A photolabile linker for the solid-phase synthesis of peptide hydrazides and heterocycles

Article abstract of DOI:10.1021/ol502219s

A photolabile hydrazine linker for the solid-phase synthesis of peptide hydrazides and hydrazine-derived heterocycles is presented. The developed protocols enable the efficient synthesis of structurally diverse peptide hydrazides derived from the standard

Full text of DOI:10.1021/ol502219s

Letter
pubs.acs.org/OrgLett
A Photolabile Linker for the Solid-Phase Synthesis of Peptide
Hydrazides and Heterocycles
Katrine Qvortrup,† Vitaly V. Komnatnyy,† and Thomas E. Nielsen*,†,‡
†Department of Chemistry, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
‡Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore 637551, Singapore
S
* Supporting Information
ABSTRACT: A photolabile hydrazine linker for the solid-phase synthesis of peptide hydrazides and hydrazine-derived
heterocycles is presented. The developed protocols enable the efficient synthesis of structurally diverse peptide hydrazides
derived from the standard amino acids, including those with side-chain protected residues at the C-terminal of the resulting
peptide hydrazide, and are useful for the synthesis of dihydropyrano[2,3-c]pyrazoles. The linker is compatible with most
commonly used coupling reagents and protecting groups for solid-phase peptide synthesis.
rganic hydrazides are versatile building blocks for the
chemical methods, and attractive for direct applications in
Osynthesis of pharmacologically relevant heterocycles and biochemical reactions, where contamination with cleavage
advanced materials.1,2 Peptide hydrazides have been widely
reagents is undesired. We now wish to report a photolabile
linker based on the o-nitro-veratryl group,11 which is capable of
releasing peptide hydrazides upon UV irradiation. The strategy
is fully orthogonal to the most commonly used protecting
groups and chemical methods in SPPS and shows excellent
compatibility with peptide composition; notably all 20 natural
occurring α-amino acid residues (including side-chain protected
analogs) are accepted in the C-terminal of the peptide
hydrazides. Furthermore, this linker unit can be applied to
synthesize combinatorial libraries of biological interesting
heterocyclic compounds, such as pyranopyrazoles.
The synthesis of the linker commenced with the alkylation of
acetovanilone with ethyl-4-bromobutyrate followed by nitra-
tion, reduction, and chlorination to yield key intermediate
benzylic chloride 2 in 70% overall yield (Scheme 1).12
Substitution of chloride with tert-butyl carbazate gave 3, and
finally, hydrolysis of the ester moiety afforded the linker 1. It
can be made in only six steps with an overall yield of 59% (+10
g scale).
utilized in bioconjugation reactions to form glycoconjugates3
and more recently emerged as powerful precursors for peptide
ligation.4 However, their applicability may be limited in two
respects: (1) peptide hydrazides remain challenging to
synthesize and require postsynthesis purification prior to
ligation and (2) current peptide hydrazide synthesis strategies
are not compatible with all amino acid residues or their side-
chain protected derivatives. Therefore, more efficient and mild
methods for peptide hydrazide synthesis are needed.
Given the importance of hydrazide derivatives in drug and
probe discovery efforts, the solid-phase synthesis of protected
hydrazides have been subject to many studies. Peptide
hydrazides may be directly accessed from solid-supported
ester-linked peptides by hydrazinolysis,5 but this strategy often
requires an excess of hydrazine, which is incompatible with a
range of protecting groups.6 The method also complicates
postcleavage purification and gives low yields of peptides
containing side-chain protected cysteine, aspartic or glutamic
acid residues.7 Wang et al. reported the synthesis of peptide
hydrazides on alkoxycarbonyl hydrazide resins.8 Recent
strategies have relied on 2-Cl-trityl-derived hydrazine resins,9
where postsynthetic release of compounds can be achieved with
more dilute TFA solutions. These linkers nevertheless face
several limitations, as C-terminal glutamine, asparagine, or
aspartic acid residues are generally not tolerated4a,10 and some
reported peptide hydrazides remain challenging to synthesize
using these methods.4f Acidic reactions can generally not be
employed to elaborate the solid-supported hydrazides, and
many protected derivatives are not compatible with acids.
Photolysis offers an exceedingly mild method for releasing
compounds from solid supports which is fully orthogonal to
Whereas the linker 1 was readily coupled13 to an amino-
functionalized solid support (ChemMatrix), synthetic difficul-
ties were encountered in the removal of the Boc-group.
Exposing the immobilized linker to standard TFA/CH2Cl2
(1:1) deprotection conditions resulted in formation of the
corresponding trifluoroacetylated derivative. By screening a
variety of acidic reaction conditions, we found that TMSOTf/
2,6-lutidine14 mediated a clean Boc-deprotection to give the
resin-bound hydrazine-derivative 4. Peptides were then
synthesized via an Fmoc-strategy using TBTU/NEM-mediated
Received: July 30, 2014
Published: August 28, 2014
© 2014 American Chemical Society
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Letter
Glu(tBu) and Asp(tBu) residues should be released just prior
to use, or stored as an acetone solution, as storage of the free
peptide hydrazides over a longer time resulted in conversion
into the corresponding 1,2-diazepane-3,7-diones and tetra-
hydropyridazine-3,6-diones, respectively.
Scheme 1. Synthesis of Boc-Protected Hydrazine Photolabile
Linker 1
Given the importance of peptide hydrazides in protein
chemical synthesis, we envisioned the application of linker 1 in
an efficient one-pot strategy, comprising solid-phase synthesis,
mild photolytic release, and the direct ligation of the crude
peptide hydrazides to cysteine-containing peptides. Peptide
hydrazide 6i (Table 1, entry 9) was easily synthesized from 4
and cleanly released as the acetone hydrazone by light. After
removal of the excess acetone by a stream of nitrogen, an
aqueous phosphate (0.2 M) buffer contaning 6.0 M
guanidinum chloride followed by H-Cys-OH was added (final
peptide concentration of 1.5 and 2.0 mM, respectively). At low
pH (3.0) and temperature (−10 °C), an aqueous NaNO2
solution was added to the ligation mixture. After 20 min, 4-
mercaptophenylacetic acid was added, the pH value was
adjusted to 7.0, and the reaction was left at room temperature
for 12 h. Analysis of the reaction by HPLC showed a clean
ligation reaction in 85% yield. Utility of the linker is illustrated
by the synthesis of the decapeptide H-Leu-Tyr-Arg-Ala-Tyr-
Cys-Lys-Tyr-Met-His-OH (8) through ligation of peptide
hydrazide 6i with peptide fragment 7 using our one-pot
protocol (Scheme 2). We found that the ligation proceeded as
cleanly as reported by Liu and co-workers4a to provide the
desired product 8 in a high yield (93%).
coupling reactions, and photolytic cleavage was routinely
carried out by irradiating for 60 min at rt with 365 nm light
using an LED UV-lamp. To avoid any side reactions associated
with the released hydrazide functionality, including reactions
with the resin-bound nitroso and ketone groups formed in the
event of photolysis, the release was performed in acetone/
CH3CN (3:2), thereby conveniently releasing the peptide
hydrazide protected as its acetone hydrazone. No separate
deprotection step was required to free the hydrazide, as the
acetone moiety rapidly dissociates under the aqueous
conditions commonly used in ligation and bioconjugation
reactions. Evaporation of the aqueous solution of acetone
hydrazone cleanly provided the free hydrazide without the need
for postcleavage purification. Rewardingly, the 20 standard
amino acid residues (including side-chain protected analogs)
were accepted in the C-terminal position of the peptide
hydrazide (Table 1). Peptide hydrazides containing C-teminal
After the successful preparation of peptide 8, we focused our
attention on the application of the same strategy to ligate the
more challenging peptides H-Leu-Tyr-Arg-Ala-Asn-NHNH2
Table 1. Synthesis and Photolytic Release of Peptide Hydrazides (6a−6ad)
C-
terminal
AA
C-
terminal
AA
a
a
entry
1
peptide hydrazide
purity
yield entry
peptide hydrazide
purity
yield
36%
Gly
naphtoyl-Phe-Gly-NHNH2 (6a)
>95%
34%
16
Fmoc-Arg(Pbf)-Ala-Asn(Trt)-NHNH2
>95%
(6p)
2
Ala
naphtoyl-Ala-NHNH2 (6b)
82%
36%
17
H-Leu-Tyr(OtBu)-Arg(Pbf)-Ala-Asn(Trt)
-NHNH2 (6q)
>95%
42%
3
Val
Leu
Ile
Boc-Phe-Val-NHNH2 (6c)
>95%
89%
44%
48%
59%
44%
47%
83%
58%
20%
18
19
20
21
22
23
24
25
Gln
Fmoc-Ala-Gln(Trt)-NHNH2 (6r)
H-Leu-Tyr-Arg-Ala-Gln-NHNH2 (6s)
Boc-Phe-Leu-Cys(StBu)-NHNH2 (6t)
Boc-Ala-Tyr(tBu)-Arg(Pbf)-NHNH2 (6u)
Boc-Val-Phe-His(Boc)-NHNH2 (6v)
naphtoyl-His-NHNH2 (6w)
90%
>95%
>95%
>95%
90%
31%
31%
42%
43%
52%
55%
55%
51%
4
Boc-Pro-Phe-Leu-NHNH2 (6d)
Boc-Phe-Ala-Ile-NHNH2 (6e)
Boc-Leu-Phe-Met-NHNH2 (6f)
naphtoyl-Leu-Phe-NHNH2 (6g)
naphtoyl-Phe-Pro-NHNH2 (6h)
H-Leu-Tyr-Arg-Ala-Tyr-NHNH2 (6i)
5
87%
Cys
Arg
His
6
Met
Phe
Pro
Tyr
87%
7
>95%
>95%
>95%
>95%
8
>95%
94%
9
Lys
Asp
Boc-Ala-Phe-Lys(Boc)-NHNH2 (6x)
10
Boc-Leu-Tyr(tBu)-Arg(Pbf)-Ala-Tyr
(tBu)-NHNH2 (6j)
Fmoc-Tyr(OtBu)-Arg(Pbf)-
Ala-Asp(OtBu)-NHNH2 (6y)
>95%
11
12
13
Trp
Ser
Boc-Phe-Ala-Trp-NHNH2 (6k)
Boc-Phe-Ser(Bzl)-NHNH2 (6l)
Boc-Phe-Leu-Thr(tBu)-NHNH2 (6m)
>95%
>95%
87%
44%
43%
42%
26
27
28
H-Leu-Tyr-Arg-Ala-Asp-NHNH2 (6z)
Boc-Phe-Asp(tBu)-NHNH2 (6aa)
85%
>95%
>95%
56%
67%
59%
Thr
H-Leu-Tyr(OtBu)-Arg(Pbf)-
Ala-Asp(OtBu)-NHNH2 (6ab)
14
15
Asn
Boc-Ala-Tyr(tBu)-Asn(Trt)-NHNH2
(6n)
92%
80%
44%
30%
29
30
Glu
H-Leu-Tyr(OtBu)-Arg(Pbf)-
Ala-Glu(OtBu)-NHNH2 (6ac)
85%
90%
60%
61%
H-Leu-Tyr-Arg-Ala-Asn-NHNH2 (6o)
Fmoc-Ala-Glu(OtBu)-NHNH2 (6ad)
a
The purity of the combined peptide hydrazide and corresponding acetone hydrazone was determined by RP-HPLC and UPLC-MS.
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Scheme 2. Application of Linker Construct 4 for the
Synthesis of a Decapeptide
Table 2. Synthesis and Photolytic Release of
Dihydropyrano[2,3-c]pyrazoles (11a−h)
(6o), H-Leu-Tyr-Arg-Ala-Gln NHNH2 (6s), and H-Leu-Tyr-
Arg-Ala-Asp-NHNH2 (6z) with H-Cys-OH. However, although
we showed the successful release of these peptides (protected
as there acetone hydrazones) in excellent yield (Table 1, entries
15, 19, 26), the acidic conditions used in the ligation resulted in
reaction mixtures containing the desired ligation product and
the side product resulting from an intramolecular cyclization
reaction between the hydrazide and the C-terminal side-chain
amide or acid group. While we were able to obtain a ligation
yield of 55% with H-Leu-Tyr-Arg-Ala-Gln-NHNH2 (6s), the
Asp C-terminal peptide hydrazide 6z resulted in a much lower
yield (<20%), and we were not able to detect any ligation
product in the case of 6o (Asn C-terminal).
To further demonstrate the synthetic potential of the linker
1, the synthesis of more elaborate heterocycles was investigated.
It was decided to target dihydropyrano[2,3-c]pyrazoles, which
constitute an important class of compounds with nummerous
biological properties, such as anticancer,15 antimicrobial,16 and
antiinflammatory17 activities. Given the easy access to β-keto
esters,18 a two-step procedure19 (Table 2) was deemed suitable
for the solid-phase synthesis of dihydropyrano[2,3-c]pyrazoles,
conveniently introducing various substituents at the 3- and 4-
positions (R1 and R2). Hydrazine-functionalized photolabile
support 4 reacted smoothly with appropriate β-keto esters to
produce 1H-pyrazol-5(4H)-one derivatives 9, which undergo a
base-catalyzed three-component reaction with an aldehyde and
malononitrile, entailing a tandem Michael addition/Thorpe−
Ziegler reaction followed by tautomerization to the dihydro-
pyrano[2,3-c]pyrazole 10. Gratifyingly, the steps of synthesis
and photolytic release of the dihydropyrano[2,3-c]pyrazoles
(11a−h) were very clean (Table 2).
In summary, we have developed a photolabile hydrazine
linker for the synthesis of peptide hydrazides on a solid support.
The synthesis strategy shows excellent compatibility with
peptide composition, notably the 20 common proteinogenic α-
amino acid residues (including side-chain protected analogs)
were accepted at the C-terminal hydrazide moiety. The linker is
compatible with most commonly used protecting groups for
SPPS and remains intact throughout multistep peptide
synthesis. Products are ultimately released as hydrazides (in
situ trapped as acetone-derived hydrazone derivatives) from the
solid support in high purity using light. In addition, we have
demonstrated the use of the linker for the generation of
pharmacologically relevant heterocycles in excellent purity.
Finally, we showed the potential of ligating crude deprotected
peptide hydrazides to Cys-functionalized peptides in high
purity and excellent yield. We expect that this one-pot strategy
a
Purity determined by UPLC.
will enable the synthesis of complex and structurally diverse
peptides in the future.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and full spectroscopic data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: ten@kemi.dtu.dk.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Danish Council for Independent Research,
Natural Sciences, Technology and Production Sciences, DSF
Center for Antimicrobial Research, the Lundbeck Foundation,
and the Technical University of Denmark for financial support.
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