Mixed-Phase synthesis of glycopeptides using a n-peptidyl-2, 4-dinitrobenzenesulfonamide-Thioacid ligation strategy

Article abstract of DOI:10.1021/ol202163e

A strategy for the solid phase peptide synthesis (SPPS) and coupling of N-peptidyl and N-glycopeptidyl 2,4-dinitrobenzenesulfonamides (dNBS) with C-terminal peptidyl thioacids has been developed. The resulting N-dDNBS peptides were coupled to generate lon

Full text of DOI:10.1021/ol202163e

ORGANIC
LETTERS
2011
Vol. 13, No. 19
5298–5301
Mixed-Phase Synthesis of
Glycopeptides Using a N-Peptidyl-2,
4-dinitrobenzenesulfonamideꢀThioacid
Ligation Strategy
Partha Karmakar, Rommel S. Talan,† and Steven J. Sucheck*
Department of Chemistry, The University of Toledo, 2801 West Bancroft Street, Toledo,
Ohio 43606, United States
Steve.Sucheck@UToledo.edu
Received August 9, 2011
ABSTRACT
A strategy for the solid phase peptide synthesis (SPPS) and coupling of N-peptidyl and N-glycopeptidyl 2,4-dinitrobenzenesulfonamides (dNBS)
with C-terminal peptidyl thioacids has been developed. The resulting N-dDNBS peptides were coupled to generate longer peptides. Ligation
reactions were complete within 15 to 20 min.
There is significant interest in developing chemoselective
amide bond forming reactions for the preparation of
peptides and glycopeptides.1 Many of these amidation
reactions involve the use of peptidyl thioesters. However,
these reactions are often slow which can lead to hydrolysis
of the thioester employed and other side reactions. For
example, the direct aminolysis of peptidyl thioesters gen-
erally requires reaction times of 48 to 96 h.2,3
ureas, dithioic acid to yield thioamides, and dithiocarba-
mates to yield thioureas.4 Since those early reports, only a few
examples of the use of this chemistry for the chemoselective
formation of amide bonds have surfaced. A putaive mechan-
ism for the amidation is shown in Figure 1.4 More recently, it
2,4-Dinitrobenzenesulfonamides (dNBS) were found by
Tomkinson et al. to undergo condensation reactions with
thioacids to form amides. In addition, dNBSs react with a
range of nucleophiles such as hydroxamic acids to yield
† Current address: Peptides International Inc.
(1) (a) Nilsson, B. L.; Soellner, M. B.; Raines, R. T. Annu. Rev.
Biophys. Biomol. Struct. 2005, 34, 91. (b) Sanki, A. K.; Talan, R. S.;
Sucheck, S. J. J. Org. Chem. 2009, 74, 1886.
(2) Chen, G.; Wan, Q.; Tan, Z.; Kan, C.; Hua, Z.; Ranganathan, K.;
Danishefsky, S. J. Angew. Chem., Int. Ed. 2007, 46, 7383.
(3) Wilkinson, B. L.; Chun, C. K. Y.; Payne, R. J. Biopolymers 2011,
96, 137.
(4) (a) Messeri, T.; Sternbach, D. D.; Tomkinson, N. C. O. Tetra-
hedron Lett. 1998, 39, 1669. (b) Messeri, T.; Sternbach, D. D.; Tomkinson,
N. C. O. Tetrahedron Lett. 1998, 39, 1673.
Figure 1. Probable mechanism for the dNBSꢀthioacid ligation
reaction proposed by Tomkinson et al.4,5
r
10.1021/ol202163e
Published on Web 09/14/2011
2011 American Chemical Society

has been suggested the Meisenheimer complex decomposes
to an arylthioester which acts as an amine acylating agent.5
In the past, thioacids have been used in combination
with N-terminal dNBS amino acid methyl esters6 to form
glycosyl, peptidoglycosyl, and peptidyl amide linkages. We
recently investigated the coupling of thioacids with N-
glycosyl-2,4-dinitrobenzenesulfonamides to afford N-gly-
cosyl amides.7 Further, Crich et al. demonstrated a solu-
tion phase peptide synthesis using combinations of
electron-deficient peptidyl benzenesulfonamides capable
of reacting with peptidyl thioacids with differential
reactivity.5 Seeking to further expand this amidation reac-
tion involving peptidyl thioacids and electron-deficient
peptidyl benzenesulfonamides we were interested in devel-
oping an Fmoc-based SPPS of N-dNBS peptides to access
longer N-dNBS peptides. We also desired to ligate the
resulting N-peptidyl sufonamide with an SPPS-derived
peptidyl thioacid to demonstrate a mixed-phase synthesis
of peptides and glycopeptides using the chemistry. We
chose to work with a glycopeptide sequence based on a
MUC1 tandem repeat based on our interest in the target.8
MUC1 tandem repeats are a heavily glycosylated
sequences that are part of the MUC1 transmembrane
protein. Aberrant MUC1 glycoforms are associated
with cancers of epithelial origin and are relevant targets
for glycopeptide-based anticancer therapeutics.9ꢀ11 The
20 amino acid tandem repeat possesses several possible
glycosylation sites at the threonine and serine residues
found along the repeating sequence NH2-PDTRPAPG-
STAPPA14H15G16VTSA-COOH, and synthetic access
to pure forms of the individual MUC1 glycoforms is
desirable. We investigated ligation at the H15-G16 and
A14-H15 sites to evaluate the dNBSꢀthioacid coupling
strategy with the simultaneous presence of multiple
unprotected functional groups, such as imidazole, car-
boxyl, and hydroxyl in the MUC1 fragments to demon-
strate the chemoselectivity of the amidation.
into an SPPS as well. To begin our study, we performed a
screening of conditions potentially useful for introducing
2,4-dinitrobenzenesulfonyl chloride (dNBS-Cl) on NH2-
Ala-Wang resin. The combination of dichloromethane
and pyridine was found optimal for introducing the dNBS
group onto peptides in solution phase.6a On solid phase
however, increased equivalents of dNBS-Cl (4 equiv) were
required for completion of the sulfonation. Other condi-
tions that were explored included CH2Cl2ꢀDIEA, DMFꢀ
pyridine, DMFꢀDIEA, and CH2Cl2ꢀpyridineꢀDMAP
(0.1 equiv), but all appeared unsatisfactory. The sulfonyla-
tion took 4 h to reach completion, and the reaction
progress was monitored by a Kaiser test.13 The dNBS-
alanine was cleaved from the resin by TFA treatment to
give the dNBS-alanine as the primary product. It was
notable that the dNBS group was stable to the acidic
cleavage condtions. After the optimization of the sufona-
tion conditions, the synthesis of longer 2,4-dinitrobenze-
nesulfonyl peptides and glycopeptides was attempted. The
peptides were manually assembled on preloaded Wang
resin using Fmoc chemistry. Coupling of the amino acids
was accomplished using PyBOP, HOBt, and DIEA in
DMF. The dNBS group was installed in the last coupling
step using 4 equiv of dNBS-Cl and 10 equiv of pyridine in
dichloromethane.
Table 1. N-Peptidyl-2,4-dinitrobenzenesulfonamides
SPPS is a common method for procuring medium sized
peptide fragments, and the coupling of o- and p- nitroben-
zenesulfonyl groups to the N-terminus of a peptide bound
to a rink amide MBHA resin has been reported.12,13 Thus,
it seemed feasible that dNBS groups could be introduced
(5) Crich, D.; Sharma, I. Angew. Chem., Int. Ed. 2009, 48, 7591.
(6) (a) Crich, D.; Sana, K.; Guo, S. Org. Lett. 2007, 9, 4423. (b) Crich,
D.; Sasaki, K.; Rahaman, M. Y.; Bowers, A. A. J. Org. Chem. 2009, 74,
3886.
(7) Talan, R. S.; Sanki, A. K.; Sucheck, S. J. Carbohydr. Res. 2009,
344, 2048.
(8) Sarkar, S.; Lombardo, S. A.; Herner, D. N.; Talan, R. S.; Wall,
K. A.; Sucheck, S. J. J. Am. Chem. Soc. 2010, 132, 17236.
(9) Gendler, S. J.; Lancaster, C. A.; Taylor-Papadimitriou, J.; Duhig,
T.; Peat, N.; Burchell, J.; Pemberton, L.; Lalani, E.-N.; Wilson, D. J.
Biol. Chem. 1990, 265, 15286.
(10) Finn, O. J.; Jerome, K. R.; Henderson, R. A.; Pecher, G.;
Domemech, N.; Magarian-Blander, J.; Barratt-Boyes, S. M. Immunol.
Rev. 1995, 145, 61.
€
(11) Brossart, P.; Schneider, A.; Dill, P.; Schammann, T.; Grunebach,
€
F.; Wirths, S.; Kanz, L.; Buhring, H.-J.; Brugger, W. Cancer Res. 2001, 61,
6846.
(12) (a) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119,
2301. (b) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1998, 120, 2690.
(13) Chan, W. C.; White, P. D. In Fmoc Solid Phase Peptide Synth-
esis: A Practical Approach; Chan, W. C., White, P. D., Eds.; Oxford
University Press: New York, NY, 2000; Chapter 3, pp 42ꢀ76.
Org. Lett., Vol. 13, No. 19, 2011
5299

The successful on-resin coupling of dNBS at a variety of
amino acids and in the presence of protected glycan
provides peptide fragments for ligation at relatively hin-
dered sites (Table 1). Cleavage of peptides from the resin
was generally effected by TFA in the presence of various
nucleophilic additives to capture highly reactive cationic
species.13 The TFAꢀTIPSꢀH2O (95:2.5:2.5) cleavage
mixture14 not only worked well as a cleavage mixture but
also acted as a global deprotecting system for these pep-
tides. The dNBS, which remains attached to the peptide
through cleavage from the resin, has not been shown to
survive TFA treatment until now. After cleavage, the
peptides were isolated by reversed phase HPLC using
C-8 column monitoring at a 254 nm wavelength.
Scheme 2. Access of Peptidyl Acid 8 from Resin and Its
Conversion into Corresponding Peptidyl Thioester 9 and
Thioacid 10a
Scheme 1. Coupling between Fmoc-Histidinyl Thioacid and dNBS-
Glycopeptide 4 Using ThioacidꢀSulfonamide Ligation Chemistry
a Compound 10 was directly taken to the next ligation step without
further purification.
Scheme 3. Coupling between Fmoc-Dipeptidyl Thioacid and
dNBS-Glycopeptide 5 Using ThioacidꢀSulfonamide Ligation
Chemistry
To demonstrate the utility of the N-dNBS peptides the
solution phase dNBSꢀthioacid ligation was first executed
with dNBS-glycopeptide 4 and Fmoc-histidine thioacid 6
(Scheme 1). The Fmoc-histidine thioacid was obtained
from Fmoc-N(trityl)-histidine by coupling with tritylthiol
in the presence of HATU/DIEA in DMF followed by
global deprotection with TFAꢀTIPSꢀCH2Cl2 (50:5:45).
The ligation was performed in the presence of cesium
carbonate at 0.5 M total concentration of the reactants
in DMF. The reaction reached completion in 20 min
(monitored by ESI-MS). Semipreparative reversed phase
HPLC purification afforded the hexapeptide 7 in 71%
yield (with respect to the dNBS-glycopeptide 4) (m/z
calculated for C52H68N9O19 is 1122.3 [MþHþ], found:
1122.5).
Dipeptide 8 was obtained from SPPS by manual assem-
bling of the amino acids on Barlos’s (chlorotrityl) resin.
Coupling of the amino acids was effected using PyBOP,
HOBt, and DIEA in DMF. Cleavage from the resin with
(14) Teixido, M.; Albericio, F.; Giralt, E. J. Peptide Res. 2005, 65,
153.
5300
Org. Lett., Vol. 13, No. 19, 2011

TFEꢀAcOHꢀDCM (2:2:6) followed by azeotroping the
filtrate with toluene afforded the dipeptidyl acid in con-
siderably pure form. We selected Barlos’ resin because the
mild conditions required to cleave the peptide chain from
this support are compatible with many common side-chain
protection groups, and maintaining these protecting groups
is necessary prior to conversion of the C-terminal acid group
to a thiotrityl ester. However, the attempt to convert dipep-
tide 8 into the corresponding thiotrityl ester 9 ended with a
maximum of a 36% yield. Hence, a more direct route to the
thioacid from the peptidyl acid appeared to be necessary.
A one-pot conversion of N-terminallyprotectedpeptidyl
acid into thioacid has recently been reported using CDI
and Na2S in acetonitrile.15 In our case, a combination of
CDI and the less basic NaHS in dichloromethane was
superior for obtaining the dipeptide thioacid 10 from 8
(Scheme 2). The peptidyl thioacid 10 was directly used in
ligation with peptide 5 without extensive purification, and
the reaction completed in 15 min (monitored by ESI-MS).
The pure glycosylated MUC1 octapeptide 11 was afforded
by reversed phase HPLC with a 67% yield (with respect
to the dNBS-glycopeptide 5) (m/z calculated for C60H80-
N11O21 is 1290.5 [MþHþ], found: 1291.1) (Scheme 3). No
epimer of 7 or 11 was identified by mass spectrometry of
isolated HPLC peaks.
The successful ligation of an SPPS derived N-arylsulfo-
nated MUC1 glycopeptide containing an unprotected
amino acid side chain and O-linked glycans using the
dNBSꢀthioacid ligation chemistry suggests that this meth-
odology is efficient and relatively chemoselective. The ease
with which we have synthesized the N-dNBS peptides on
the solid phase and coupled them with peptidyl thioacids,
we believe, facilitates the adoption of this chemistry for
mixed-phase peptide synthesis.
Acknowledgment. This work was supported by the
National Institutes of Health (GM094734).
Supporting Information Available. Experimental de-
tails of synthesis, NMR spectra, and HPLCs of com-
pounds 1ꢀ9 and 11. This material is available free of
charge via the Internet at http://pubs.acs.org.
(15) Assem, N.; Natarajan, A.; Yudin, A. K. J. Am. Chem. Soc. 2010,
132, 10986.
Org. Lett., Vol. 13, No. 19, 2011
5301