Scope and limitation of side-chain assisted ligation

Article abstract of DOI:10.1002/psc.1336

Side-chain assisted ligation is an auxiliary-mediated ligation strategy in which a thiol bearing cyclohexane or cyclopentane is attached to the side-chain of Asp, Glu, Ser or Thr to function in a similar manner to Cys in a native chemical ligation. Follow

Full text of DOI:10.1002/psc.1336

Protocol
Received: 5 August 2010
Revised: 20 October 2010
Accepted: 1 November 2010
Published online in Wiley Online Library: 3 February 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1336
Scope and limitation of side-chain assisted
ligation
Liat Spasser, K. S. Ajish Kumar and Ashraf Brik∗
Side-chain assisted ligation is an auxiliary-mediated ligation strategy in which a thiol bearing cyclohexane or cyclopentane is
attached to the side-chain of Asp, Glu, Ser or Thr to function in a similar manner to Cys in a native chemical ligation. Following
c
the ligation step, the auxiliary is removed, without product isolation, under alkaline conditions. Copyright ꢀ 2011 European
Peptide Society and John Wiley & Sons, Ltd.
Keywords: peptide ligation; aminoacyl transfer; protein synthesis; native chemical ligation; removable auxiliary
REACTION SCHEME
O
O
H
N
Auxiliary
SR
CONH
L-Y-R-A
R = -(CH
H2N
O
O
O
2
)
NH
O
2 2
2
Auxiliary
O
O
2
NH
O
S
Thiol exchange
H2N
O
S
H3N
A-R-Y-S
COOH
H
N
H
N
L-Y-R-A
O
6a
H2N
A-R-Y-S COOH
N
H
=
O
Auxiliary
cyclohexyl
O
Auxiliary
O
O
2
NH
O
HO
2
Acyl transfer
HS
O
pH~10
H
N
H2N
L-Y-R-A
COOH
A-R-Y-S
O
N
N
H
H
H
H2N L-Y-R-A
N
A-R-Y-S COOH
O
O
N
N
H
H
O
O
GENERAL OPTIMIZED PROCEDURE
General procedure for side-chain assisted ligation: Unprotected peptide segments (1.00 eq of peptide 6a and 1.20 eq of LYRAG-SR) (Reaction
Scheme) were dissolved in 0.2 M phosphate buffer (pH 8.0, purged with argon for 10 min) containing 6 M Gn·HCl (denaturated reaction
conditions) to a final concentration of 5 mM and incubated at 37 °C. The ligation attained completion after ~8 h and the auxiliary was
removed by adjusting the pH of the ligation mixture to pH~10 using 1N NaOH, and was kept for 5 min at rt. The reaction was monitored
using analytical reverse-phase HPLC on a C18 column (Jupiter 5 micron, 300A, 150 × 4.60 mm) and a linear gradient (0-90%B) over 15 min.
Purification by preparative reverse phase HPLC on a C18 column (Jupiter 10 micron, 300A, 250 × 21.20 mm) using identical gradient afforded
the unmodified ligation product 8 in 68 % isolated yield.
Scope and Comments
and chemoselective S–N acyl transfer. After completion of ligation,
in situ hydrolysis of the auxiliary (pH ∼10) furnishes the unmodified
Native chemical ligation (NCL) continues to lead as the method
of choice for chemical and semi-synthesis of proteins [1]. It also
peptide. Similar to SAL, the addition of one amino acid after the
residue to which the auxiliary is attached facilitates the ligation as
remains the basis for the quest of new chemical ligation methods
the reaction proceeds faster through 14/15 membered transition
state (TS) compared to the 12-membered TS. The attachment of
mercaptoacetic acid as an auxiliary reduces the rate of ligation
significantly [13], emphasizing the role of the cyclic auxiliary to
[2–6], including the auxiliary-mediated ligation strategies, which
show great potential in chemical protein synthesis [7–12]. We
have recently reported a removable auxiliary-assisted cysteine-
free ligation method so-called side-chain assisted ligation (SCAL)
[13]. SCAL relies on principles similar to sugar-assisted ligation
(SAL) [14,15], which is employed in glycopeptide ligation. In SCAL,
a thiol bearing cyclohexane or a cyclopentane auxiliary is attached
to the side-chain of Asp, Glu, Ser or Thr residues through an ester
bond to mimic the function of N-terminal cysteine in NCL. The
auxiliary assists ligation by allowing the capture of the thioester
peptide through a transthioesterification step followed by a rapid
∗
Correspondence to: Ashraf Brik, Department of Chemistry, Ben-Gurion Univer-
sity of the Negev, Beer Sheva 84105, Israel. E-mail: abrik@bgu.ac.il
Department of Chemistry, Ben-Gurion University of the Negev, Beer Sheva
84105, Israel
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J. Pept. Sci. 2011; 17: 252–255
Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd.

SIDE-CHAIN ASSISTED LIGATION
position the nucleophilic amine in a close proximity to the acyl
group.
1MEPVDPRLEP WKHPGSQPKT ACTNCYCKKC
CFHCQVCFIT KALGISYGRK KRRQRRRAPQ
GSQTHQVSLS KQPTSQSRGD PTGPKE86
SCAL is an efficient method for peptide ligation due to the
commercial availability of the template, easiness to couple to the
side-chain of amino acid and the facile removal of the auxiliary
without prior product isolation. The rate-limiting step in the SCAL
is S–N acyl transfer; hence, it is possible to perform ligations under
low peptide concentrations, which increases its potential in the
synthesis of proteins.
O
O
S
O
O
H
+
N
Tat 38-60
HN
NH
SR
N
O
H
Tat 63-86
HN
O
O
HS
Limitations
NH2
We attempted the total chemical synthesis of the regulatory
protein HIV-1 Tat (86 residues) assisted by SCAL [16]. As shown
in Scheme 1, SCAL was used to link the middle fragment, HIV-
1 Tat(37–60), to the C-terminal fragment, HIV-1 Tat(61–86).
The resultant ligation product was exposed to methoxylamine
to covert the N-terminal Thz to Cys [17] affording HIV-1
Tat(37–86). Subsequently, employing NCL between fragments
HIV-1 Tat(37–86) and HIV-1 Tat(1–36), the N-terminal fragment,
yielded the full-length protein with auxiliary. Although the
construction of the fragments using SCAL (60% isolated yield
for the ligation and Thz removal steps) and NCL (70% isolated) was
very successful, the removal of the auxiliary from the polypeptide
was not fully accomplished. This could be due to a specific
conformation that the polypeptide adopts under these conditions,
which could mask the ester bond from saponification. A possible
mean to overcome this issue is to use such an auxiliary that
would allow ligation at pH 7 and could be detached from the
side-chain through preferred intramolecular cyclization at basic
pH. For example, an auxiliary that is derived from pipecolic acid
would yield quinolizidone (fused bicyclic six-membered ring) in
contrary to the cyclohexane-derived auxiliary, which could yield a
less preferred seven–six fused bicyclic system. Another limitation
is formation of aspartimide by-products, in particular when Asp
is used to anchor the auxiliary, which could occur throughout
the ligation and/or during the auxiliary removal steps. However,
aspartimide formation is considerably reduced when the amino
acid subsequent to Asp is sterically hindered. In principle, the use
of backbone protection or cleavage of the ligation product with
HF would also eliminate this side reaction.
1. SCAL
2. Thz-Cys conversion
O
HN
HS
H2N
-Gly-Ser(Aux)-
Tat 38-60
Tat 63-86
NCL
Tat 1-36
O
S(CH2)2C(O)OMe
-Cys-
-Gly-Ser(Aux)-
Tat 63-86
Tat 1-36
Tat 38-60
Scheme 1. The use of SCAL in the synthesis of HIV-1 Tat.
Synthesis of 3
Compound 2 (1.10 g, 2.55 mmol) was added to a solution of
Fmoc-Asp-OAll (0.80 g, 2.04 mmol) with DIC (0.35 ml, 2.30 mmol)
and catalytic amount of DMAP in dry DCM (20 ml) at 0 ◦C. The
reaction mixture was stirred at room temperature for 12 h. The
organic solvent was concentrated and extracted with ethyl acetate
(3 × 20 ml). The combined organic layer was washed with brine,
dried over MgSO4, concentrated and purified using flash column
chromatography (hexane/ethyl acetate 3 : 1) to give 1.20 g of
3 in 72% yield. Rf 0.27 [EtOAc–Hex (1 : 2)]. 1H NMR (500 MHz,
CDCl3) δ 0.94–1.45 (m, 4H), 1.52–1.99 (m, 4H), 2.70–3.25 (m,
4H), 3.62–3.82 (m, 1H), 4.10–4.27 (m, 2H), 4.27–4.42 (m, 1H),
4.42–4.58 (m, 1H), 4.58–4.75 (m, 3H), 5.23 (d, J = 10.7 Hz, 1H),
5.31 (d, J = 17.1 Hz, 1H), 5.80–5.94 (m, 1H), 7.06–7.64 (m, 19H),
7.57–7.70 (m, 2H), 7.76 (d, J = 7.4 Hz, 2H); 13C NMR (125 MHz,
CDCl3) δ 23.7, 23.9, 24.1, 30.7, 30.7, 30.7 31.3, 36.1, 36.2, 37.0,
37.0, 47.0, 47.1, 50.2, 50.6, 51.7, 52.2, 66.2, 66.4, 67.3, 67.4, 67.8,
67.9, 75.5, 75.8, 118.7, 118.8, 119.9, 125.2, 125.3, 127.0, 127.6,
127.9, 128.1, 129.5, 131.4, 131.5, 141.2, 143.8, 144.0, 155.9, 156.2,
168.2, 168.3, 170.1, 170.2, 170.6; MALDI-TOF (matrix: α-Cyano-4-
hydroxycinnamic acid): calcd. for C49H48N2O7S.Na+ [M + Na]+:
831.31 (monoisotope); C49H48N2O7S.K+ [M +K]+ (monoisotope):
847.42, found: 831.45, 847.40.
Experimental Procedure
Synthesis of Building Block 4
Synthesis of 2
Compound 1 (1.00 g, 6.38 mmol) was added to a solution of S-
trityl-2-mercaptoacetic acid (2.13 g, 6.38 mmol) with HBTU (2.53 g,
6.66 mmol) and DIEA (3.70 ml, 21.05 mmol) in dry DMF (15 ml). The
reaction mixture was stirred at room temperature for overnight,
diluted with ethyl acetate (40 ml) and washed with water and
brine. The organic layer was dried over MgSO4, concentrated and
purified over flash column chromatography (CHCl3/MeOH 20 : 1),
to give 2 (1.30 g) in 50% yield. Rf 0.73 [CHCl3 –MeOH (10 : 1)];
1H NMR (500 MHz, CDCl3) δ 0.92–1.35 (m, 4H), 1.55–1.75 (m,
3H), 1.9 (m, 1H), 2.79 (s, 2H), 3.09–3.20 (m, 2H), 3.28–3.38 (m,
1H), 6.07 (d, J = 6.8 Hz, 1H), 7.18–7.35 (m, 9H), 7.35–7.43 (m,
6H); 13C NMR (125 MHz, CDCl3) δ 23.8, 24.3, 30.9, 34.0, 35.9, 55.9,
68.0, 74.8, 127.1, 128.1, 129.4, 143.9, 170.0; MALDI-TOF (matrix: α-
Cyano-4-hydroxycinnamic acid): calcd. for C27H29NO2S.Na+, [M
+ Na]+: 454.18 (monoisotope); C27H29NO2S.K+ [M + K]+: 470.29
(monoisotope), found: 454.20, 470.16.
Synthesis of 4
Compound 3 (1.20 g, 1.50 mmol) was dissolved in THF (25 ml);
N-methylaniline (1.58 ml, 15.00 mmol) and Pd(PPh3)4 (0.17 g,
0.15 mmol) were added subsequently. The reaction mixture
was stirred at room temperature for 1 h. After removing the
solvent under reduced pressure, the residue was subjected to
column chromatography (MeOH/CH3Cl 20 : 1) to give 4 in 90%
yield (1.03 g). Rf 0.55 [CHCl3 –MeOH (10 : 1)]; H NMR (500 MHz,
CDCl3) δ 1.14–1.64 (m, 8H), 2.76–3.24 (m, 4H), 3.79–3.95 (m,
1H), 4.12–4.24 (m, 1H), 4.24–4.38 (m, 2H), 4.55–4.69 (m, 1H),
1
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J. Pept. Sci. 2011; 17: 252–255 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

SPASSET, AJISH KUMAR AND BRIK
4.8–4.97 (m, 1H), 5.96–6.14 (m, 1H), 6.44–6.59 (m, 1H), 7.02–7.4
(m, 19H), 7.66–7.70 (m, 2H), 7.75 (d, J = 7.4 Hz, 2H)]; 13C NMR
(125 MHz, CDCl3) δ 20.2, 20.5, 22.9, 23.3, 27.3, 27.3, 28.1, 28.3,
36.1, 37.1, 37.3, 47.1, 47.1, 48.9, 49.1, 50.5, 50.5, 67.3, 67.3, 67.8,
72.7, 72.8, 119.9, 125.2, 127.0, 127.6, 128.1, 128.5, 128.6, 129.4,
131.1, 131.9, 132.1, 132.2, 132.3, 141.3, 143.7, 143.7, 144.0, 156.1,
156.1, 168.5, 168.5, 169.9, 173.0, 173.1; MALDI-TOF (matrix: α-
Cyano-4-hydroxycinnamic acid): calcd. for C46H44N2O7S.Na+ [M +
Na]+: 791.28 (monoisotope); C46H44N2O7S.K+ [M + K]+: 807.39
(monoisotope), found: 791.39, 807.38.
SPPS of Peptides with an Auxiliary Anchored to Ser or Thr
H-Ser(tBu)-2-chlorotrityl resin (0.49 mmol/g) was used for the
synthesis of peptide 7. Amino acids and HBTU were used in
fivefold excess of the initial loading of the resin. DIEA was used in
tenfold excess. Peptide coupling was performed for 30 min. Fmoc
deprotection was achieved by treatment of the resin with 20%
piperidine (3 ×3 min). Coupling of unprotected Fmoc-Ser-OH was
achieved using DIC and HOBt (each in fivefold excess). Boc-Gly-OH
was used as the N-terminal amino acid. Peptide resin was treated
with 10% N2H4 in methanol (2 ×1.5 h) to hydrolyze any undesired
ester on the side-chain of serine. Next, commercially available
Fmoc-trans-1,2-aminocyclohexane carboxylic acid (Aldrich) was
coupled to the side-chain using EDCI (fivefold excess) in the
presence of catalytic amount of DMAP in dry DCM. The Fmoc
protecting group was then removed with 20% piperidine followed
by coupling the S-Trt-mercaptoacetic acid (fivefold excess) using
HBTU/DIEA(fivefold/tenfoldexcess)couplingconditions.Cleavage
from the resin and work-up were done as described previously to
give the desired product in 60% isolated yield.
O
TrtS
Fmoc-(Asp)-OAll
DIC, DMAP
OH
OH
OH
NH2
O
NH
HBTU, DIEA
(+)
1
TrtS
2 (2a: SS)
(2b: RR)
O
O
O
O
NH
Pd(PPh3)4
NMA
O
O
NH
C(O)OH
TrtS
OH
O
1.
FmocHN
OH
O
NH
TrtS
NHFmoc
EDCI,DMAP
O
OAll
Boc
H
O
FmocHN
N
G-S-A-R-Y-S
HS
2. 20% piperidine
3. S-Trt-mercaptoacetic acid,
HBTU,DIEA
O
H
4 (4a: SSS)
(4b: RRS)
3 (3a: SSS)
(3b: RRS)
N
COOH
G-S-A-R-Y-S
H
4. TFA/H O/TIS (95:2.5:2.5)
2
7
SPPS of Peptides with an Auxiliary Anchored to Asp or Glu
Synthesis of Peptide Thioester H2N-LYRAG-SR
SPPS of peptides 5a-6a
MBHA resin (0.59 mmol/g) was washed (5 × DMF, 10 min 10%
DIEA/DMF, 5 × DMF). A solution of HBTU (fivefold excess) in DMF
was added to 3-(tritylthio)propanoic acid followed by the addition
of DIEA (tenfold excess), after 5 min the reaction mixture was
added to the resin. After 1 h, the resin was washed with DMF and
the trityl was removed using a mixture of TFA, triisopropylsilane
and water (90 : 5 : 5) (3 × 1 min). Amino acids and HBTU were used
in fivefold excess while DIEA in tenfold excess of the initial loading
of the resin. The peptide coupling was performed for 30 min. Boc
deprotection was achieved by treatment of the resin with TFA
(3 × 1 min).
H-Ser(tBu)-2-chlorotrityl resin (0.49 mmol/g) was used for the
synthesis of peptides 5a-6a. Amino acids [including the Fmoc-
Asp(auxiliary)-OH and HBTU] were used in fivefold excess of the
initial loading of the resin. DIEA was used in tenfold excess.
Peptide coupling was performed for 30 min. Fmoc deprotection
was achieved by treatment of the resin with 20% piperidine
(3 × 3 min).
Cleavage from the resin
A mixture of TFA, triisopropylsilane and water (95 : 2.5 : 2.5) was
added to the dried resin peptide. After 2 h, the resin was washed
with TFA (4 × 4 ml).
Cleavage
A mixture of TFMSA, TFA and thioanisole (2 : 8 : 1) was added to
the resin. After 1.5 h, the resin was washed with TFA (4 × 4 ml).
Work-up
The combined solutions were concentrated in vacuo. The residue
was dissolved in water, purified by preparative reverse-phase
HPLC on a C18 column (Jupiter 10 micron, 300A, 250 × 21.20 mm)
using a linear gradient (0–90%B) over 20 min (buffer A: 0.1% TFA
in water; buffer B: 0.1% TFA in acetonitrile). The fractions were
analyzed by MALDI-TOF/MS (RefleX IV, Bruker) (matrix: α-Cyano-4-
hydroxycinnamic acid). The desired fractions were collected and
lyophilized to give the product in 55–60% isolated yield.
Work-up
The combined solutions were concentrated. The residue was
dissolved in water, purified by preparative reverse-phase HPLC on
a C18 column (Jupiter 10 micron, 300A, 250 × 21.20 mm) using
a linear gradient (0–90%B) over 30 min and analyzed by MALDI-
TOF/MS (RefleX IV, Bruker) (matrix: α-Cyano-4-hydroxycinnamic
acid).
O
O
5a: AA1 = Gly
General Procedure for Chemical Ligation
O
O
5b: AA1 = Ala
5c: AA1 = Asp
5d: AA1 = His
6a: AA1 = Gly
O
NH
O
NH
Fmoc-SPPS
The ligation of unprotected peptide segments was performed as
follows: 0.2 M phosphate buffer (pH 8.0) containing 6 M Gn·HCl
was degassed with Argon for 10 min before use. Peptides were
dissolved (1 eq of peptide auxiliary and 1.20 eq of peptide
OH
TrtS
HS
FmocHN
O
H2N-AA1-XX-Ala-Arg-Tyr-Ser-COOH
4
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wileyonlinelibrary.com/journal/jpepsci Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2011; 17: 252–255

SIDE-CHAIN ASSISTED LIGATION
thioester) in the degassed buffer to a final concentration of
5 mM and incubated at 37 ◦C until the reaction shows completion
on analytical HPLC analysis. Before analytical analysis, tris(2-
carboxyethyl)phosphinehydrochloride(TCEP)(50 mM)wasadded
to reduce any disulfide bonds. The reaction was monitored using
analytical reverse-phase HPLC on a C18 column (Jupiter 5 micron,
300A, 150×4.60 mm) and a linear gradient (0–90%B) over 15 min.
4
5
Crich D, Banerjee A. Native chemical ligation at phenylalanine. J. Am.
Chem. Soc. 2007; 129: 10 064–10 065.
Chen J, Wan Q, Yuan Y, Zhu J, Danishefsky SJ. Native chemical
ligation at valine: a contribution to peptide and glycopeptide
synthesis. Angew. Chem. Int. Ed. Engl. 2008; 47: 8521–8524.
Harpaz Z, Siman P, Ajish Kumar KS, Brik A. Protein synthesis assisted
by native chemical ligation at leucine. ChemBioChem 2010; 11:
1232–1235.
Canne LE, Bark SJ, Kent SBH. Extending the applicability of native
chemical ligation. J. Am. Chem. Soc. 1996; 118: 5891–5896.
Offer J, Boddy CNC, Dawson PE. Extending synthetic access to
proteins with a removable acyl transfer auxiliary. J. Am. Chem.
Soc. 2002; 124: 4642–4646.
6
7
8
General Procedure for Auxiliary Hydrolysis
After the completion of ligation, the pH of the ligation mixture
was adjusted to pH ∼10 using 1N NaOH and was kept for 5 min
at room temperature. The hydrolysis reaction was monitored by
analytical HPLC as described above and purified as described for
peptides 5a-6a. The ligation followed by in situ removal of auxiliary
afforded the desired product in 60–70% isolated yield (ligation
product without auxiliary).
9
Dawson PE, Dirksen A. Expanding the scope of chemoselective
peptide ligations in chemical biology. Curr. Opin. Chem. Biol. 2008;
12: 760–766.
10 Low DW, Hill MG, Carrasco MR, Kent SBH, Botti P. Total synthesis of
cytochrome b562 by native chemical ligation using a removable
auxiliary. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6554–6559.
11 Kawamaki T, Aimoto S. A photoremovable ligation auxiliary for use
in polypeptide synthesis. Tetrahedron Lett. 2003; 44: 6059–6061.
12 Hojo H, Ozawa C, Katayama H, Ueki A, Nakahara Y, Nakahara Y. The
mercaptomethyl group facilitates an efficient one-pot ligation at
Xaa-Ser/Thr for (Glyco)peptide synthesis. Angew. Chem. Int. Ed. Engl.
2010; 49: 5318–5321.
Acknowledgement
We are grateful to the Israel Science Foundation for their financial
support.
13 Lutsky MY, Nepomniaschiy N, Brik A. Peptide ligation via side chain
auxiliary. Chem. Commun. 2008; 10: 1229–1231.
14 Brik A, Yang YY, Ficht S, Wong C-H. Sugar-assisted glycopeptide
ligation. J. Am. Chem. Soc. 2006; 128: 5626–5627.
15 Brik A, Wong C-H. Sugar-assisted ligation for the synthesis of
glycopeptides. Chem. – Eur. J. 2007; 13: 5670–5675.
References
1
Dawson PE, Muir TW, Clark-Lewis I, Kent SBH. Synthesis of proteins
by native chemical ligation. Science 1994; 266: 776–779.
Yan LZ, Dawson PE. Synthesis of peptides and proteins without
cysteine residues by native chemical ligation combined with
desulfurization. J. Am. Chem. Soc. 2001; 123: 526–533.
16 Ajish Kumar KS, Harpaz Z, Haj-Yahya M, Brik A. Side-chain assisted
ligation in protein synthesis. Bioorg. Med. Chem. Lett. 2009; 19:
3870–3874.
2
17 Bang D, Makhatadze GI, Tereshko V, Kossiakoff AA, Kent SB. Total
chemical synthesis and X-ray crystal structure of
a protein
3
Haase C, Rohde H, Seitz O. Native chemical ligation at valine. Angew.
Chem. Int. Ed. Engl. 2008; 47: 6807–6810.
diastereomer[d-Gln35]Ubiquitin. Angew. Chem., Int. Ed. Engl. 2005;
44: 3852–3856.
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J. Pept. Sci. 2011; 17: 252–255 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci