Access o Ni ogen−ni ogen Bond-Con aining He e ocycles
Th ough Subs a e P omiscui y o Pipe aza e Syn hases
Yongxin Li, Angelina Osipyan, Niels A.W. de Kok, Simon Sch öde , Ma ia Foun i, Pe e Fod an,
Ronald an Me ke k, A u Maie , Di k Tischle , and Sandy Schmid *
Ci e This: ACS Ca al. 2025, 15, 8846−8854
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ABSTRACT: The ni ogen−ni ogen (N−N) bond mo i com-
p ises an impo an class o compounds o d ug disco e y.
Syn he ic me hods a e p ima ily based on he modi ica ion o N−
N o N�N p ecu so s, whe eas selec i e me hods o di ec N−N
coupling o e ad an ages in e ms o a om economy and yield. In
his con ex , enzymes such as pipe aza e syn hases (PZSs), which
na u ally ca alyze he N−N cycliza ion o L-N5-hyd oxyo ni hine o
he cyclic hyd azine L-pipe aza e, may allow an expansion o he
cu en na ow ange o chemical app oaches o N−N coupling. In
his s udy, we demons a e ha PZSs a e able o ca alyze he
con e sion o a ious N-hyd oxyla ed diamines, which a e di e en
om he na u al subs a e. The N-hyd oxyla ed diamines we e
ob ained in si u using N-hyd oxyla ing monooxygenases (NMOs),
allowing subsequen cycliza ion by PZS, ul ima ely o ming he N−N bond o yield a ious N−N bond-con aining he e ocycles.
Using bioin o ma ic ools, we iden i ied NMO and PZS homologues ha exhibi dis inc ac i i y and s e eoselec i i y p o iles. The
sc eened panel yielded 17 hyd oxyla ed diamines and mo e p omiscuous NMOs, he eby expanding he subs a e ange o NMOs,
esul ing in he o ma ion o p e iously poo ly accessible N-hyd oxyla ed p oduc s as subs a es o PZS. The in es iga ed PZSs led
o a se ies o 5- and 6-membe ed cyclic hyd azines, and he mos p omiscuous ca alys s we e used o scale up and op imize he
syn hesis, yielding he desi ed N−N bond-con aining he e ocycles wi h up o 45% isola ed yield. O e all, ou da a p o ides essen ial
insigh s in o he subs a e p omiscui y and ac i i y o NMOs and PZSs, u he enhancing he po en ial o hese bioca alys s o an
expanded ange o N−N coupling eac ions.
KEYWORDS: bioca alysis, N-he e ocycles, diamines, N-hyd oxyla ing monooxygenases, pipe aza e syn hases,
ni ogen−ni ogen bond- o ming enzymes
■INTRODUCTION
The ni ogen−ni ogen (N−N) bond is p esen in a wide
a ie y o building blocks and is a highly aluable mo i in he
pha maceu ical and ine chemical indus ies. In pa icula , N−
N bond-con aining he e ocycles ha e shown ema kable
biological ac i i y and a e used as an i-in lamma o y d ugs
(e.g., phenylbu azone 1),
1
p o ease and kinase inhibi o s o
he ea men o a a ie y o diseases associa ed wi h he
a ge ed p o eases and kinases (e.g., DB7461 2),
2
clinical
eagen s (e.g., Le osimendan 3),
3
and as angio ensin-
con e ing enzyme inhibi o s (e.g., Cilazap il 4)
4
(Figu e
1A). Mo e han 300 na u al me aboli es con aining N−N
bonds ha e been isola ed om a a ie y o o ganisms and ha e
po en ial as he apeu ic agen s and p ecu so s o he syn hesis
o biologically ac i e molecules.
5−8
Despi e ad ances in o ganic chemis y, he accessibili y o
N−N bond-con aining compounds emains a signi ican
challenge, and syn he ic me hods a e mainly based on he
modi ica ion o N−N o N = N p ecu so s such as hyd azine
and diazo compounds.
9−12
Di ec N−N coupling may p o ide
a mo e con e gen syn hesis s a egy, allowing g ea e
e osyn he ic lexibili y. Howe e , di ec N−N coupling
emains di icul . Fo example, he con en ional syn hesis o
cyclic hyd azines, such as pipe azic acid (sys ema ic name: (S)-
hexahyd opy idazine-3-ca boxylic acid; abb e ia ed as Piz)
equi es a leas nine s eps,
13,14
g oup p o ec ion and
dep o ec ion, ul ima ely esul ing in low o e all yields (a ound
20%).
15−17
Mo eo e , N−N coupling o en equi es ac i a ion
o ni ogen-con aining molecules. One o he me hods used by
na u e is he N-hyd oxyla ion o amine g oups. N-Hyd ox-
yla ion o amino acids and diamines a e c ucial o he
Recei ed: Feb ua y 17, 2025
Re ised: Ap il 29, 2025
Accep ed: May 1, 2025
Published: May 12, 2025
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syn hesis o alue-added me aboli es and ni ogen-con aining
compounds bu emains challenging due o low yields and
ins abili y.
18−20
Enzymes as en i onmen ally iendly ca alys s
could expand he cu en na ow ange o chemical app oaches
o N−N coupling wi hou he need o me al ca alys s o ha sh
eac ion condi ions. Howe e , he bioca alys s capable o
o ming N−N bond-con aining molecules, such as hyd azines,
N−ni oso- and diazo-compounds, ha e only ecen ly been
elucida ed.
21−31
Among hem a e he enzymes in ol ed in he
biosyn hesis o Piz 7a in Ku zne ia sp. 744, which allow he
hyd oxyla ion o he N5ni ogen in o ni hine 5a, ca alyzed by a
la in-dependen N-hyd oxyla ing monooxygenase (NMO),
namely K zI, o L-N5-OH-o ni hine (OH-O n) 6a. Subse-
quen ly, a heme-dependen enzyme, he pipe aza e syn hase
Figu e 1. (A) Rep esen a i e examples o N−N bond-con aining pha maceu icals. (B) Biosyn hesis o L-pipe azic acid in Ku zne ia sp. 744
ca alyzed by he N-hyd oxyla ing monooxygenase (NMO) K zI and he pipe aza e syn hase (PZS) K zT. (C) O e iew o he bioca aly ic syn hesis
owa d N−N-bonded he e ocycles, comp ising N-hyd oxyla ion ca alyzed by an N-hyd oxyla ing monooxygenase (NMO) and N−N bond
o ma ion ca alyzed by a pipe aza e syn hase (PZS). Glucose dehyd ogenase (GDH) is used o egene a e he cosubs a e NADPH.
Figu e 2. Mechanism o PZS-ca alyzed con e sion o L-N5-hyd oxy-L-o ni hine (L-6a) o L-pipe azic acid (L-7a) and po en ial deamina ion side
eac ion.
33
Figu e adap ed om Re .
31
.
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(PZS) K zT, ca alyzes he N−N cycliza ion o L-N5-OH-O n
o he cyclic hyd azine Piz 7a (Figu e 1B and Figu e 2).
32,33
This compound is hen inco po a ed in o non ibosomal
pep ide syn he ase (NRPS) o NRPS-polyke ide syn hase
(PKS) hyb id pa hways.
21
Piz o i s congene s such as 5-
hyd oxy-, 5-chlo o-, and dehyd o-pipe azic acid can be
in eg a ed in o mo e complex s uc u es, e.g., ku zne ides
(e.g., 8, Figu e 1B), pipe azimycins, padanamides, himas a ins,
o monamycines.
34,35
NMOs ca alyze he N-hyd oxyla ion o diamines o diamino
acids such as 5a, gene a ing he eac i e in e media es o he
o ma ion o di e se N−O o N−N linkages. In his con ex ,
K zT
21
and i s iden i ied PZS homologues
36
a e p omising
candida es o he coupled syn hesis o a ious N−N bond-
con aining compounds oge he wi h NMOs. Recen s udies
ha e shown ha PZS-ca alyzed N−N coupling is mo e likely o
p oceed ia di e gen pa hways ha may o igina e om a
common ni enoid in e media e ha e e ses he nucleophil-
ici y o he hyd oxylamine ni ogen in 6a (Figu e 2).
33,37
In e es ingly, a s uc u ally di e en hyd oxylamine (N-
benzylhyd oxylamine) was shown o unde go a deamina ion
eac ion ins ead o N−N bond o ma ion compa ed o he
na u al subs a e 6a. Ini ial a emp s we e made o unde s and
he subs a e scope o PZS by s udying de i a i es o he
na u al subs a e 6a wi h sho e o longe chain leng hs (L-N4-
OH-diaminobu y ic acid (DABA) and L-N6-hyd oxylysine)
21,33
o subs a es wi h a e minal hyd oxy g oup ins ead o an
amino g oup (e.g., 2-amino-5-hyd oxy ale ic acid).
38
No ably,
he subs a e ange appea s o be limi ed o he o ma ion o 5-
and 6-membe ed α-hyd azino acids. In addi ion, mechanis ic
s udies ha e e ealed a side deamina ion eac ion and
spon aneous C−N bond o ma ion.
33
Recen s udies also
p o ide e idence o he easibili y o Piz p oduc ion h ough
he use o a chime ic NMO-PZS enzyme in enginee ed
ac inobac e ia, suppo ing he po en ial u ili y o NMO-
coupled eac ions in he syn hesis o aluable N−N bond-
con aining he e ocycles.
39
In his s udy, we demons a e ha PZSs can ca alyze he
con e sion o a ious N-hyd oxyla ed diamines ha a e
di e en om he na u al K zT subs a e 6a. The N-
hyd oxyla ed diamines we e ob ained in si u using NMOs,
allowing subsequen cycliza ion by PZSs, ul ima ely o ming
he N−N bond o yield a ious N−N bond-con aining
he e ocycles (Figu e 1C). Using bioin o ma ic ools, we
iden i ied no el NMO and PZS homologues ha exhibi
di e en ac i i y and s e eoselec i i y p o iles. The sc eened
panel yielded 17 hyd oxyla ed diamines and a new
p omiscuous NMO (SgNMO), he eby expanding he
subs a e scope o poo ly accessible hyd oxyla ed p oduc s.
We es ed hem agains he panel o PZSs and iden i ied 5
commonly accep ed subs a es. The mos p omiscuous
ca alys s, K zT, SspMPZS and AspPZS, we e used o scale
up and op imize he syn hesis, yielding he desi ed N−N bond-
con aining he e ocycles wi h up o 45% isola ed yield. The
s udied enzymes also exhibi ed an in e se enan iome ic
p e e ence, making hem p omising candida es o u u e
enzyme enginee ing e o s. O e all, ou da a p o ides essen ial
insigh s in o he subs a e p omiscui y and ac i i y o NMOs
and PZSs, u he enhancing he po en ial o hese bioca alys s
o an expanded ange o N−N coupling eac ions.
■RESULTS AND DISCUSSION
Enzyme Selec ion. All known pipe azic acid-con aining
na u al p oduc s iden i ied o da e, such as padanamides,
ma lys a ins, and himas a in, p edominan ly o igina e om
ac inomyce e bac e ia.
40
To in es iga e he p omiscui y o
Figu e 3. Sequence simila i y ne wo k (SSN) o p o eins assigned o he pipe aza e syn hase (PZS) amily. Nodes colo ed in g een ep esen he
selec ed pu a i e PZSs. Blue nodes ep esen he enzymes ha con ain a pu a i e monooxygenase (PF13434) in he gene neighbo hood. The SSN
was cons uc ed using he EFI-EST Web se e , employing an E alue o 5 and alignmen sco e o 52. The SSN was isualized using Cy oscape
(Ve sion 3.10.3). *P o eins did no exp ess in he soluble ac ion.
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PZSs, we ini ia ed a sequence simila i y ne wo k (SSN)
analysis o his p o ein amily using he cha ac e ized K zT as
a s a ing poin (Unip o ID: A8CF72, p o ein amily numbe
PF04299, Figu e 3), which was cons uc ed wi h 12,801
sequences. To educe he numbe o en ies, he h eshold o
p o ein leng h was se a 200 o 300 amino acids. In mos
cases, NMOs and PZSs a e obse ed o colocalize in gene
clus e s con aining NRPS assembly genes. In he pas , pu a i e
PZS genes ha e o en been o e looked in da abases due o
hei anno a ion as ansc ip ional egula o s and la in-binding
p o eins.
21
Hence, we iden i ied h ee SSN clus e s ep esen -
ing PZSs along wi h hei pu a i e NMOs based on genome
neighbo hood analysis (EFI-GNT, Figu e S3). F om he
iden i ied clus e s, 18 genes encoding 8 pu a i e NMOs and
10 pu a i e PZSs om o ganisms known o syn hesize
pipe aza e-con aining me aboli es we e selec ed o in es iga e
hei N−N bond- o ming capabili ies (Table S1). Conse-
quen ly, he selec ed genes o NMOs and PZSs we e cloned
in o an exp ession ec o and he e ologously exp essed in
Esche ichia coli s ain BL21 (DE3) wi h an N- e minal o C-
e minal his idine ag, espec i ely, and pu i ied wi h he
excep ion o wo pu a i e NMOs (Unip o ID: A0A1U9K2D1
and A0A5S4H6S6) and wo pu a i e PZSs (Unip o IDs:
A0A557ZX56, A0A2S8QH93), which could no be p oduced
in soluble o m in E. coli (Figu e S2). In addi ion, one pu a i e
NMO was ound o be uns able unde he expe imen al
condi ions (Unip o ID: A0A2T5KZR9).
Subs a e Scope o he Hyd oxyla ion Reac ion. We
hen s a ed explo ing he p omiscui y o NMOs by
in es iga ing a panel o subs a es ha included diamines,
diamino acids and hei de i a i es (Figu e 4A). Ini ially, he
selec ed pu i ied NMOs we e sc eened o cosubs a e
deple ion in 96-well mic opla es in he p esence o NADH
and NADPH, espec i ely. No ably, NAD(P)H deple ion was
also obse ed in he absence o a subs a e o when he
subs a e was no con e ed (Figu e S4), indica ing he
p esence o nonp oduc i e eac ions, also e e ed o as oxygen
uncoupling.
41
The e o e, ca alase was added o he eac ions o
p e en he accumula ion o H2O2. Reac ions con aining
enzymes and subs a es ha showed inc eased NAD(P)H
deple ion compa ed o con ols in he absence o subs a es
we e hen subjec ed o liquid ch oma og aphy−mass spec-
ome y (LC-MS) analysis o iden i y he co esponding
p oduc o med. To de e mine he amoun o N-hyd oxyla ed
p oduc by he selec ed NMOs, he Csaky assay
42
was used o
assess he deg ee o N-hyd oxyla ion agains a s anda d
(hyd oxylamine). NMOs a e gene ally known o be highly
subs a e-speci ic, ei he o hyd oxyla ing he amino g oup o
diamines o amino acids. Fu he mo e, epo ed NMOs a e
mos ly NADPH-speci ic, and we hus only used NADPH as a
cosubs a e in u he expe imen s.
43−45
We ound ha wo
NMO homologues om S ep omyces g iseoch omogenes
(SgNMO) and S ep omyces spongiae (SsNMO) accep he
na i e subs a e o K zI 5a, and SgNMO is also capable o
ca alyzing he N-hyd oxyla ion o se e al diamines (Figu e 4B).
In pa icula , SgNMO is ac i e on a a ie y o diamines wi h
di e en ca bon chain leng hs, such as 9b (3%), 9e (15%) and
9h (23%), compa ed o i s na u al subs a e 5a (82%). To ou
su p ise, SsNMO was no ac i e agains L-lysine (5b), bu
showed ac i i y on lysine de i a i es (5d, 42%) and o ni hine
me hyl es e (5c, 5%). We hus decided o also include a
known NMO, namely Go A, which was p e iously cha ac-
e ized by Esuola e al.,
44
and he a ian L237R in ou
subs a e scope sc eening. In addi ion o he known subs a es
(9a and 9d) ha we e p e iously epo ed o be con e ed by
Go A, we iden i ied se e al addi ional subs a es o be accep ed
by Go A o i s a ian (9b, 9c, 9g, 9j −9n) (Figu e 4B, Table
S2). Also in line wi h ou expec a ions, no double hyd oxyla ed
Figu e 4. (A) Panel o subs a es o p omiscui y sc eening. Diamines 9a−9n and diamino acids 5a−5d a y in he ca bon chain leng h; diamine
analogs (9b, 9c, 9e, 9g−9n) con ain di e en subs i u ions; na u al subs a es 9a,9d, and 9 o Go A; de i a i es o diamino acids (5c and 5d). (B)
Csaky assay hea map o de e mine N-hyd oxyla ion o a ious NMOs owa d all subs a es. Reac ion condi ions: 1 mM subs a e, 50 mM NaPi
bu e pH 8.0, 10 U/mL glucose dehyd ogenase (GDH), 10 mM glucose, 1 mM NADP+, 1 mg/mL ca alase, 30 μM NMO, 100 μL eac ion
olume, incuba ion a 25 °C, 1 h. Da a we e ob ained om iplica e measu emen s by means o Csaky assay esul s using hyd oxylamine as a
s anda d o a calib a ion cu e.
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p oduc s we e de ec ed by LC-MS o any o he NMOs
es ed. O e all, mos o he diamines and diamino acids
in es iga ed, excep o L-lysine (5b), we e accep ed by he
selec ed NMO panel.
Subs a e Scope o he Cycliza ion Reac ion. We hen
sough o in es iga e he in amolecula N−N bond o ma ion
by coupled enzyma ic cycliza ion o he ac i a ed subs a e. To
s udy his, we indi idually coupled he NMO-subs a e pai s
wi h he highes obse ed con e sion o each subs a e wi h
each PZS. The subs a e scope o PZS comp ises he
accep ance o 6a and unsubs i u ed diamine de i a i es 10a
−10e (Figu e 5). Howe e , no ac i i ies we e ound agains
subs a es 10 −10n and hyd oxyla ed 5b −5d o any o he
in es iga ed homologues. K zT appea s o be he mos
p omiscuous ca alys , and he mos op imal o he syn hesis
o 7a,11a,11c. Fo he o ma ion o 11b and 11e,SspMPZS
showed he highes ac i i y and was selec ed oge he wi h
K zT o he enan iome ic a io (e. .) analysis. The o he PZS
homologues ha e a na owe subs a e ange o lowe ac i i y
compa ed o K zT and SspMPZS. Fo example, AwPZS and
PspPZS a e unable o accep 6a, which may be due o lowe
sequence iden i y o K zT (34% and 25%, espec i ely).
Figu e 5. (A) O e iew o selec ed NMOs o gene a ing he N-hyd oxyla ed subs a es o PZSs. **The p epa a ion o compound 7a has only
been ca ied ou on an analy ical scale (B) Hea map indica ing N−N bond- o ming ac i i y o es ed PZSs. Reac ion condi ions: 20 mM
NH4HCO3pH 8.5, 1 mM subs a e (9a−9e,5a), 1 mM NADP+, 0.05 mM FAD, 10 mM glucose, 10 U/mL GDH, 1 mg/mL ca alase, 30 μM
NMOs and 5 μM heme-con aining PZS, 100 μL eac ion olume, incuba ion a 25 °C, 1 h. Ex ac ed ion ch oma og am (EIC) o expec ed mono/
bis-Fmoc we e in eg a ed (11a−11e and 7a), assuming he ioniza ion o all compounds a e he same. NC is he con ol wi hou he addi ion o
PZS.
Figu e 6. Ex ac ed ion ch oma og am (EIC) om LC-MS analysis o syn hesized cyclic hyd azines in coupled NMO-PZS eac ions,
co esponding o Bis-Fmoc-11a (A), Bis-Fmoc-11b (B), Bis-Fmoc-11c (C), Bis-Fmoc-11d (D), and Bis-Fmoc-11e (E). Reac ion condi ions: 20
mM pH 8.5 NH4HCO3, 1 mM subs a e (9a−9e), 1 mM NADP+, 0.05 mM FAD, 10 mM glucose, 10 U/mL GDH, 1 mg/mL ca alase, 30 μM
NMO (Go A o 9a,9c, and 9d, Go A L237R o 9b, and SgNMO o 9e) and 5 μM heme-con aining K zT/SspMPZS/AspPZS, 100 μL eac ion
olume, incuba ion a 25 °C, 1 h. The e e ence compounds a e indica ed in pu ple, he con ols in he absence o PZS a e ep esen ed in blue. The
m/z alues a e heo e ical. The en i e cascade is shown in g een. All assays we e conduc ed in duplica es.
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SspPZS belongs o he same clus e bu displayed a a he
limi ed subs a e scope compa ed o K zT. AspPZS, howe e ,
showed he highes ac i i y o he o ma ion o 11d (Table
S3). I is no ewo hy ha PZSs ha e been desc ibed o ca alyze
a deamina ion eac ion a he han N−N bond o ma ion,
33
which should lead o he p oduc ion o an imine (wi h
subsequen hyd olysis o aldehyde) as a byp oduc in he
con ex o non-na u al subs a es. The o ma ion o he
p oposed i e-membe ed byp oduc s was only obse ed wi h
subs a es 9d and 9e, consis en wi h p e ious esul s (Figu e
S6),
33
bu he expec ed masses co esponding o aldehydes and
ou -membe ed imines we e no de ec ed o subs a es 9a −
9c, likely due o hei inhe en ins abili y o low ioniza ion
e iciency unde he gi en analy ical condi ions.
The e alua ion o he coupled eac ion mix u e was
pe o med h ough a compa ison o he enzyma ic cascade
wi h he chemically syn hesized e e ences and he con ol
eac ions wi hou PZSs. Fo he linea subs a es 9a and 9d,
he new p oduc s demons a ed he same e en ion ime and
iden ical mass (m/z517.2122, m/z531.2278) as he syn he ic
e e ence compounds 11a and 11d (Figu e 6A, D). This
con i ms ha he PZS homologues can o m he N−N bond-
con aining p oduc s e en in he absence o he ca boxyl g oup
p esen in hei na u al subs a e 6a. Howe e , when subs a e
9j was used in he Go A-PZSs coupled cascade, no p oduc
wi h he expec ed mass was iden i ied. Ins ead, he p esence o
he double bond in he ca bon chain a o s deamina ion o he
subs a e wi h subsequen o ma ion o 1H-py ole (Figu e
S7). The use o b anched p opylenediamines (9b,9c,9e) in
his cascade esul ed in he o ma ion o he co esponding
p oduc s 11b (m/z531.2278, Figu e 6B), 11c (m/z545.2435,
Figu e 6C) and 11e (m/z545.2435, Figu e 6E), which possess
a chi al cen e . To de e mine he s e eoselec i i y o PZSs, we
pe o med he ull cascade eac ion wi h ei he he acemic
subs a e o an a ailable pu e enan iome o 9b, 9c and 9e
using K zT and SspMPZS, espec i ely, oge he wi h Go A
L237R, Go A and SgNMO. As a esul , he (S)-enan iome o
11e was ob ained enzyma ically using (S)-9e as a subs a e,
demons a ing a conse a ion o he s e eocen e in he
SgNMO-K zT-coupled cascade (Figu e S17, en y 4). Mo e-
o e , he expe imen pe o med wi h he subs a e 9e
demons a ed a sligh p e e ence o K zT owa d he (S)-
enan iome , while SspMPZS yielded a acemic p oduc (Figu e
S17, en ies 2 and 3). In e es ingly, he eac ion wi h acemic
9b using SspMPZS esul ed p edominan ly in he o ma ion o
(R)-11b, whe eas he eac ion wi h K zT yielded mainly he
(S)-11b (Figu e S15). Consequen ly, i is concluded ha K zT
shows a p e e ence o accep ing he (S)-con igu ed subs a es,
which is consis en wi h he esul s o he p e ious s udies.
21
In
con as , SspMPZS shows a subs a e p e e ence o he (R)-
con igu a ion in he con e sion o 9b and a lack o
s e eoselec i i y in he con e sion o 9c and 9e (Figu e S15,
en y 3; Figu e S16, en y 3; Figu e S17, en y 2).
We we e in igued by he appa en di e ence in enan io-
me ic p e e ence be ween K zT and SspMPZS in he
con e sion o chi al subs a es 9b and 9e, and hus a emp ed
o iden i y po en ial s uc u al explana ions by compa ing
AlphaFold models o bo h enzymes and pe o ming subs a e
docking (Figu e S18). Alignmen o he s uc u es e ealed
comple e conse a ion among esidues lining he ac i e si e,
Figu e 7. Tempe a u e, bu e concen a ion, bu e and pH op imiza ion, and sal ole ance (NaCl) o he bioca aly ic syn hesis o N−N bond-
con aining he e ocycles using NMO and PZS. (A) Tempe a u e op imiza ion. (B). 10−200 mM NH4HCO3bu e pH 8.5 was used o analysis o
op imal bu e concen a ion. (C) The ollowing bu e s we e in es iga ed: 50 mM NaPi (pH 6.5−8.0), MOPS (pH 6.5−7.5), T is (pH 7.5−8.5),
NH4HCO3(pH 8.5−9.5), NaHCO3(pH 9.0−9.5), and CHES (pH 8.5−9.5). No NaCl was added. (D) Sal ole ance o he cascade compa ing
NaCl concen a ions ( om 20 o 350 mM). The assay mix u es o abo e expe imen s con ained 20 mM NH4HCO3(excep o panel B and C),
no NaCl (excep o panel D), 1 mM NADP+, 0.05 mM FAD, 1 mg/mL ca alase, 10 mM glucose, 10 U/mL GDH, and 1 mM 9a; he eac ion was
ini ia ed by adding 30 μM Go A and 2 μM K zT, and was incuba ed o 3 h a 25 °C (excep o panel A) in a o al eac ion olume o 100 μL.
Samples we e analyzed by HPLC by in eg a ing he a ea o Bis-Fmoc-11a. Resul s a e de i ed om iplica e measu emen s.
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hus no explaining enan iome ic p e e ence wi hin he i s
shell. While he ac i e si e appea s e y la ge and exposed, he
o e all shape o he ca i y is modeled o be sligh ly di e en o
bo h enzymes, sugges ing ha emo e a ia ions could ha e an
impac on molecula dynamics and subs a e p e e ence. Due
o he la ge size and good accessibili y o he ac i e si e,
subs a e docking expe imen s we e also inconclusi e. The
diamine subs a es appea o p ea ange in a la e
con o ma ion compa ed o he diamino acids (6a), possibly
p omo ing he ing closu e and compensa ing o he weake
coo dina ion by he enzyme (Figu e S19).
Op imiza ion o he Reac ion Condi ions and
Upscale. To op imize he ca aly ic sys em o he syn hesis
o he cyclic hyd azines, we de e mined he op imal condi ions
o he o ma ion o 11a using he Go A-K zT coupled
eac ion, aking in o accoun he e ec s o empe a u e, bu e
ype, bu e concen a ion and pH. The eac ion achie es
highes subs a e con e sion a mode a e empe a u e and
bu e concen a ion, speci ically a ound 25 °C and 20 mM
(Figu e 7A, B). The highes ac i i y was obse ed a he pH o
8.5 (Figu e 7C), sugges ing ha he dep o ona ion o he N-
hyd oxyla ed subs a e acili a es nucleophilic a ack. Fu he -
mo e, he highes amoun o 11a obse ed in he p esence o
NH4HCO3bu e compa ed o he o he bu e s in es iga ed
(Figu e 7C). No ably, he o ma ion o 11a was signi ican ly
educed in he p esence o addi ional NaCl (Figu e 7D). This
obse a ion is consis en wi h he esul s o p e ious esea ch
indica ing ha K zT has a p e e ence o lowe sal
concen a ion.
32
In he ime-cou se expe imen , he highes
amoun o 11a was o med a e incuba ing he eac ion o 3
h, p esumably due o he ins abili y o he p oduc in aqueous
phase (Figu e S8).
In o de o e i y he s uc u e o he p oduc s o med, he
eac ions we e ca ied ou on a mg scale unde he op imal
condi ions de e mined. The eac ion was pe o med using 2
mM subs a e 9a −9e in a o al olume o 15 mL. To achie e
ull con e sion, he NMO concen a ion was inc eased o 50
μM. Despi e he signi ican o ma ion o deamina ion p oduc s,
SspMPZS and AspPZS exhibi ed he highes p oduc yields o
11d and 11e and we e he e o e selec ed as ca alys s o
p epa a i e scale syn hesis (Figu e S6). Ex ac ion o he cyclic
hyd azines p o ed o be a signi ican challenge, esul ing in low
isola ed yields due o hei high solubili y in he aqueous phase.
To o e come his p oblem, he eac ion p oduc s we e
de i a ized wi h Fmoc p io o ex ac ion wi h e hyl ace a e.
Subsequen ly, he de i a ized p oduc s we e isola ed and he
s uc u es we e con i med by NMR and HRMS wi h
compa ison o chemically syn hesized s anda ds. The isola ed
yields o i e-membe ed p oduc s 11a −11c anged om 33%
o 45% (Figu e 8). No ably, six-membe ed p oduc s 11d and
11e we e ob ained wi h signi ican ly lowe yields (9 −13%),
likely due o p edominan side eac ions o he co esponding
hyd oxylamine in e media es.
■CONCLUSIONS
N−N bond-con aining he e ocyclic sca olds ha e high
syn he ic alue o he p epa a ion o mo e complex unc ion-
alized molecules. In his s udy, we es ablished he bioca aly ic
syn hesis o cyclic hyd azines by exploi ing he subs a e
p omiscui y o bo h NMOs and PZSs. Ou genome-mining
s a egy led o he disco e y o a no el NMO (SgNMO) wi h
he abili y o accep bo h diamines and diamino acids,
expanding he known NMO subs a e scope. In addi ion,
SspMPZS was shown o exhibi he opposi e s e eoselec i i y
o K zT. Howe e , he achie able p oduc scope o wild- ype
PZSs appea s o be limi ed o i e- o six-membe ed
he e ocycles, which cu en ly appea s o be he main
bo leneck in he di e si y o cyclic hyd azines ha can be
o med using nonenginee ed PZSs. To iden i y he op imal
eac ion condi ions, we in es iga ed he in luence o a ious
eac ion pa ame e s, including bu e ype, pH, sal concen-
a ion, and empe a u e. To u he inc ease he yield o cyclic
hyd azines, p o ein enginee ing o supp ess he unwan ed
deamina ion eac ion o in si u p oduc emo al s a egies
could be explo ed. In addi ion, he de ec ion o N−N bond-
con aining he e ocycles is cu en ly dependen on LC-MS due
o he low s abili y and high pola i ies o he esul ing
molecules. In ligh o hese obse a ions, he de elopmen o a
sui able high- h oughpu spec oscopy-based de ec ion me hod
would acili a e u he esea ch in o he o ma ion o N−N
bond-con aining molecules using PZS. In summa y, he ange
o compounds accessible by PZS is expanding, allowing access
o mo e complex N−N bond-con aining molecules. I is
expec ed ha his bioca aly ic syn hesis will be u he
ex ended in he u u e o enable he bioca aly ic syn hesis o
Figu e 8. P oduc scope ob ained wi h he NMO-PZS ca alysis app oach owa d N−N bond-con aining he e ocycles. R = H, Me, E ; n= 0, 1.
Reac ion condi ions: 20 mM NH4HCO3bu e pH 8.5, 2 mM subs a e (9a−9e), 1 mM NADP+and 0.05 mM FAD, 10 mM glucose, 10 U/mL
GDH, 1 mg/mL ca alase, 50 μM NMO (Go A o 11a,11c, and 11d; Go A L237R o 11b; and SgNMO o 11e), 2 μM PZS, 15 mL eac ion
olume, incuba ion a 25 °C, 5 h. The enan iome ic a io (e. .) was calcula ed by (S)- o (R)-enan iome . The isola ed yields a e epo ed o he
enzymes, showing he highes ac i i y acco ding o he hea map.
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a wide ange o cyclic hyd azines wi h di e en s uc u al
di e si y using enginee ed NMO/PZS couples.
■ASSOCIATED CONTENT
*
sı Suppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/acsca al.5c01237.
Addi ional ables and igu es demons a ing enzyme
ac i i y, NMR spec a, and HPLC da a (PDF)
■AUTHOR INFORMATION
Co esponding Au ho
Sandy Schmid −Depa men o Chemical and
Pha maceu ical Biology, G oningen Resea ch Ins i u e o
Pha macy, Uni e si y o G oningen, G oningen 9713 AV,
The Ne he lands; o cid.o g/0000-0002-8443-8805;
Email: [email p o ec ed]
Au ho s
Yongxin Li −Depa men o Chemical and Pha maceu ical
Biology, G oningen Resea ch Ins i u e o Pha macy,
Uni e si y o G oningen, G oningen 9713 AV, The
Ne he lands
Angelina Osipyan −Depa men o Chemical and
Pha maceu ical Biology, G oningen Resea ch Ins i u e o
Pha macy, Uni e si y o G oningen, G oningen 9713 AV,
The Ne he lands
Niels A.W. de Kok −Depa men o Chemical and
Pha maceu ical Biology, G oningen Resea ch Ins i u e o
Pha macy, Uni e si y o G oningen, G oningen 9713 AV,
The Ne he lands
Simon Sch öde −Depa men o Chemical and
Pha maceu ical Biology, G oningen Resea ch Ins i u e o
Pha macy, Uni e si y o G oningen, G oningen 9713 AV,
The Ne he lands; o cid.o g/0009-0002-2778-6392
Ma ia Foun i −Depa men o Chemical and Pha maceu ical
Biology, G oningen Resea ch Ins i u e o Pha macy,
Uni e si y o G oningen, G oningen 9713 AV, The
Ne he lands
Pe e Fod an −Depa men o Chemical and Pha maceu ical
Biology, G oningen Resea ch Ins i u e o Pha macy,
Uni e si y o G oningen, G oningen 9713 AV, The
Ne he lands
Ronald an Me ke k −Depa men o Chemical and
Pha maceu ical Biology, G oningen Resea ch Ins i u e o
Pha macy, Uni e si y o G oningen, G oningen 9713 AV,
The Ne he lands
A u Maie −Facul y o Biology and Bio echnology, Mic obial
Bio echnology, Ruh Uni e si y Bochum, Bochum 44780,
Ge many
Di k Tischle −Facul y o Biology and Bio echnology,
Mic obial Bio echnology, Ruh Uni e si y Bochum, Bochum
44780, Ge many; o cid.o g/0000-0002-6288-2403
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acsca al.5c01237
No es
The au ho s decla e no compe ing inancial in e es .
■ACKNOWLEDGMENTS
This wo k was suppo ed by he China Schola ship Council
(pe sonal ellowship Yongxin Li, CSC no. 202206750031),
The Ne he lands O ganiza ion o Scien i ic Resea ch (VI.V-
idi.213.025), he Eu opean Resea ch Council (ERC, g an
ag eemen no. 101075934), and he Eu opean Union’s
Ho izon Eu ope esea ch and inno a ion p og amme (unde
he Ma ie Skłodowska-Cu ie g an ag eemen no. 101073065).
A.M. was suppo ed by he Ge man Resea ch Council (DFG)
wi hin he amewo k o GRK 2341 (Mic obial Subs a e
Con e sion; no. 321933041), which was awa ded o D.T. The
Go A and Go A a ian (L237R) we e kindly p o ided by Di k
Tischle ’s esea ch g oup. We would also like o hank
Zhengyang Wu o his help wi h he syn hesis o e e ence
compounds, D . Hen ik Te holsen o his insigh ul guidance
and assis ance wi h da a analysis, and Alexande A gy ou o
his help wi h p oo eading he manusc ip .
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