Hydrazine-sensitive thiol protecting group for peptide and protein chemistry

Article abstract of DOI:10.1021/ol1028755

In the search for a new Cys side-chain protecting group that is compatible to the solid-phase peptide synthesis yet can be removed under mild conditions, the Hqm and Hgm groups that are readily deprotected by using aqueous hydrazine have been developed. The utility of these groups for peptide and protein chemistry is tested by the total synthesis of a peptide antibiotic trifolitoxin and the human neutrophil defensin hNP2.

Full text of DOI:10.1021/ol1028755

ORGANIC
LETTERS
2011
Vol. 13, No. 4
568–571
Hydrazine-Sensitive Thiol Protecting
Group for Peptide and Protein Chemistry
Fei Shen, Zhi-Ping Zhang, Jia-Bin Li, Yun Lin, and Lei Liu*
Department of Chemistry, University of Science and Technology of China, Hefei 230026,
China, and Department of Chemistry, Tsinghua University, Beijing 100084, China
lliu@mail.tsinghua.edu.cn
Received November 8, 2010
ABSTRACT
In the search for a new Cys side-chain protecting group that is compatible to the solid-phase peptide synthesis yet can be removed under
mild conditions, the Hqm and Hgm groups that are readily deprotected by using aqueous hydrazine have been developed. The utility of these
groups for peptide and protein chemistry is tested by the total synthesis of a peptide antibiotic trifolitoxin and the human neutrophil defensin
hNP2.
Chemically synthesized peptides1 and proteins2 are im-
portant tools in chemical biology. The development of
more efficient methods for the preparation of increasingly
complex peptides and proteins still provides important
challenges.3 One such challenge is the synthesis of multiple
Cys-containing peptides, where an often encountered dif-
ficulty is unambiguous formation of several disulfide
bridges.4 To solve this difficulty there is often a need to
conduct orthogonal protection of Cys residues for the
stepwise formation of disulfide bonds.5 Second, for chemical
synthesis of proteins without suitable Xaa-Cys ligation
site(s), there is a need to use the recently developed “ligation-
desulfurization” approach6 in which the desulfurization
step is performed in the presence of side chain protected
Cys.7 Finally, peptide-protein bioconjugation may also
need selective protection and deprotection of active Cys side
chains under mild conditions.8
In this context, it is important to stress that very few
good-quality protecting groups are available for the Cys
side chain.9 The popular trityl (Trt) group9 cannot survive
the peptide cleavage step in SPPS (solid-phase peptide
synthesis), whereas the disulfide protection (e.g., StBu)10 is
not stable to the reductive conditions often used to handle
(1) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
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Danishefsky, S. J. J. Am. Chem. Soc. 2009, 131, 5438.
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923. (b) Nilsson, B. L.; Soellner, M. B.; Raines, R. T. Annu. Rev. Biophys.
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2008, 47, 1553. (d) Kent, S. B. H. Chem. Soc. Rev. 2009, 38, 338.
(4) Pazgier, M.; Li, X.; Lu, W.; Lubkowski, J. Curr. Pharm. Design
2007, 13, 3096.
(5) (a) Liu, H.; Boudreau, M. A.; Zheng, J.; Whittal, R. M.; Austin,
P.; Roskelley, C. D.; Roberge, M.; Andersen, R. J.; Vederas, J. C. J. Am.
Chem. Soc. 2010, 132, 1486. (b) Schulz, A.; Kluver, E.; Schulz-Maronde, S.;
Adermann, K. Biopolymers 2005, 80, 34.
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ꢀ
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r
10.1021/ol1028755
Published on Web 01/14/2011
2011 American Chemical Society

developed thiol protecting groups9 either cannot be removed
by hydrazine or are unstable to SPPS.
To solve the above problems, we conjecture that the new
protecting group can exploit the Acm strategy by using an
acylamidomethyl group that is stable to SPPS. As a con-
sequence, the challenge becomes to design a hydrazine-
based approach to rupture the amide bond in the acylami-
domethyl moiety. Noting the difficulty of amide bond
cleavage under mild conditions, we turn to the idea of
using intramolecular acyl transfer to promote the depro-
tection reaction. Previous studies by the groups of Suggs16
and Hansen17 provide interesting examples for rapid cleav-
age of unactivated amide bonds at neutral pH (Figure 1).
Accordingly, we designed Hgm and Hqm groups (nomen-
clature for Hgm and Hqm is explained in the Supporting
Information) for the protection of a thiol (Figure 1), in
which the OAc moiety is removed by aqueous hydrazine to
trigger the deprotection event.
Figure 1. Design of new thiol protecting groups.
peptides. The tBu and MeBzl (4-methylbenzyl) protecting
groups have been shown to be useful in the stepwise
formation of disulfide bridges,5,11 but their deprotection
requires harsh conditions. For practical purposes, the
method of choice is the use of an acetamidomethyl (Acm)
group7,12 that is compatible with both tert-butoxycarbonyl
(Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc) SPPS.
However, the deprotection of Acm either needs touse toxic
heavy metals7,12 or may cause side reactions (e.g., iodina-
tion at the Trp and Tyr residues).13 Thus, new protec-
tion groups for the Cys side chain are still needed not
only to overcome the problems associated with Acm
but also to permit a more adaptable manipulation of
Cys residues.
Scheme 1. Synthesis of Hqm-Protected Cys
In our studies on protein chemical synthesis,14 we ques-
tion the possibility of developing a new Cys side chain
protecting group that can be removed by aqueous hydra-
zine. Previous studies on protein chemistry have shown
that the aqueous hydrazine condition provides a mild and
operationally simple method for the removal of ester protec-
tion of alcohols.15 The hydrazine method is also compatible
with SPPS and ligation chemistry as demonstrated in the
synthesis of full-length glycoproteins such as diptericin.15
Thus, we envisage that a hydrazine-sensitive, yet stable-to-
SPPS, thiol protecting group would be highly useful for
peptide and protein chemistry. However, the previously
The synthesis of Hqm-protected Cys is illustrated in
Scheme 1. The previously made 117,18 (as a racemic mixture)
is converted to 2 through TBS protection and N-alkyla-
tion. Then a key step is the reaction of 2 with formaldehyde
to form an N-(hydroxymethyl)acylamide intermediate,
which without separation is converted to the correspond-
ing acylamidomethyl acetate. Nucleophilic substitution of
the OAc group by Boc-Cys-OtBu affords 3, which is con-
verted to 4 through standard protecting group transforma-
tions. Finally, 4 is convertedtoBoc-Cys(Hqm)-OH (5) and
Fmoc-Cys(Hqm)-OH (6). Note that 3-6 are synthesized
as inseparable diastereomeric mixtures. Below we will show
that these diastereomeric mixtures do not interfere with
their applications in peptide and protein chemistry. Besides,
(11) (a) Musiol, H. J.; Moroder, L. Angew. Chem., Int. Ed. 2010, 49,
7624. (b) Kl€uver, E.; Schulz-Maronde, S.; Scheid, S.; Meyer, B.; Forssmann,
W. G.; Adermann, K. Biochemistry 2005, 44, 9804.
(12) Veber, D. F.; Milkowski, J. D.; Varga, S. L.; Denkewalter, R. G.;
Hirschmann J. Am. Chem. Soc. 1972, 94, 5456. (b) Okamoto, R.; Souma,
S.; Kajihara, Y. J. Org. Chem. 2009, 74, 2494.
(13) (a) Mullen, D. G.; Weigel, B.; Barany, G.; Distefano, M. D.
J. Pept. Sci. 2010, 16, 219. (b) Wu, Z.; Alexandratos, J.; Ericksen, B.;
Lubkowski, J.; Gallo, R. C.; Lu, W. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
11587.
(14) (a) Zheng, J. S.; Cui, H. K.; Fang, G. M.; Xi, W. X.; Liu, L.
ChemBioChem 2010, 11, 511. (b) Fang, G. M.; Cui, H. K.; Zheng, J. S.; Liu,
L. ChemBioChem 2010, 11, 1061.
(16) Suggs, J. W.; Pires, R. M. Tetrahedron Lett. 1997, 38, 2227.
(17) Fernandes, N. M.; Fache, F.; Rosen, M.; Nguyen, P. L.; Hansen,
D. E. J. Org. Chem. 2008, 73, 6413.
(18) Fache, F. Synlett 2004, 15, 2827.
(19) Breil, B. Y.; Ludden, P. W.; Triplett, E. W. J. Bacteriol. 1993,
175, 3693.
(15) (a) Tan, Z.; Shang, S.; Halkina, T.; Yuan, Y.; Danishefsky, S. J.
J. Am. Chem. Soc. 2009, 131, 5424. (b) Wu, B.; Chen, J.; Warren, J. D.;
Chen, G.; Hua, Z.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2006, 45, 4116.
(c) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J. A.;
Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121, 11684.
Org. Lett., Vol. 13, No. 4, 2011
569

group (Figure 2). The deprotection is complete in approxi-
mately 8 h with a HPLC yield of 93%. Note that 12 is a
diastereomericmixture due totheuseofracemic mixtureof
1 in the beginning of synthesis. Nonetheless, both the dia-
stereomers transform to 13 with a similar speed.
Table 1. Solid-Phase Peptide Synthesis Using Hgm- and Hqm-
Protected Cys
Compared to Hqm, the deprotection of Hgm is much
slower. For model peptide 11, use of the above conditions
to remove the Hgm group requires three days to complete
the reaction to give the target peptide in 91% yield. This
observation is consistent with the previous findings by
Suggs et al.16 and Hansen et al.17 shown in Figure 1. Thus,
for practical purposes, the Hqm group is more useful.
Furthermore, we have also tested the deprotection of Hqm
in peptide 12 by using the traditional I2 and AgOAc methods
a Isolated yields.
chiral resolution of 1 can be considered if enantiomerically
pure 5 or 6 is needed. By using a similar synthetic route we
have also prepared Boc-Cys(Hgm)-OH (7) and Fmoc-
Cys(Hgm)-OH (8) (Supporting Information).
With the protected Cys 5-8 in hand, we next examined
their compatibility with SPPS by synthesizing model pep-
tides 9-12. Both the Boc and Fmoc methods are tested
(Table 1), and the results indicate that the Hgm and Hqm
groups can well tolerate the standard SPPS conditions.
Subsequently, we examined the deprotection of the Hqm
group in model peptide 12. At pH 8.5 and 37 °C, addition
of 5% (v/v) hydrazine to the solution of 12 in Gn HCl
3
(guanidine hydrochloride) causes the removal of the Hqm
Figure 3. Synthesis of trifolitoxin and the corresponding analytic
HPLC data. Observed and calculated masses for each peptide
are as follows: 16 obsd = 4706.5 Da, calcd = 4704.6 Da; 17
obsd = 4674.6 Da, calcd = 4672.5 Da; trifolitoxin obsd =
4406.9 Da, calcd = 4406.2 Da. More details can be found in the
Supporting Information.
Figure 2. Deprotection of Hqm.
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Org. Lett., Vol. 13, No. 4, 2011

developed for Acm.12,13 Both these methods can deprotect
Hqm to give 13 in over 90% yields in about 30 min. On the
contrary, our test shows that the Acm group in Boc-
Cys(Acm)--OH is completely inert to the hydrazine con-
ditions. Thus, Hqm is partially orthogonal to Acm, but
Hqm is more flexible than Acm in the deprotection.
“ligation-desulfurization” approach for protein chemical
synthesis.6
Having shown that Hqm can replace Acm in protein
ligation chemistry, we also want to emphasize that Hqm
can provide complementary utility that is not achievable
with Acm alone. This feature can be demonstrated by the
synthesis of human neutrophil defensin hNP2, which is a
29-mer peptide with three disulfide bridges.20 A previous
chemical synthesis of hNP2 by Raj et al.21 used three
orthogonal thiol protecting groups, namely, Trt, StBu,
and Acm. In our new synthesis, we use Trt, Hqm, and
Acm as the thiol protecting groups (Figure 4). The Trt
groups on Cys8and Cys28are removed whenthe peptide is
cleaved from the resin. The Cys8-Cys28 disulfide bridge is
thenformedbyusingtheDMSO oxidation in70% isolated
yield determined by weight of the peptide. Subsequently,
the Hqm groups on Cys1 and Cys29 are removed by using
aqueous hydrazine. This step is followed by DMSO oxida-
tion to generate the Cys1-Cys29 disulfide bridge in 76%
isolated yield. Finally, the Acm groups on Cys3 and Cys18
are removed by using iodine and during this process the
Cys3-Cys18 disulfide bridge is generated in 65% isolated
yield. The final product compares identical with the com-
mercial hNP2 in both HPLC and MS analysis.22 The
overall yield of the three steps for disulfide formation is
34%. By comparison, in Raj’s synthesis21 the three di-
sulfide bonds were formed in 68%, 58%, and 48% yields
with an overall yield of 19%.
To test the application of the Hqm group in protein
ligation chemistry, we chose trifolitoxin as the synthetic
target which is a 42-mer peptide antibiotic.19 Trifolitoxin
has a single Cys40 residue very close to C-terminal so that
this Cys is not suitable for the ligation. Therefore, we have
to use the “ligation-desulfurization” approach6 by chang-
ing Ala23 to Cys23 first. After the ligation is accomplished,
Cys23 must be converted back to Ala23 and during this
process Cys40 should remain intact. In our synthesis,
Cys40 is protected by the Hqm group (Figure 3) in the
peptide fragment 15 that is prepared through Fmoc SPPS.
Native chemical ligation of 15 with trifolitoxin[1-22]-
Rthioester 14 generates a 42-mer peptide 16 in 90% yield.
Desulfurization is then conducted to convert Cys23 back
to Ala23 in 85% yield by using the metal-free reduction
protocol developed by Danishefsky et al.7 Finally, the
desulfurized product 17 is treated with aqueous hydrazine
at pH 8.5 for 12 h to produce the designed full-length
trifolitoxin in 88% yield. Thus, the Hqm group provides
another useful protecting group to be used in the
In summary, in the search for a new Cys side chain
protecting group that is compatible to the solid-phase
peptide synthesis yet can be removed under mild conditions,
we developed the Hqm and Hgm groups that are readily
deprotected by using aqueous hydrazine at pH 8.5. These
groups provide more options for the manipulation of Cys
residues in complex peptides and proteins, as demonstrated
by the synthesis of a 42-mer peptide antibiotic trifolitoxin
through the “ligation-desulfurization” approach. We also
showed a new and high-yielding synthesis of human neutro-
phil defensin hNP2 using Hqm through stepwise formation
of three disulfide bridges. These results show the value and
potential of developing a hydrazine-sensitive yet stable-to-
SPPS thiol protecting group. Further optimization and
applications of the hydrazine-sensitive thiol protecting
groups are being explored in our laboratory to improve
the technology for complex peptide and protein synthesis.
Acknowledgment. This study was supported by NSFC
(Nos. 20932006 and 20802040).
Supporting Information Available. Experimental de-
tails. This material is available free of charge via the
Internet at http://pubs.acs.org.
(20) Tam, J. P.; Wu, C. R.; Liu, W.; Zhang, J. W. J. Am. Chem. Soc.
1991, 113, 6657.
(21) Raj, P. A.; Antonyraj, K. J.; Karunakaran, T. Biochem. J. 2000,
347, 633.
(22) Note that the deprotection of Hqm at pH 8.5 may cause
disulfide-bond isomerization. Our HPLC analysis indicated that this
did not occur in the synthesis of hNP2. Detailed enzymatic degradation
analysis on this problem will be reported in our ensuing studies.
Figure 4. Use of Hqm in the synthesis of hNP2.
Org. Lett., Vol. 13, No. 4, 2011
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