Synthesis of human growth hormone-releasing hormone via three-fragment serine/threonine ligation (STL)

Article abstract of DOI:10.1039/c3ob41027g

The synthesis of human growth hormone-releasing hormone (hGH-RH), by the chemoselective serine/threonine ligations (STLs) of three unprotected peptide fragments, is reported. To allow for the multiple-fragment ligation, we chose the Msz (p-(methylsulfinyl

Full text of DOI:10.1039/c3ob41027g

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Synthesis of human growth hormone-releasing
hormone via three-fragment serine/threonine
ligation (STL)†
Cite this: Org. Biomol. Chem., 2013, 11,
5584
Received 17th May 2013,
Accepted 9th July 2013
Yinfeng Zhang, Tianlu Li and Xuechen Li*
DOI: 10.1039/c3ob41027g
www.rsc.org/obc
The synthesis of human growth hormone-releasing hormone
(hGH-RH), by the chemoselective serine/threonine ligations (STLs)
of three unprotected peptide fragments, is reported. To allow for
the multiple-fragment ligation, we chose the Msz (p-(methylsulfi-
nyl)benzyloxycarbonyl) group, which is compatible with the
preparation of peptide salicylaldehyde esters via Fmoc-SPPS and
readily removed by reductive acidolysis, to protect the serine and
threonine residue at the N-terminus.
Revolutionary work in the development of chemical ligation
methods enabling protein chemical synthesis1 includes native
chemical ligation (NCL),2 which relies on the N-terminal
cysteine residue to mediate a chemoselective union reaction of
two side-chain unprotected peptide segments with a natural
peptidic linkage (Xaa-Cys) at the conjunction site. Since
cysteine is one of the lowest abundant amino acids (1.7%
occurrence)1c in natural polypeptides and proteins, many
efforts have been devoted to searching for the chemoselective
ligation at other amino acid sites, including a menu of comp-
lementary cysteine-free ligation approaches.3–5 These methods
have provided important additions to the toolbox of chemical
ligation.
In our own efforts we have recently reported a serine/threo-
nine ligation (STL) enabling peptide/protein convergent syn-
thesis.6 A side-chain unprotected peptide segment with a
C-terminal salicylaldehyde ester chemoselectively reacts with
another side-chain unprotected peptide segment containing
an N-terminal serine or threonine residue to afford an N,O-
benzylidene acetal linked product. The resulting acetal group,
upon treatment with acid, is removed to restore the natural
peptide bond (Xaa-Ser/Thr) at the ligation site (Fig. 1). As
serine and threonine residues are very abundant in natural
polypeptides and proteins (12.7% occurrence in nature),1c
Fig. 1 Serine/threonine ligation (STL).
serine/threonine ligation (STL) may offer new opportunities for
convergent protein synthesis.
In this report, we describe the synthesis of human growth
hormone-releasing hormone (hGH-RH) via serine/threonine
ligation. hGH-RH is produced in the arcuate nucleus of the
hypothalamus, and it stimulates growth hormone (GH)
secretion by binding to the GH-RH receptor.7 The acylated
GH-RH analog tesamorelin has been approved by the US FDA
in 2010 for the treatment of lipodystrophy in HIV patients
under highly active antiretroviral therapy. Thus, we chose to
synthesize acetylated hGH-RH in this study: Ac-YADAIFTN-
SYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL-NH2. Issues
associated with the conventional stepwise synthesis of longer
peptide therapeutics include the low overall yield and, more
importantly, difficulties in obtaining the product in high
purity under reproducible conditions, because the deletion
sequences and residual protecting groups can cause contami-
nation.8 Thus, convergent synthesis via chemical ligation may
offer advantages in this regard.9 The synthesis of hGH-RH also
served as a model to probe the conditions to realize C → N
multi-fragment serine/threonine ligations.
Department of Chemistry, The University of Hong Kong, Hong Kong, P. R. China.
E-mail: xuechenl@hku.hk; Fax: +86 85228571586
†Electronic supplementary information (ESI) available: Details of experimental
procedures. See DOI: 10.1039/c3ob41027g
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Fig. 3 Synthesis of peptide SAL-esters via Fmoc-SPPS.
(p-(methylsulfinyl)benzyloxycarbonyl)14 in the serine/threo-
nine chemical peptide ligation. Msz for amino group protec-
tion is stable under both basic and acidic conditions, but
readily cleavable under reductive acidolysis conditions via the
Fig. 2 Retro-synthetic scheme of hGH-RH via STL.
A
three-fragment ligation was devised to synthesize Mtz group (p-(methylthio)benzyloxycarbonyl). According to the
hGH-RH by ligating three segments at Leu17–Ser18 and Met27– general procedure, we prepared the peptide SAL ester (Fig. 3).
Ser28, respectively (Fig. 2). We encountered a critical issue and Surprisingly, when we checked the quality of the peptide
challenge: a C → N multi-fragment ligation strategy required (Msz-SARKLLQDIM)-Nbz prior to the on-resin phenolysis, a signi-
the N-terminal Ser/Thr of the middle fragment to be tempor- ficant amount of the peptide product without Msz but
arily protected. Such a temporary “good” protecting group with a molecular weight 16 Da higher was observed, which
should be orthogonal to the solid phase peptide synthesis was presumed to be compound 4. It is likely that the Msz
(SPPS) of the peptide salicylaldehyde esters, and readily re- group underwent oxygen exchange with the methionine
movable for the subsequent ligation. Furthermore, a hydrophilic residue (2 ⇌ 3),15 thereby leading to the removal under acido-
protecting group would be advantageous to help solubilize the lysis (Fig. 4).
peptide.
To avoid the abovementioned problem, we decided to use a
According to the retrosynthetic plan (Fig. 2), we first chose methionine sulfoxide residue for the synthesis of the peptide
to use the Fmoc group as the N-terminal serine residue pro- (18–27)-SAL ester. Thus, peptide Msz-SARKLLQDIM(O) SAL-
tecting group, although its hydrophobicity made it not ideal. ester 5 was synthesized with ease via on-resin phenolysis of
To prepare the necessary peptide salicylaldehyde (SAL)-ester, peptide Nbz. Next, peptide SAL ester 5 and peptide 6
we adopted an on-resin phenolysis of the Fmoc-SPPS generated SRQQGESNQERGARARL-NH2 were dissolved in pyridine
peptide N-acyl-benzimidazolinone (Nbz).10 Such a strategy could acetate buffer (1 : 1, mole : mole). The progress of the reaction
provide peptide SAL-esters efficiently and cleanly, without was monitored by LC-MS. After 13 h at room temperature, the
observing epimerization (Fig. 3).6b However, when we syn- pyridine acetate was removed and the reaction mixture was
thesized peptide N-acyl-benzimidazolinone (Nbz) with N-termi- treated with TFA–H2O to afford hGH-RH (18–44) (Fig. 5).
nal serine protected with an Fmoc group prior to the on-resin
Next, we explored the condition to remove the Msz group.
phenolysis, we observed a significant amount (∼25%) of Some commonly used conditions (i.e. NH4I–TFA–DMS,16
the peptide product without the N-terminal Fmoc group. TMSCl–DMS,17 and TMSBr–thioanisole18) gave either a messy
It was likely that the Fmoc group was removed by the excess or no reaction. Gratifyingly, a cocktail of TMSOTf (1.0 M)–
of DIEA used during the cyclization step.11,12 Thus, the thioanisole (1.0 M)–TFA19 cleanly removed the Msz group;
Fmoc group was not a proper terminating group to serve our at the same time, methionine sulfoxide was also reduced.
purpose.
When TMSOTf was reduced to 0.2 M, the cocktail was still
We next started with using an azide group as the temporary effective. As a control, 0.2 M TMSOTf in TFA did not work.
protecting group (N3-SARKLLQDIM-SAL ester). The azide Thus peptide 7 was obtained in 33% yield over two steps
group has been previously used in peptide synthesis.13 It is (ligation and reductive acidolysis) after one reverse-phase
rather stable, while readily removable under reducing con- HPLC purification.
ditions. However, during the synthesis of hGH-RH (18–27)
with N3-Ser18(tBu)-OH, two peaks with the desired molecular GARARL-NH2) in hand, we continued to ligate it with hGH-RH
weight were observed, likely due to the epimerization.
(1–17) (Ac-YADAIFTNSYRKVLGQL SAL-ester) between Leu and
Having hGH-RH (18–44) (SARKLLQDIMSRQQGESNQER-
As both Fmoc and azide were not “good” temporary pro- Ser to afford the full-length hGH-RH (Fig. 5) in 25% after
tecting groups, we continued to explore the utility of Msz reverse-phase HPLC purification.
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Fig. 5 Synthesis of hGH-RH.
Notes and references
Fig. 4 Methionine reducing Msz cleavage.
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Conclusions
In summary, we have synthesized human growth hormone-
releasing hormone via C → N three-fragment serine ligation.
Msz (p-(methylsulfinyl)benzyloxycarbonyl), which is compati-
ble with the preparation of peptide SAL esters, is found to be a
proper temporary protecting group for the N-terminal serine
and threonine residues of the middle peptide fragment.
Furthermore, its hydrophilic properties provide advantage in
the purification of the peptide. The current studies pave the
way for the application of serine/threonine ligation in the
synthesis of peptides/proteins with increased complexity and
length.
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Acknowledgements
We thank the General Research Fund (HKU703811P, 707412P)
of the Research Grants Council of Hong Kong, the Peacock
Program-Project Development Fund (KQC201109050074A), and
the National Basic Research Program of China (973 Program,
2013CB836900) for financial support. We thank Ms Helen
Farrants of the University of Edinburgh for suggestions on
editing.
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