Examination of mercaptobenzyl sulfonates as catalysts for native chemical ligation: Application to the assembly of a glycosylated Glucagon-Like Peptide 1 (GLP-1) analogue

Article abstract of DOI:10.1039/c4cc09502b

3/4-Mercaptobenzyl sulfonates were investigated as aryl thiol catalysts for native chemical ligation (NCL). Whilst catalysing NCL processes at a similar rate to 4-mercaptophenyl acetic acid (MPAA), the increased polarity and solubility of 3-mercaptobenzyl

Full text of DOI:10.1039/c4cc09502b

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Examination of mercaptobenzyl sulfonates as
catalysts for native chemical ligation: application
to the assembly of a glycosylated Glucagon-Like
Peptide 1 (GLP-1) analogue†
Cite this: Chem. Commun., 2015,
51, 3208
Received 27th November 2014,
Accepted 5th January 2015
Ben Cowper,a Tsz Mei Sze,a Bhavesh Premdjee,a Aileen F. Bongat White,b
DOI: 10.1039/c4cc09502b
Andrew Hackingb and Derek Macmillan*a
www.rsc.org/chemcomm
3/4-Mercaptobenzyl sulfonates were investigated as aryl thiol catalysts
for native chemical ligation (NCL). Whilst catalysing NCL processes at a
similar rate to 4-mercaptophenyl acetic acid (MPAA), the increased
polarity and solubility of 3-mercaptobenzyl sulfonate in particular may
favour its selection as NCL catalyst in many instances.
Native chemical ligation (NCL), where peptide thioesters and
cysteinyl peptides combine to form native peptide linkages,1,2 has
benefited significantly from the use of 4-mercaptophenyl acetic
acid (MPAA) as a catalyst.3 MPAA (1, Scheme 1) is relatively non-
malodorous, has good water solubility under most commonly
employed NCL reaction conditions, and catalyses NCL through
formation of a reactive aryl thioester intermediate (3).4,5
These advantageous properties have resulted in the near
ubiquitous adoption of MPAA in NCL reactions. However, MPAA is
rather hydrophobic, has poor solubility below pH 7, and it can often
co-elute with NCL products during HPLC purification.6 In contrast
sodium 2-mercaptoethane sulfonate (MESNa, 4), whilst less reactive,
is significantly more polar and elutes with buffer components.
Consequently, in the absence of established MPAA alternatives, we
examined 4-mercaptobenzyl sulfonate (4-MBSA, 5) and 3-mercapto-
benzyl sulfonate (3-MBSA, 6) as catalysts (Scheme 2).7 Ideally,
they would possess the desirable polarity profile of MESNa yet, as
aryl thiols, exhibit similar potential for catalysis to MPAA.
5 and 6 were readily prepared on gram scale from the corres-
ponding nitrobenzyl chlorides (7/8) in four steps (Scheme 3).8,9
Notably, throughout the synthesis no flash column chromatogra-
phy was required.
Scheme 1 In situ thiol–thioester exchange during NCL converts the
2-mercaptoethanesulfonate (MESNa) thioester 2 to the highly reactive
MPAA thioester 3.
Scheme 2 MPAA analogues investigated as NCL catalysts.7
With 5 and 6 in hand we were pleased to observe that they
both eluted 10 minutes earlier than MPAA on a C18 analytical
reverse-phase column10 and we next examined application in
NCL. To compare their performance as catalysts model ligations
were conducted using H-LYRAG-SCH2CH2SO3Na (15) and H-CRAFS-
OH (16) over a pH6–pH8 range (Fig. 1).11 The thioester component
was conveniently prepared from H-LYRAGC-OH (17) in near quanti-
tative yield via N-S acyl shift.12–17NCL progress was then monitored
by analytical reverse-phase HPLC and confirmed by LC-MS.
At this stage the anticipated instability of 4-MBSA (5) became
evident.19 Whilst preparing 0.25 M aqueous stock solutions of
4-MBSA for immediate use at 50 mM final concentration in NCL
reactions allowed us to evaluate it as a catalyst, at higher
concentrations 4-MBSA rapidly precipitated and/or polymerised.
a Department of Chemistry, University College London, 20 Gordon Street, London,
WC1H 0AJ, UK. E-mail: d.macmillan@ucl.ac.uk
b Dextra Laboratories Ltd, Science and Technology Centre, Earley Gate,
Whiteknights Road, Reading, RG6 6BZ, UK
† Electronic supplementary information (ESI) available: Experimental procedures
for the synthesis of 5 and 6. See DOI: 10.1039/c4cc09502b
3208 | Chem. Commun., 2015, 51, 3208--3210
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Scheme 3 Synthesis of mercaptobenzyl sulfonates 5 and 6.
Scheme 4 (a) Sequence of glycosylated GLP-1(7-36) 18. (b) Simply
glycosylated analogue 19. (c) Preparation of 19 from thioester 20 and
cysteinyl glycopeptide 21 employing 50 mM 5 or 0.1 M 6.
production of NCL components 20 and 21 we successfully
assembled 19 in 8 M Urea using 50 mM 4-MBSA as catalyst
(Scheme 4c). In this case we observed similar catalytic perfor-
mance to 50 mM MPAA.21 However, the poor stability of 5 at
neutral pH led us to focus on 3-MBSA (6) since 1 M solutions
were fully soluble at pH7 and compatible with 6 M guanidine
hydrochloride (Fig. 2).
The 0.1 M 3-MBSA catalysed reaction was successful but
appeared to proceed significantly more slowly than the corres-
ponding MPAA catalysed process (Fig. 2a and b), requiring an extra
2 h reaction time to reach a similar level of conversion. It is clear
Fig. 1 (a) Preparation of LYRAG thioester 15 and reaction with 16 in NCL
reactions in the presence of thiol catalyst. (b) 5 and 6 are compared with
MPAA as NCL catalysts at pH 6.18
Consequently no reliable kinetic data could be obtained for the
5-mediated ligation between 15 and 16 at typical working con-
centrations (0.1–0.2 M) at pH 6 or above.
Interestingly 5 also precipitated from all solutions prepared
in 6 M guanidine hydrochloride, a common NCL component
yet did not precipitate when identical solutions were prepared
in 8 M urea, allowing 4-MBSA mediated ligation reactions
between peptides of low solubility to take place. We took
advantage of this to assemble a glycosylated Glucagon-Like
Peptide-1 (GLP-1) analogue (Scheme 4). GLP-1 (residues 7-36)
has gained attention as a therapeutic for the treatment of type 2
diabetes and affects glucose control by stimulating insulin
secretion. Although GLP-1 is not naturally glycosylated, a recent
study showed that the addition of carbohydrate moieties at N34
(18, Scheme 4a) prolonged the half-life of the peptide in vivo,
thus increasing its potential for therapeutic applications.20 We
envisaged that GLP-1 analogues of 18 could be reached via
simply glycosylated 19 (Scheme 4b). Following straightforward
Fig. 2 Analytical HPLC analysis of (a) 3-MBSA and (b) MPAA catalysed
ligation between 20 and excess 21 (0.82 mM 20, 1.4 mM 21, 0.1 M catalyst,
6 M guanidine hydrochloride, 0.1 M Na phosphate buffer; pH 7, 35 mM
TCEP). (c and d) Comparison of semi-preparative HPLC purification for
(c) the 3-MBSA catalysed reaction and (d) the MPAA catalysed reaction.
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application in processes relevant to N-S acyl transfer and
peptide transamidation reactions where MPAA has already
been employed advantageously, despite its low solubility under
these conditions.17,28
The Authors acknowledge financial support from EPSRC
(EP/J007560/1) and Dextra Laboratories UK. We are also grate-
ful to Dextra Laboratories UK for providing tetrasaccharide
oxazoline 23.
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´
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3210 | Chem. Commun., 2015, 51, 3208--3210
This journal is ©The Royal Society of Chemistry 2015