Nγ-Hydroxyasparagine: A Multifunctional Unnatural Amino Acid That is a Good P1 Substrate of Asparaginyl Peptide Ligases

Article abstract of DOI:10.1002/anie.202108125

Peptidyl asparaginyl ligases (PALs) are powerful tools for peptide macrocyclization. Herein, we report that a derivative of Asn, namely N^γ-hydroxyasparagine or Asn(OH), is an unnatural P1 substrate of PALs. By Asn(OH)-mediated cyclization, we prepared cyclic peptides as new matrix metalloproteinase 2 (MMP2) inhibitors displaying the hydroxamic acid moiety of Asn(OH) as the key pharmacophore. The most potent cyclic peptide (K_i=2.8±0.5 nM) was built on the hyperstable tetracyclic scaffold of rhesus theta defensin-1. The Asn(OH) residue in the cyclized peptides can also be readily oxidized to Asp. By this approach, we synthesized several bioactive Asp-containing cyclic peptides (MCoTI-II, kB2, SFTI, and integrin-targeting RGD peptides) that are otherwise difficult targets for PAL-catalyzed cyclization owing to unfavorable kinetics of the P1-Asp substrates. This study demonstrates that substrate engineering is a useful strategy to expand the application of PAL ligation in the synthesis of therapeutic cyclic peptides.

Full text of DOI:10.1002/anie.202108125

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How to cite:
International Edition: doi.org/10.1002/anie.202108125
German Edition: doi.org/10.1002/ange.202108125
Cyclic Peptides
Ng-Hydroxyasparagine: A Multifunctional Unnatural Amino Acid
That is a Good P1 Substrate of Asparaginyl Peptide Ligases
Yiyin Xia, Janet To, Ning-Yu Chan, Side Hu, Heng Tai Liew, Seetharamsing Balamkundu,
Xiaohong Zhang, Julien Lescar, Surajit Bhattacharjya, James P. Tam,* and Chuan-Fa Liu*
Abstract: Peptidyl asparaginyl ligases (PALs) are powerful
tools for peptide macrocyclization. Herein, we report that
a derivative of Asn, namely Ng-hydroxyasparagine or Asn-
(OH), is an unnatural P1 substrate of PALs. By Asn(OH)-
mediated cyclization, we prepared cyclic peptides as new
matrix metalloproteinase 2 (MMP2) inhibitors displaying the
hydroxamic acid moiety of Asn(OH) as the key pharmaco-
phore. The most potent cyclic peptide (Ki = 2.8 Æ 0.5 nM) was
built on the hyperstable tetracyclic scaffold of rhesus theta
defensin-1. The Asn(OH) residue in the cyclized peptides can
also be readily oxidized to Asp. By this approach, we
synthesized several bioactive Asp-containing cyclic peptides
(MCoTI-II, kB2, SFTI, and integrin-targeting RGD peptides)
that are otherwise difficult targets for PAL-catalyzed cycliza-
tion owing to unfavorable kinetics of the P1-Asp substrates.
This study demonstrates that substrate engineering is a useful
strategy to expand the application of PAL ligation in the
synthesis of therapeutic cyclic peptides.
are cysteine endopeptidases that typically recognize and
cleave peptide bonds after an Asn or Asp residue.[4] Some
members of the AEP family catalyze transpeptidation at the
asparaginyl peptide bonds with little hydrolase activity, which
qualifies them as peptidyl asparaginyl ligases (PALs).[3b,5]
Most PALs have a strong preference for P1-Asn over P1-
Asp substrates. In fact, the rate of cyclization reactions for
these two types of substrates can differ by 100 to 800 folds.[3b,6]
Therefore, most applications of PAL-mediated cyclization
reported to date have been limited to P1-Asn substrates.[5,7]
Applications of PALs will be expanded if one can broaden
their substrate scope.
The optimal pH for the hydrolysis of Asp/Asn–peptide
bonds by AEPs is around 4.5–5.5.[4,5b,c] Structural character-
ization of AEP–aspartyl peptide inhibitor complexes shows
g
that the hydroxy group of side chain COOH of P1-Asp acts
as a hydrogen bond donor to bind to a key residue in the S1
pocket of the enzyme.[8] Given the low pKa value of the
M
acrocyclization is an
effective strategy to
restrict the conformations
of linear peptides.[1] The
large surface areas of
cyclic peptides confer high
binding affinity and selec-
tivity to inhibit protein–
protein interactions that
are often not easily drug-
gable using small-molecule
compounds.[2] Responsible
for cyclotide synthesis in
plants, asparaginyl endo-
peptidases (AEPs) are
a useful tool to cyclize
synthetic peptides.[3] AEPs
Figure 1. Modeled H-bond interactions between the S1 pocket of AEPs and the P1 amino acid of the
substrates at neutral pH. Residues in the S1 pocket and P1 amino acids of the substrates are shown in blue
and black, respectively.
carboxyl group, an acidic pH is required to maintain the
neutral, protonated form needed for enzyme binding
(Figure 1). However, at acidic pH, the N-terminal amine of
an acyl-acceptor substrate is mostly protonated, making it
largely unavailable in a ligation reaction. This dilemma is
likely the main reason that limits the rate of PAL-catalyzed
ligation at aspartyl bonds.[7h] In this study, we used the
asparagine analog Ng-hydroxyasparagine or Asn(OH) as the
P1 amino acid of PAL substrates (Figure 1). Because the Ng-
hydroxy group has a higher pKa than COOH, it can remain
protonated as a hydrogen bond donor at near neutral pH
which favors the ligation reaction. We show that P1-Asn(OH)
peptides are well recognized by butelase-1 and VyPAL2 in the
cyclization reaction (Table 1, Figure S2). This unnatural Asn-
[*] Y. Xia, Dr. J. To, N.-Y. Chan, S. Hu, H. T. Liew, Dr. S. Balamkundu,
Dr. X. Zhang, Dr. J. Lescar, Dr. S. Bhattacharjya, Dr. J. P. Tam,
Dr. C.-F. Liu
School of Biological Sciences, Nanyang Technological University
60 Nanyang Drive, Singapore 637551 (Singapore)
E-mail: jptam@ntu.edu.sg
cfliu@ntu.edu.sg
Dr. S. Balamkundu
Singapore-MIT Alliance for Research and Technology
Singapore 138602 (Singapore)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202108125.
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Table 1: Kinetics of peptide cyclization by butelase-1 and VyPAL2
of Asn(OH) is involved in H-
bonding to the S1 pocket of PALs
(Figure 1).
Sequence
k
cat [sÀ1
]
Km [mM]
kcat/Km [MÀ1/sÀ1
]
AIYRRGRLYRRNHV 1[a]
AIYRRGRLYRRN(OH)HV 2[a]
AIYRRGRLYRRDHV 3[a]
AIYRRGRLYRRNSL 4[b]
AIYRRGRLYRRN(OH)SL 5[b]
AIYRRGRLYRRDSL 6[b]
14.86Æ0.34
3.46Æ0.27
67.5Æ2.8
98.8Æ17.8
629.7Æ19.5
42.5Æ8.83
119.9Æ19.2
2069Æ407
220148Æ10429
35020Æ6876
721Æ39
Asn(OH) contains a hydroxa-
mic acid moiety that has interesting
properties,[9,10] including the ability
to bind to metal ions[9a,b,10] and
reactivity toward an S-aryl thio-
0.454Æ0.02
4.56Æ0.24
107294Æ22996
7857Æ1432
281Æ70
0.942Æ0.082
0.581Æ0.09
ester for peptide ligation.[9c]
A
[a] Peptides for cyclization by butelase-1. [b] Peptides for cyclization by VyPAL2. The cyclization reactions
were performed at 378C in 20 mM PBS (pH 6.5) with various substrate concentrations. Cyclization was
monitored by analytical RP-HPLC.
variety of hydroxamic acid-derived
inhibitors of metalloenzymes have
been designed as pharmacologic
agents.[9b,10] Our method provides
(OH) residue can function as a good chelator for metal
cations to inhibit metalloenzymes or be further converted to
natural Asp in a mild oxidation reaction (Scheme 1).
a convenient approach to prepare hydroxamic acid displaying
cyclic peptides as potential inhibitors of pathogenic metal-
loenzymes such as matrix metalloprotease 2 (MMP2).[11] We
used peptide 8, a b-amyloid precursor protein derived
inhibitor peptide (APP-IP),[12] to demonstrate the utility of
this method (Figure 2). APP-IP 8 is an MMP inhibitor and is
largely selective for MMP2 (Ki ꢀ 60 nM).[12a] Asp6 in APP-IP
8 mediates Zn2+ bonding. Simple replacement of Asp6 with
Asn(OH) resulted in a 2.6-fold improvement in inhibitory
activity (Figure 2b) that could be attributed to the stronger
Zn2+-chelating ability of the Asn(OH) hydroxamate group
compared to the carboxylate in Asp.[10] Nevertheless, the
linear APP-IP peptide 8 has poor proteolytic stability and its
half-life in human serum is short.[12c] Cyclization is a useful
strategy to improve the stability and selectivity of MMP2
inhibitory peptides.[10] Therefore, we performed head-to-tail
cyclization of APP-IP peptides of various lengths using the
Asn(OH) ligation method. However, the cyclized products,
peptides 10–15, had decreased inhibitory activities toward
MMP2 (Table S3, Figure 2b), likely because simple cycliza-
tion restrained the peptides into undesirable conformations.
We then grafted Ile1Ser2Tyr3, Leu8 and Asn(OH)6 from
Scheme 1. Cyclization of P1-Asn(OH) peptide substrates as catalyzed
by AEPs and subsequent oxidation of Asn(OH) to Asp.
Compounds 1–6 (Table 1 and S1) were prepared by Fmoc
solid-phase peptide synthesis (SPPS) and used as substrates
for butelase-1 or VyPAL2-catalyzed cyclization. As expected,
the P1-Asn(OH) peptides were good substrates of these PALs
and exhibited a much higher binding affinity (Km) and
catalytic turnover (kcat
)
than the native P1-Asp
peptides (Table 1). The
catalytic efficiency of bute-
lase-1 on the N(OH)HV
substrate 2 is only approx-
imately 6-fold lower than
that on the NHV peptide 1,
but 48-fold higher than
that on the DHV peptide
3 (Table 1). Similarly, the
catalytic
efficiency
of
VyPAL2 on the N(OH)SL
peptide 5 is approximately
14-fold lower than that on
the NSL peptide 4, but 28-
fold higher than that on the
DSL peptide 6 (Table 1).
As a control, the P1-Asn-
(Me) peptide 7 was not
recognized by butelase-
1 (Table S1 and Figure S3).
Therefore, we conclude
that the Ng-hydroxy group
Figure 2. Design of MMP2 peptide inhibitors based on APP-IP and theta defensin. a) HPLC and ESI-MS
monitoring of butelase-1-catalyzed cyclization of the linear precursor CLCRRGVCRCICTISYCN(OH)-AL for the
preparation of 17 (reduced form). b) Ki values of peptides 8–17 towards MMP2.
2
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APP-IP peptide 9 into rhesus theta defensin-1 (RTD-1) 16
NaNO2 oxidation can also be used to convert Asn(OH) to
(Figure 2) to form Asn(OH)-RTD1 17. RTD-1 has a highly
compact, tetracyclic Cys-ladder structure.[13] Asn(OH)-RTD1
17 was successfully cyclized at the Asn(OH) residue by
incubating the linear substrate with butelase-1 (Figure 2a,
Figure S4A). RP-HPLC monitoring indicated that the reac-
tion was nearly completed in 1 h. After enzymatic cyclization,
the cyclic product (reduced form) was oxidatively refolded
into the mature form by incubation in 0.1 M Tris·HCl buffer
(pH 8.5, 2 M urea) at 48C for 8 h. Enzyme inhibition results
showed that the folded backbone-cyclic Asn(OH)-RTD1
peptide 17 had a Ki value of (2.8 Æ 0.5) nM toward MMP2
(Figure 2b, Figure S4), representing an 8.2-fold improvement
over the linear Asn(OH)-APP-IP peptide 9.
Asp.[9c] Asn-RTD1 19 was prepared in the same manner as
Asn(OH)-RTD1 17 by cyclization at the Asn residue using
butelase-1. Although peptides 17, 18, and 19 have the same
conformation and nearly the same sequences (Figure S6,
Table S4), MMP2 inhibition assay shows that Asn(OH)-
RTD1 17 has a 2.3- and 13-fold improvement in binding
affinity over Asp-RTD1 18 and Asn-RTD-1 19, respectively
(Table S4, Figure S4). This result further corroborates that
Asn(OH) is an excellent pharmacophore for metalloenzyme
inhibition.[10] All three peptides are more potent MMP2
inhibitors than the native RTD-1 16 (Table S4).
Similar to APP-IP 8, Asn(OH)-RTD1 17 is also selective
for MMP2 over MMP9 (ca. 1,000-fold difference in Ki)
(Table S4). The stability of the linear APP-IP and the cyclic
Asn(OH)-RTD1 were examined by incubating the peptides in
human serum at 378C. The cyclic peptide 17 remained intact
after 16 h, while over 50% of the linear APP-IP 8 was
degraded within 32 min (Figure S7). We also did not observe
the loss of the Ng-hydroxy group on Asn(OH), which is
different from what observed by the Heinis group on their
bicyclic peptide.[10] This Ng-hydroxy group may be stabilized
by the highly constrained conformation of the tetracyclic
peptide 17. The high selectivity and stability of the Asn(OH)-
RTD-1 peptide 17 thus makes it a useful pharmacological tool
to study the physiological and pathological roles of MMP2.
Facile conversion of Asn(OH) to Asp makes Asp
cyclization more attainable through PAL ligation, albeit
indirectly. To show the general applications of this method-
ology, we further performed cyclization of P1-Asn(OH)
peptides of 6–34 amino acids, followed by NaIO4-mediated
oxidation of Asn(OH) to Asp (Table 2). The first peptide
prepared was MCoTI-II. Isolated from Momordica cochin-
chinensis seeds, MCoTI-II is a potent trypsin inhibitor.[15]
Cyclization of MCoTI-II has been achieved using various
methods.[15b,16–19] Linear MCoTI-II-N(OH)IV 20 was pre-
pared by SPPS and oxidatively folded. Backbone cyclization
of acyclic, folded MCoTI-II-N(OH)IV 20 was done by
incubation with butelase-1 (0.01 equiv) for 3 h at 378C
(Figure S8a,b). After enzymatic cyclization, the unnatural
Asn(OH) residue was then converted to Asp in 10 min by
NaIO4 oxidation at 08C (Figure 4). To study the folding states,
NMR results showed that peptide 17 has the same 3D
structure as RTD-1 16 (PDB:1HVZ).[13] The Tyr3 and Asn-
(OH)5 residues in peptide 17 are constrained in a b-sheet
structure with the two side chains pointing parallel in the
same direction (Figure 3, Figure S5), similar to the orientation
Figure 3. Superposed 20 low-energy structures of peptide 17 (PDB:
7F32; N-OH not shown) highlighting the spatial disposition of the
side chains of residues.
adopted by Tyr3 and Asp6 of APP-IP 8 in binding to
MMP2.[12b] This preorganized conformation of peptide 17 may
have contributed to its improved inhibitory activity. For
comparison studies, Asp-RTD1 18 was obtained by oxidation
of Asn(OH)-RTD1 17 with NaIO4[14] (1 equiv) in 20 mM PBS
(pH 7.2) at 08C, which quickly converted Asn(OH) to Asp in
5–10 min. Previously, Pentelute and co-workers showed that
Table 2: Peptide cyclization at the P1 site (residue in bold) and oxidation of Asn(OH) to Asp.[a]
Peptide
Sequence
Cyclization
time [h]
Yield [%]
Oxidation
time [min]
Yield [%]
MCoTI-II-(OH)IV 20
MCoTI-II-DIV 21[c]
kB2-N(OH)IV 22
kB2-DIV 23
SFTI-N(OH)IV 24
SFTI-DIV 25
GGVCPKILKKCRRDSDCPGACICRGNGYCGSGSN(OH)IV[b]
GGVCPKILKKCRRDSDCPGACICRGNGYCGSGSDIV[b]
GLPVCGETCFGGTCNTPGCSCTWPICTRN(OH)IV[d]
GLPVCGETCFGGTCNTPGCSCTWPICTRDIV[d]
GRCTKSIPPICFPN(OH)IV[b]
3
3
1
2
3
3
1
1
95
ND
80
ND
85
ND
85
10
NA
10
NA
10
NA
10
>95
NA
>95
NA
>95
NA
>95
>95
GRCTKSIPPICFPDIV[b]
RGD-N(OH)HV 26
RGD-defensin-N(OH)SL 27
FLaRGN(OH)HV[e]
ACRCLCRRGDCRCICRGN(OH)SL[d]
95
15
[a] Cyclization reactions were performed at 378C with 400 mM of the peptide substrate and butelase-1 or VyPAL2 (0.01 equiv) in 20 mM PBS (pH 6.5)
for 1–3 h. Peptide 27 was cyclized by VyPAL2, and the other peptides were cyclized by butelase-1. Asn(OH) oxidation was performed in 20 mM PBS
(pH 7.4) at 08C, with NaIO4 (1 equiv). [b] Backbone cyclization was conducted after oxidative refolding. [c] Cyclization was also conducted at pH 4.5 or
6.5 at room temperature for overnight, and no cyclic product was observed. [d] Backbone cyclization was conducted before oxidative refolding.
[e] Cyclic dimer [FLaRGN(OH)-FLaRGN(OH)] was formed. ND=not detectable; NA=not applicable.
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Asp peptides. A wide range of cyclic peptides were prepared
to demonstrate the utility of our method for P1-Asn(OH)-
enabled cyclization and oxidative Asn(OH)-to-Asp conver-
sion. The structure and bioactivity of these peptides were
confirmed by CD spectroscopy, NMR and enzyme inhibition
assays. More importantly, we demonstrate that cyclization of
Asn(OH)-containing peptides is well-suited for generating
inhibitors that have high potency and selectivity toward
MMP2 as well as proteolytic stability. Taken together, the
engineered Asn(OH) serves three functions: 1) mediator of
PAL-catalyzed ligation and a site of peptide cyclization, 2) a
chelator for metal ions to inhibit metalloenzymes, and 3) an
Asp precursor. Our study shows that substrate engineering
can expand the substrate scope of PALs and increase the
therapeutic value of the cyclized peptides.
Acknowledgement
This research was supported by Academic Research Fund
(AcRF) Tier 3 (MOE2016-T3-1-003) grants to J.P.T., J.L., and
C.-F.L. laboratories and by AcRF Tier 1 (2019-T1-002-100)
and NTUitive Gap grant NGF-2019-07-029 to C.-F.L. from
the Singapore Ministry of Education (MOE).
Figure 4. Conversion of Asn(OH) into Asp by NaIO4-mediated oxida-
tion. RP-HPLC and ESI-MS monitoring of the oxidation reaction of
Asn(OH)-MCoTI-II.
CD spectra for the linear MCOTI-II, acyclic MCoTI-II
(folded), and cyclic MCoTI-II (folded) were collected (Fig-
ure S8c). The spectra for acyclic and cyclic MCoTI-II (both
folded) are comparable to the reported CD spectrum.[20,21]
Butelase-1 cyclized P1-Asn(OH) peptides (20, kB2 22,[22]
and SFTI 24[23]) within 1–3 h at 378C despite a sterically
hindered Asn(OH)-Ile bond (Table 2). In contrast, direct
cyclization of the corresponding P1-Asp peptides 21, 23 and
25 by butelase-1 was extremely inefficient as cyclic products
were not detectable after 3–24 h (Figures S9–S11). This is
most likely because 1) Asp is a poor P1-amino acid, 2) Ile is
a bulky P1’ residue and 3) the N-terminal motifs in 21 and 25
(GG- and GR-) are unfavorable nucleophilic substrates of
butelase-1. Nevertheless, all these problems were overcome
by the higher reactivity of P1-Asn(OH) during cyclization.
We also prepared cyclic peptides containing RGD (for
integrin targeting)[24] using Asp as the cyclization site
(Table 2). The RGD-theta-defensin linear precursor 27 was
cyclized in 1 h by VyPAL2 (Figures S12 and S17). Interest-
ingly, FLaRGN(OH)HV 26 was converted to a cyclic dimer in
1 h, likely because the high angle strain prevented the
formation of the small six-residue ring, similar to a previous
finding by Hemu et al.[25]
Next, trypsin inhibition assays were performed (Table S5
and Figure S21). The Ki values of the synthetic MCoTI-II and
SFTI toward trypsin were 0.18 and 0.44 nM, respectively,
which are consistent with reported values.[16,26] Therefore,
both cyclic trypsin inhibitor peptides prepared by our method
have the expected biological activities.
In conclusion, we have used an unnatural amino acid,
Asn(OH), as the P1 substrate for PAL-catalyzed peptide
cyclization. The P1-Asn(OH) residue is an excellent mimic of
P1-Asn in PAL substrates, and the P1-Asn(OH) peptides
exhibit a much higher affinity and turnover rate than the P1-
Conflict of Interest
The authors declare no conflict of interest.
Keywords: amino acids · cyclic peptides · enzymes · inhibitors ·
macrocyclization
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Accepted manuscript online: August 15, 2021
Version of record online: && &&, &&&&
Angew. Chem. Int. Ed. 2021, 60, 1 – 6
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Cyclic Peptides
An unnatural amino acid, Asn(OH),
mimics P1-Asn in the substrates of
asparaginyl ligases, enabling efficient
cyclization of P1-Asn(OH) peptides. The
hydroxamic acid functionality in Asn(OH)
is a metal-ion chelator for metalloenzyme
inhibition, and Asn(OH) can be con-
verted into native Asp by periodate oxi-
dation (see scheme).
Y. Xia, J. To, N.-Y. Chan, S. Hu, H. T. Liew,
S. Balamkundu, X. Zhang, J. Lescar,
S. Bhattacharjya, J. P. Tam,*
C.-F. Liu*
&&&& — &&&&
Ng-Hydroxyasparagine: A Multifunctional
Unnatural Amino Acid That is a Good P1
Substrate of Asparaginyl Peptide Ligases
6
www.angewandte.org
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 1 – 6
These are not the final page numbers!