a a-like BGC, an ATP-dependen diazo ase homologue
o A aA6 was ound, named CmaA6. This enzyme ca alyzes
he diazo iza ion o 3-aminocouma ic acid (3-ACA, 105),
o ming he in e media e 3-diazocouma ic acid (3-DCA, 106).
I is no ewo hy ha CmaA6 exhibi ed diazo iza ion ac i i y
wi h 3-AAA (102), he subs a e o A aA6, wi h signi ican ly
highe e iciency han A aA6. Following diazo iza ion,
analogous o he biosyn hesis o 95,106 unde goes a
deni i ica ion ca alyzed by CmaA7, eleasing N2and o ming
104 (Scheme 11G). The eason behind he e olu ion o such a
specialized biosyn he ic pa hway in he seconda y me abolism
o ac inomyce es, which includes diazo iza ion and deni i ica-
ion, emains unclea . This pa hway is u ilized o syn hesize a
common me aboli e, such as 104, despi e he lack o a clea
selec i e ad an age.
139
Diazo g oups can also be u he ans o med in o py idazine
mo i s, as seen in he azame one (84) biosyn hesis. This NP is
a py idazine-con aining compound isola ed om he ma ine
S ep omyces sp. CNQ-766, which belongs o he napy adio-
mycin class o NPs.
207
Win e e al. hypo hesized ha he BGC
esponsible o he biosyn hesis o 84 mus be simila o he
napy adiomycin BGC (nap), p e iously iden i ied in S ep o-
myces sp. CNQ-525 and S ep omyces aculeola us NRRL
18442.
193,208
This g oup also conduc ed eeding s udies,
which sugges ed he po en ial in ol emen o 49 as a ni ogen
Scheme 12. Biosyn hesis o Azame one (84) ia he nap BGC
193,207
,
a
a
The N−N unc ional g oup di ec ly o med by he NNzyme o in e es is highligh ed in blue, while o he N−N bonds a e highligh ed in pu ple.
Scheme 13. SznF/S zF, Essen ial Residues o Ac i i y, and SznF/S zF Mechanism in he Biosyn hesis o S ep ozocin
a
a
A, C ys al s uc u e o SznF/S zF (PDB ID: 6VZY), ep esen ed as homodime ic p o ein.
215
The di e en domains a e depic ed wi h
dis inguished colo s. In blue, he N- e minal domain is depic ed, in g een he HDO cen al domain and in ed he C- e minal cupin domain. The
o ange sphe es ep esen he i on a oms in he ac i e si es. B, Essen ial esidues o ac i i y H407, H409, H448 and Ty 459 in he cupin domain
( op) and E215, H225, E281, H311, D315, and H318 in he mul inuclea cen al domain (bo om). C, P oposed mechanism o SznF/S zF in he
biosyn hesis o s ep ozocin (5).
140,215
The N−N unc ional g oup di ec ly o med by he NNzyme o in e es is highligh ed in blue.
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sou ce o he dis al ni ogen a om in he diazo-con aining
p ecu so o 84, designa ed as SF2415A1 (107).
193
This
hypo hesis was u he suppo ed by he disco e y o C eE and
C eD homologues o ming an ope on a a di e en locus om
he pu a i e azame one BGC.
192
This disco e y implies ha
he diazo moie y obse ed in he in e media e s ages o he
biosyn hesis o 84 could po en ially be o med h ough
enzyma ic p ocesses. Win e and colleagues ha e hypo hesized
ha he pu a i e amino ans e ase NapB3 (NCBI ID:
ABS50480.1) may acili a e he ans e o an amino g oup
o he a oma ic ing o SF2415B1 (108), he eby in oducing
he ini ial ni ogen a om equi ed o subsequen N−N bond
o ma ion. Subsequen ly, an unknown NNzyme can ans e
ano he ni ogen a om o o m he diazo g oup-con aining
p ecu so 107.
25,193
La e on, Waldman e al. iden i ied a
C eM homologue wi hin he BGC o 84 ha could po en ially
be his elusi e NNzyme. To u he in es iga e his, we
conduc ed a BLAST sea ch que ying C eM in he genome o
nap BGC-con aining o ganisms S ep omyces aculeola us and
S ep omyces sp. CNQ-525. This sea ch unco e ed he C eM
homologues (43% iden i y) named NapB4 in bo h o ganisms
(Scheme 12). Rega ding he py idazine ing p esen in he
s uc u e o 84, i has been pos ula ed ha his ing is o med
ia an oxida i e ea angemen o a diazo in e media e.
193,207
In gene al, gi en he eac i i y o diazo g oups, hey a e
ypically p oposed as elusi e eac ion in e media es and in
o ganic syn hesis, as chemical p obes o he modi ica ion o
p o eins and nucleic acids, and as building blocks in he
biosyn hesis o pha maceu ically ele an compounds. The
po en ial o NNzymes o c ea e eac i e diazo in e media es in
syn he ic applica ions was ecen ly demons a ed in i o o
CmaA6. This enzyme o med he diazo g oup ha hen was
used o gene a e phenyldiazene de i a i es ia C−N bond
o ma ion.
209
Apa om he use o diazo compounds as
eac i e in e media es, he p esence o diazo g oups in NPs,
such as 20 and 21, con e s upon hese molecules he abili y o
in e cala e DNA, he eby ende ing hem p omising candida es
o an icance he apies.
4.3. N-Ni oso Func ional G oup. 4.3.1. Ni osamide (N-
Ni osou ea) Func ional G oup. S ep ozocin (5) (s ep ozo-
ocin, o ade name Zanosa ) is a N-ni osou ea-con aining
NP ha was i s isola ed in he la e 1950s om S ep omyces
ach omegenes subsp. s ep ozo icus and documen ed as a new
an ibio ic.
140,210,211
Nowadays, he comme cial o mula ion is
used as an an ineoplas ic d ug o ea panc ea ic cance .
13,212
Al hough 5has been in use o mo e han hal a cen u y, i s
BGC was no iden i ied un il 2019 by he Balskus and Ryan
g oups. Balskus’s g oup sequenced and mined he genome o
S ep omyces ach omegenes subsp. s ep ozo icus NRRL 2697
and ound he szn BGC ha encodes he NNzyme SznF
(UniP o ID: A0A411MR89, Scheme 13 A and B).
140
The
g oup o Ryan iden i ied he esponsible NNzyme, named in
his s udy S zF, in he genome o S ep omyces ach omegenes
subsp. s ep ozo icus NRRL 3125, and named he BGC s z.
116
A e u he in es iga ion o he clus e and in i o
cha ac e iza ion o SznF/S zF i was ound ha his NNzyme
ac s syne gis ically wi h an a ginine-guanidino me hyl ans e -
ase, SznE/S zE (UniP o ID: A0A411EW25), o accep Nω-
me hyl-L-a ginine (109) as a na i e subs a e. SznF/S zF
hyd oxyla es sequen ially bo h o he unme hyla ed ni ogen
a oms o he guanidine g oup, o ming wo hyd oxyl
in e media es, Nδ-hyd oxy-Nω-me hyl-L-a ginine (110) and
Nδ-hyd oxy-Nω-hyd oxy-Nω-me hyl-L-a ginine (111). This di-
hyd oxyla ed in e media e unde goes a ea angemen esul -
ing in he p oduc ion o a N-ni osou ea in e media e (112),
which is hen con e ed u he o 5 ia glycosyla ion, likely
media ed by he enzymes SznH, SznJ, and SznK. The
in e media e 112 can also unde go a nonenzyma ic deg ada-
ion, p oducing ni ic oxide (113) and he deg ada ion
p oduc s 114 and 115 (Scheme 13C).
116,140
I is no ewo hy
ha SznF/S zF is he only NNzyme ha has been iden i ied o
cons uc he N-ni osou ea unc ional g oup. Al hough i was
p e iously specula ed ha he N-ni oso g oup o igina ed om
he in amolecula inco po a ion o 68, simila o all known in
i o N-ni osa ions,
213,214
eeding expe imen s demons a ed
ha S ep omyces ach omogenes subsp. s ep ozo icus inco po-
a es he in ac guanidine g oup o 109 in o he N-ni osou ea
p oduc 112 wi hou u ilizing 15N-labeled ni i e, ni a e, o
ammonium sal s o gene a e he N-ni oso moie y.
140
Conside ing all o he abo e-men ioned, SznF/S zF exhibi s
a unique enzyma ic ac i i y, pe o ming bo h hyd oxyla ion
and N−N bond o ma ion h ough an in amolecula
ea angemen eac ion wi hou equi ing a coupled N-
hyd oxylase o he use o 68. In ligh o he obse a ion ha
112 is o med in he biosyn he ic pa hway o 5, i can be
hypo hesized ha he in e media es o med by SznF/S zF may
ac as dono s o he N-ni osou ea subuni used in
chemo he apeu ic d ugs such as ca mus ine (11) o lomus ine
(12). In addi ion o ha , SznF/S zF comp ises one o he
NNzymes wi h a esol ed c ys al s uc u e (PDB IDs: 6M9R,
6XCV, 6VZY, 6M9S).
215
The X- ay c ys allog aphy e ealed
ha his NNzyme is a homodime composed o h ee domains:
an N- e minal domain, esponsible o in amolecula in e -
ac ions du ing dime iza ion, a heme-oxygenase−like dii on
oxidase and oxygenase (HDO) cen al domain, which ca alyzes
wo consecu i e N-hyd oxyla ions and a C- e minal monoi on
cupin domain, which ca alyzes he inal ea angemen and he
o ma ion o he N−N bond (Scheme 13A).
140,215
The
enzyme is known o possess wo ac i e si es, one in each
i on-con aining domain. Subs i u ion o any o he me al-
binding esidues (E215, H225, E281, H311, D315, o H318)
in he mul inuclea cen al domain wi h alanine esul ed in
aboli ion o SznF/S zF ac i i y (Scheme 13B). Simila ly,
subs i u ions o esidues H407, H409 and H448 in he cupin
domain led o he accumula ion o he dihyd oxyla ed
in e media e 111, wi hou any p oduc o ma ion.
140
In
addi ion, compu a ional analyses and mechanis ic s udies
conduc ed by Chen’s g oup on he HDO and he cupin
domain o SznF/S zF p o ide a basis o u he explo a ion o
his bioca alys .
216,217
Mechanis ic analysis o he HDO cen al
domain ac i i y and he hyd oxyla ion p ocess e ealed ha he
a e-limi ing s ep in he o ma ion o he monohyd oxyla ed
in e media e 110 is a hyd oxyl ebound, whe eas o he
second hyd oxyla ion and he o ma ion o in e media e 111, i
is a hyd ogen abs ac ion.
216
Addi ionally, analysis o he
cupin-media ed ea angemen indica ed ha he esidue
Ty 459 acili a es a p o on ans e essen ial o his ea ange-
men s ep. These indings highligh he c i ical ole o hese
esidues in he enzyme’s ca aly ic ac i i y.
217
4.3.2. Ni osohyd oxylamine Func ional G oup. In he
biosyn hesis o se e al NPs, including L-alanosine (22),
g amibac in (23), (−)- agin (24), aldiazen (116) and
chalkophomycin (25), speci ic genes ha e been iden i ied
ha encode bioca alys s wi h he po en ial o unc ion as
NNzymes. These enzymes a e hypo hesized o ca alyze he
o ma ion o a ni osohyd oxylamine g oup, which is
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subsequen ly inco po a ed in o he inal s uc u e o he
espec i e NPs.
25,146,218
Al hough he ac i i y o hese
bioca alys s emains unde in es iga ion, he s uc u al
simila i y hey sha e wi h SznF/S zF
116
suppo s he
hypo hesis ha hey may indeed unc ion as NNzyme in
each biosyn he ic pa hway.
L-Alanosine (22), a noncanonical amino acid wi h an i i al
and an i umo p ope ies, was o iginally isola ed in 1966 om
S ep omyces alanosinicus ATCC 15710.
219
The BGC espon-
sible o he biosyn hesis o 22 was ecen ly iden i ied and
designa ed as ala
142
and aln.
141
The aln BGC includes he
enzyme Aln A, a pu a i e NNzyme ea u ing a cupin domain
and an A aC-like DNA-binding domain. Aln Ais hypo hesized
o ac as ei he a ansc ip ional egula o o o ca alyze N−N
bond o ma ion ia he cupin ac i e si e, possibly a ge ing N3-
hyd oxy-L-diaminop opionic acid (117) o o m he ni o-
sohyd oxylamine g oup in 22. The hyd oxyla ion o L-
diaminop opiona e (118) is hypo hesized o be ca alyzed by
a pu a i e la in-dependen acyl-CoA dehyd ogenase, AlaD/
Aln G(UniP o ID: A0A6H1Z5U0/A0A6B9JBY3) (Scheme
14A). The o igin o he dis al Nin 22 emains unde deba e
due o con lic ing iso ope eeding s udies sugges ing ei he he
ANS pa hway in ol ing C eD and C eE homologues (AlaJ/
Aln Nand AlaI/Aln M) (UniP o ID: A0A6B9JDZ4 and
A0A6H1Z626) o NOxspecies p oduced by ni a e-ni i e
educ ases as possible sou ces.
141,142
This la e hypo hesis is
u he suppo ed by ecen disco e ies, as newly iden i ied aln
BGCs in o he S ep omyces species lacking ANS pa hway
genes sugges ha he ni a e and ni i e educ ases p esen
may p o ide he dis al ni ogen necessa y o N−N bond
o ma ion.
218
Simila ly, G bD, which possesses a HDO and a C- e minal
cupin domain, is likely esponsible o ca alyzing he N−N
bond o ma ion in he biosyn hesis o 23 using hyd oxy-
a ginine as a subs a e (Scheme 14B).
143
G amibac in (23)
was isola ed in 2018 om Pa abu kholde ia g aminis and
con ains L-g aminine (119) moie y which is used as p ecu so
o cons uc he inal NP.
144
G bE (UniP o ID: B1G5G8),
sha ing homology wi h known a ginine hyd oxylases such as
AglA/AlpD,
220,221
Mh 24,
220−222
and DcsA,
220,222,223
is
specula ed o hyd oxyla e 120 o p oduce he p ecu so
119.
143
The au ho s also hypo hesized ha he N−N bond is
o med be ween Nδand Nωo he guanidinium g oup o
120,
143
in con as o p e ious s udies ha iden i ied L-
o ni hine as he p ecu so .
25
The ole o G bE in 120
hyd oxyla ion and G bD in he oxida i e ea angemen o
hyd oxy-a ginine in he o ma ion o he N−N bond in 119 is
s ill unde esea ch. In e es ingly, G bD and G bE ha e been
used as que ies o iden i y no el BGCs esponsible o he
biosyn hesis o o he g aminine-con aining side opho es, such
as is ellabac ins A and B, which ea u e G bD and G bE
homologues in hei BGCs.
224
The same app oach led o he
disco e y o o he ni osohyd oxylamine-con aining com-
Scheme 14. Biosyn hesis o NPs wi h a Ni osohyd oxylamine Func ional G oup
a
a
A, Biosyn hesis o L-alanosine (22).
141
B, Biosyn hesis o g amibac in (23).
25,144
C, Biosyn hesis o (−)- agin (24) and aldiazen (116).
146
D,
Biosyn hesis o CuII-chalkophomycin (25).
103
.
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pounds like g amibac in B, megapolibac in, plan a ibac in and
gladiobac in.
145
(−)-F agin (24), ano he ni osohyd oxylamine-con aining
NP wi h an i ungal and an ibac e ial ac i i y,
146
is likely also
cons uc ed by he in ol emen o an N-oxygenase (HamC,
UniP o ID: B4EHM6) ha hyd oxyla es L- aline 73 o 121, as
well as a HDO p o ein, HamA, and a polyke ide cyclase o
dehyd a ase, HamE, ha a e hypo hesized o acili a e N−N
bond o ma ion, yielding he key ni osohyd oxylamine
p ecu so 122. HamB, a cupin domain p o ein, migh also
play a ole in he biosyn hesis o 24. In addi ion, he BGC also
encodes genes ha a e in ol ed in aldiazen’s (116)
p oduc ion, a molecule simila o 24 (Scheme 14C). Howe e ,
u he s udies a e needed o con i m hese oles and also he
sou ce o he dis al ni ogen, which since now is hypo hesized
o de i e om NO2−.
25,146
Ano he NP whose biosyn hesis ollows hyd oxyla ion o an
amino acid p ecu so ca alyzed by an N-oxygenase wi h
subsequen ni osohyd oxylamine o ma ion ca alyzed by a
SznF/S zF homologue is CuII-chalkophomycin (25).
103
I s
po en ial applica ions include neu odegene a i e disease ea -
men and CuII-based an i umo he apeu ics.
225
In 2024, he
Balskus g oup cha ac e ized 25 om S ep omyces anula us
ATCC 11523 and elucida ed i s BGC.
226
ChmM and ChmN
(UniP o ID: A0A7H0NKC5) a e key enzymes ha ha e been
s udied o hei po en ial in ol emen in he o ma ion o he
diazeniumdiola e ion. ChmN is a heme-dependen guanidine
N-oxygenase ha con e s 120 o dihyd oxyguanidine (123).
The SznF/S zF homologue ChmM, possesses an HDO
domain bu lacks he occupancy o all he conse ed amino
acids, and a C- e minal cupin domain ha is hypo hesized o
ca alyze he subsequen ea angemen o 123 o he inal N−
N bond o 119. This in e media e is likely u he con e ed o
25 (Scheme 14D). The homology o ChmM and ChmN wi h
G bD and G bE s eng hens he hypo hesis ha he
in e media e o 119 is in ol ed in i s biosyn hesis.
103
Howe e ,
in i o s udies o ChmM agains ee hyd oxya ginine
de i a i es ha e no con i med i s ac i i y, equi ing u he
biochemical cha ac e iza ion in he u u e.
226
Al hough he
ni osohyd oxylamine g oup is cu en ly o limi ed syn he ic
in e es , i s chela ing p ope ies could p o e a aluable asse in
he de elopmen o bioca aly ically a ailable p o ein inhibi o s
ha could ha e po en ial in d ug disco e y.
4.4. T iazene Func ional G oup. The only known g oup
o NPs con aining an N-hyd oxy iazene moie y a e he
iacsins (17), which ep esen a dis inc i e unc ional g oup
wi h p onounced acyl-CoA syn he ase inhibi o p ope ies.
68
In 2018, Twigg e al. disco e ed he BGC esponsible o he
biosyn hesis o 17 in S ep omyces sukubensis NRRL 18488.
178
La e , in 2021, Del Rio Flo es e al. de e mined ha he
enzymes T i28 (Spb40 homologue, 75% sequence simila i y)
and T i17 (C eM homologue, 40% sequence simila i y) o
Ki asa ospo a au eo aciens ATCC 31442 a e esponsible o he
o ma ion o he i s and second N−N bonds, espec i ely, in
he biosyn hesis o 17.
68
The au ho s also iden i ied ANS’
pa hway homologues T i21 and T i16, wi hin he i BGC and
demons a ed hei ole in gene a ing he 68 ha se es as he
dono o he hi d ni ogen a om.
68
As men ioned in Sec ion
4.1.2.1, he hyd azine in e media e 27 in he biosyn hesis o 17
is cons uc ed by he hyd azine syn he ase T i28. In he la e
s eps o his pa hway, he hyd azine moie y 50 is ans o med
o a hyd azone (124) ha ac s as a subs a e o he NNzyme
T i17. This enzyme ca alyzes an ATP-dependen conjuga ion
o 68 and he hyd azone in e media e 124 o gene a e a N-
hyd oxy iazene (125) ha is u he con e ed o he amily o
iacsins (17) (Scheme 15).
68
Though no ha ing been s udied
exhaus i ely ye , he p oposed mechanism desc ibes T i17 i s
ac i a ing 68 by adenyla ion, allowing o nucleophilic a ack
by he dis al hyd azone-ni ogen.
178
Subsequen ly, au ome i-
za ion o he o med N-ni ohyd azide would yield he N-
hyd oxy iazene.
178
Ini ially, T i17 was shown o be speci ic
ega ding he hyd azone moie y and he acyl chain leng h, bu
less selec i e o di e en acyl chain modi ica ions (e.g.,
con e ing undeca-2,4-dien-1-ylidenehyd azine o 17),
68
lead-
ing o i s designa ion as a p omiscuous N-ni osylase. I was
la e shown by Del Rio Flo es e al. ha T i17 is also capable
o o ming azides om alkylhyd azones, hyd azines, py oli-
dines, pipe idines, a ylamines and a ylhyd azines, as well as
diazo compounds om anilines, simila o Aha11 and
C eM.
227
In conclusion, T i17 is a highly e sa ile enzyme ha accep s
a a ie y o unc ional g oups and subs a es, sugges ing i s
po en ial o he bioca aly ic syn hesis o pha maceu ical
compounds. The ecen de e mina ion o he c ys al s uc u e
o T i17 (PDB ID: 9BQ0, 8TF7), has p o ided u he insigh s
in o i s subs a e coo dina ion and ca aly ic mechanism,
he eby opening up he po en ial o u he exploi a ion o
i s p omiscui y.
227
4.5. N−N Bond-Con aining A oma ic He e ocycles.
The mechanisms unde lying he o ma ion o he e ocyclic N−
N bonds in na u e emain ela i ely unde explo ed, hough
hey a e cu en ly he ocus o ongoing esea ch. The
biosyn hesis o hese N−N bond-con aining he e ocycles
commonly ollows a pa hway ha in ol es a hyd azine
Scheme 15. Biosyn hesis o T iacsins (17) ia he i BGC
68
a
a
The N−N unc ional g oup di ec ly o med by he NNzyme o in e es is highligh ed in blue, while o he N−N bonds a e highligh ed in pu ple.
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332
in e media e. This s a egy has been obse ed in he syn hesis
o nona oma ic he e ocycles, such as ac inopy idazinones
(55,56)
125,180
and a oma ic he e ocycles, like he py azole
sca old ound in na u al p oduc s such as py azomycin (1)
8
and o mycin (16)
67
(see Sec ion 4.1.2.1). Despi e he absence
o con i med NNzymes capable o di ec ly o ming a oma ic
he e ocycles such as py azoles, e azoles, py idazines and so
o h, po en ial NNzymes o iazole cons uc ion ha e
ecen ly been iden i ied and will be discussed in g ea e de ail
he ein.
4.5.1. T iazole Func ional G oup. Cu en ly, se e al
pu a i e NNzymes ha e been iden i ied as po en ial candida es
o di ec ly ca alyzing N−N bond o ma ion in a iazole
moie y. These enzymes (P nB o 8-AzgE) a e pos ula ed o be
esponsible o he N−N bond o ma ion in he biosyn hesis o
8-azaguanine (126),
147,148
also known as pa hocidin, a
compound ha was o iginally epo ed as a syn he ic guanine
an agonis and subsequen ly isola ed as a NP syn hesized by
S ep omyces albus subsp. pa hocidicus (also known as
S ep omyces pa hocidini).
228,229
This compound is no able o
i s s uc u e as a guanine analogue, ea u ing a a e na u ally
occu ing 1,2,3- iazole used wi h a py imidine ing. As a
pu ine analogue, 126 unc ions as an an ime aboli e, displaying
a wide a ay o biological ac i i ies, including an icance ,
an i i al and an i ungal p ope ies.
228,230
As p e iously
men ioned, he mos common p ecu so s o N−N bond
o ma ion a e hyd oxylamines and ni ous acid. Howe e , Zhao
e al. e ealed ha ni ogen a om o he iazole moie y can
also be p o ided by a bac e ial ni ic oxide syn hase (NOS),
named P nF (UniP o ID: A0A6G9KI63), ound in he 8-
azaguanine (126) BGC p n.
147
NOS con e s 120 o L-
ci ulline, eleasing ni ic oxide (113).
147
Rega ding he N−N
bond cons uc ion, he au ho s sugges ed ha a NO-de i ed
eac i e ni ogen species migh be esponsible o he assembly
o he iazole moie y in a nonenzyma ic ashion.
147
Bu ,
despi e demons a ing ha his 1,2,3- iazolopy imidine
sca old can be assembled nonenzyma ically, he possibili y o
he exis ence o an NNzyme in he BGC was no excluded. I
was p oposed ha P nB, a small p o ein wi h no close
homologues o p edic ed unc ional domains, may be he
NNzyme, gi en i s classi ica ion as an i on-binding me al-
lop o ein.
147
As seen in p e ious sec ions, me allop o eins ha e
been linked o N−N bond o ma ion in he biosyn hesis o
a ious compounds including s ep ozocin (5),
140
s56-p1
(15),
66
Piz (L-4)
76
and py azomycin (1).
8
I is specula ed
ha P nB may ac owa d 127 using NO, in o de o cons uc
he iazole moie y in molecule 128, howe e , i s ac i i y has
no ye been con i med in i o, necessi a ing u he
in es iga ion o elucida e i s ole (Scheme 16).
147
Hou e al.
iden i ied as well he BGC esponsible o he biosyn hesis o
126 in S ep omyces pa hocidini (8-azg BGC) and ag eed ha a
mo e e icien enzyma ic pa hway should exis o he syn hesis
o he iazole moie y and named his pu a i e NNzyme 8-
AzgE.
148
This BGC has also been iden i ied in o he 8-
azaguanine p oducing species like S ep omyces mo ookaense
DSM 40503
231
and S ep omyces hoyna ensis KCTC 29069,
al hough no 8-azaguanine- ype p oduc has been iden i ied so
a om his s ain.
148
Fu he s udies a e equi ed o con i m
he in ol emen o hese clus e s in he biosyn hesis o 126. In
conclusion, he bioca aly ic po en ial o iazole- o ming
enzymes may be conside able i u he esea ch can
demons a e a b oade applicabili y o a oma ic diamines in
o ho posi ion. This is pa icula ly ele an gi en he ole o
iazoles as building blocks in d ug disco e y, as e idenced by
hei use in he nucleoside analog icag elo .
232
5. CHALLENGES AND OPPORTUNITIES FOR
NNZYMES
Fo a b oad syn he ic applica ion o NNzymes, a la ge
subs a e scope including di e en ca bon backbone s uc u es
and unc ionaliza ions would be ideal. In con as , he subs a e
po olio o mos NNzymes p esen ed in his e iew, i s udied
a all, indica e ha only compounds closely ela ed o he
na i e subs a es a e accep ed.
68,76,78,125,130,135,138
Fo ins ance,
in addi ion o AHA (91) (Scheme 11C), Aha11 also con e s
ela ed es e s o sho e me hyl and bu yl alcohol uni s, bu no
ela ed compounds wi hou a pa a-hyd oxy g oup o 2-amino-
5-hyd oxybenzoic acid. Ne e heless, he na u al subs a e
scope o enzymes in ol ed in N−N bond o ma ion can be
ex ended by enzyme enginee ing, as has been shown o AzoC
in azoxymycin biosyn hesis,
89
making enzyma ic ou es mo e
a ac i e o syn he ic applica ions. In his con ex , i is wo h
men ioning ha wo ecen s udies in es iga e ni o educ ases
and nonspeci ic pe oxygenases (UPOs), espec i ely, o he
gene a ion o he eac i e ni oso and hyd oxylamine
in e media es equi ed o he spon aneous o ma ion o
azoxy g oups.
90,233
In addi ion, ni o educ ases could also be
used o p oduce azobenzene compounds unde pho oca aly ic
condi ions. Howe e , acco ding o ou de ini ion, hey canno
be classi ied as NNzymes, as he inal N−N bond is o med
spon aneously. Ne e heless, he po en ial o AzoC, ni o-
educ ases and UPOs o he syn hesis o azoxy g oups is wo h
men ioning, as hese enzymes a e well cha ac e ized and
eadily applicable o syn he ic applica ions compa ed o mos
Scheme 16. P oposed Biosyn hesis o 8-Azaguanine (126)
147
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NNzymes. To achie e he same le el o applicabili y o
NNzymes, hey need o be enginee ed o a b oade subs a e
scope and highe ca aly ic ac i i y. Howe e , in-dep h
s uc u al and mechanis ic knowledge o he enzymes is
equi ed o (semi)- a ional enginee ing. Cu en ly, only he
c ys al s uc u e o he K zT homologue PipS, T i17, and
SznF/S zF is a ailable, which has made i possible o iden i y
esidues c ucial o ac i i y and o elucida e he eac ion
mechanism wi h which N−N bond o ma ion is ca a-
lyzed.
79,140,215,227
Fo o he enzymes such as VlmO
130
o
Py N,
82
o which no c ys al s uc u es a e a ailable, AlphaFold
models we e c ea ed o iden i y he esidues in ol ed in
ca alysis. Elucida ion o he esidues in ol ed in NNzyme
ac i i y is he i s s ep o gain mechanis ic insigh s ha a e
c ucial o a ional enginee ing o enzymes owa d new
subs a es. The e o e, AlphaFold can be a aluable ool o
p omo e u he mechanis ic s udies when no c ys al s uc u e
is a ailable. Howe e , o some NNzymes such as FzmP, KinJ,
P nB o Ady6, whe e he homology o well-cha ac e ized
enzymes is low o e en he ca alyzed eac ion is unce -
ain,
45,126,132,147
u he s uc u al and biochemical s udies a e
equi ed o gain mechanis ic insigh s. The gene al lack o
mechanis ic in o ma ion makes i di icul o expand he
subs a e ange o NNzymes and could explain why o he
K zT homologue PAI2, none o he se en designed mu an s
could ex end he subs a e scope o he enzyme.
78
In addi ion
o enzyme enginee ing, he na u al di e si y o enzymes can
also be used o access new subs a es. Fo example, Ma suda e
al. iden i ied eigh binding pocke esidues in na u ally
occu ing cupin/Me RS-like enzymes ha speci y hei
subs a es as ei he Gly, Ala, Se , Glu o Ty . These enzymes
can be used o syn hesize a ious hyd azine in e media es 48
(Scheme 5),
82
highligh ing how enzyme mining could help
expand he ange o accessible NNzymes’ p oduc s.
Se e al pha maceu ically in e es ing N−N bond-con aining
p oduc s, such as N-ni osamine de i a i es,
89,119,132
long-
chain alipha ic N-hyd oxy iazenes
68,178
and α-hyd azino acids,
a e al eady accessible ia NNzymes. Howe e , o he impo an
subs a e classes, such as N−N bond-con aining a oma ic
he e ocycles, azoxy compounds, linea azines and alipha ic
diazo compounds a e no ye accessible, al hough hey occu in
na u e (see Figu e 2).
147,234−236
In addi ion o enzyme
enginee ing, u he biochemical cha ac e iza ion and iden i-
ica ion o NNzymes could help o ob ain he abo e-
men ioned unc ional g oups. To ep esen a eal al e na i e
o he chemical syn hesis o N−N bonds, NNzymes mus also
ha e high ac i i y, good soluble exp ession and s abili y in
o de o educe he amoun o enzyme equi ed. Howe e ,
kine ic s udies ha e so a only been ca ied ou o K zT, he
K zT homologue PAI2, T i17 and A aA6.
68,76,78,138
K zT
shows a kca /kM alue o 57.5 s−1mM−1, which is e y
p omising o syn he ic applica ions, while PAI2, T i17 and
A aA6 wi h kca /kM alues o 0.12, 2.25, and 0.03 s−1mM−1,
espec i ely, would need o be u he imp o ed, e.g. by
enzyme enginee ing. Consequen ly, in addi ion o enzyme
enginee ing, enzyme mining, kine ic in es iga ions, eac ion
enginee ing and ecycling s a egies will also be necessa y o
pa e he way o enzyma ic N−N bond o ma ion beyond
labo a o y scale. As men ioned abo e, NNzymes cu en ly s ill
ha e hei limi a ions and chemical syn hesis emains an
in aluable echnique o he syn hesis o N−N bond-
con aining compounds, howe e , he e a e ce ain limi a ions
o chemical syn hesis ha could po en ially be o e come wi h
bioca aly ic app oaches.
25
Fo example, he con en ional
chemical syn hesis o con o ma ionally cons ained molecules
such as Piz (L-4) equi es he implemen a ion o ex ensi e
p o ec ion and dep o ec ion s eps, which ul ima ely esul s in a
educ ion in o e all yields.
237
Fu he mo e, biologically ac i e
compounds o en possess complex s e eochemis y, necessi a -
ing me iculous con ol o e he o ma ion o chi al cen e s.
238
Chemical syn hesis equen ly encoun e s di icul ies in
a aining he desi ed le el o enan iome ic and dias e eome ic
pu i y.
239
The p esence o mul iple unc ional g oups can also
esul in unin ended side eac ions, whe eas enzymes ypically
exhibi high speci ici y o he subs a es o in e es .
240
In
addi ion, chemical syn hesis o en equi es he use o oxic o
haza dous eagen s (e.g., n-bu ylli hium and o ganoaluminum
compounds) and sol en s (e.g., chlo ina ed sol en s such as
ca bon e achlo ide o 1,2-dichlo oe hane, e he s such as
u an, hyd oca bons such as benzene o o-xylene),
241
which
inhe en ly pose en i onmen al and sa e y isks, while by-
p oduc s o bio echnological p ocesses a e equen ly bio-
deg adable.
242
In his ega d, gene ic and me abolic enginee -
ing o e s a dis inc ad an age o e con en ional chemical
syn hesis, as i can be employed o op imize pa hways and
ob ain he inal p oduc s wi h high e iciency.
243
One
illus a i e example is he e men a i e p oduc ion o L-4 in
a gene ically modi ied Au eobasidium melanogenum s ain
exp essing K zT.
80
The o ma ion o mo e han 10 g o L-4
in 5 days in a 10-L eac o was demons a ed s a ing om 120
g o glucose. This me hod does no equi e o ganic sol en s,
in e media e pu i ica ion s eps o o he chemical p ecu so s.
This example highligh s ha NNzymes ep esen one o many
p omising a eas whe e enzyma ic eac ions can complemen
syn he ic chemis y o access N−N bond-con aining p oduc s
in an e icien manne .
6. CONCLUDING REMARKS
The elucida ion o he a ious pa hways and eac ion
mechanisms leading o he o ma ion o N−N bonds in NPs
has become an a ea o eme ging in e es in he pas decade.
Thus, i is no su p ising ha a la ge numbe o NPs con aining
a a ie y o N−N bond-con aining unc ional g oups ha e been
iden i ied ecen ly. To da e, he e a e se e al unc ional g oups
ha a e known o p edic ed o be accessible h ough
NNzymes. These g oups a e cyclic and linea hyd azines,
ni oso- and diazo-compounds, iazenes and iazoles, which
a e also p esen ed in his e iew in he con ex o he
con en ional me hods o hei syn hesis. Ne e heless, ou
cu en unde s anding o how NNzymes acili a e N−N bond
o ma ion emains la gely elusi e, despi e se e al o hem
ha ing been conclusi ely linked o N−N bond- o ming
eac ions h ough in i o cha ac e iza ion, and s uc u al as
well as mechanis ic in o ma ion is a ailable. Examples include
he PZS, in ol ed in he syn hesis o cyclic hyd azines
79
and
he HDO/cupin-domain con aining enzyme SznF/S zF,
in ol ed in he biosyn hesis o ni osamines.
215
This is likely
due o he di e si y o enzymes and eac ion mechanisms used
in na u e o o m N−N bonds, and he di icul ies ela ed o
he o en low le el o p o ein sequence iden i y and co ac o
dependency. Mo eo e , N−N bond o ma ion is o en no
di ec ly enzyme-ca alyzed, bu occu s spon aneously, e.g. by
adical ecombina ion,
89
a e enzyma ic o ma ion o he
eac i e in e media es. Recen ly, i has been shown ha in
addi ion o enzymes ha di ec ly ca alyze N−N bond
o ma ion, so-called hyd azine ans e ases ca alyze he
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