Transcript The Twisted Case of QWFGLM b6+

Disfavoring Macrocycle b Fragment by Constraining Torsional
+
Freedom: The Twisted Case of QWFGLM b6
Marcus Tirado1; Xian Chen1; Alfred Yeung1; Jeffery D. Steill2; Jos Oomens2; and Nick C. Polfer1
1
University of Florida, Gainesville, FL
2FOM Institute “Rijnhuizen”, Niewegein, The Netherlands
Methods
Overview
 The sequence motif QWFGLM and analogues were investigated to determine whether some
amino acid residues are capable of suppressing macrocycle formation in the corresponding
b6+ fragments.
IRMPD Results
Reference Experiment
(A) 0.7
Effect of Proline upon Oxazolone Formation
(cyclo)QWFGLM
 Proline and Aminomethylbenzoic acid (Amb) were chosen because they possess limited
torsional flexibility and may impede macrocycle formation.
(B)
(b7) QPWFGLMPG
(b6) QWFGLMPG
 A CEM Discover System was utilized to synthesize 0.1 mmol scale of the corresponding
peptide.
(b6) QPFGLMPG
0.6
(cyclo) QPFGLM
Yield
 The underlying CID chemistry of protonated peptides is governed by nucleophilic attacks
giving rise to b fragments with a C-terminal oxazolone ring.1,2
 The oxazolone ring could be subjected to a nucleophilic attack from the N-terminus ,
which then results in a “head-to-tail” macrocycle.
0.4
0.3
Results and Discussion
Stictly Macrocycle Formation
 Figure A: Sequence motif QWFGLM b6+ ion is strictly macrocyclic.
0.2
 Figure B : Proline Experiments: Figure B b7+ fragment exhibits a pronounced oxazolone band
and b6+ ion a minor oxazolone band with Proline in the second position.
0.1
 Recently, our group has demonstrated that for oligioglycines and Leu-enkephalin , mid
sized b fragment ions exist as a mixture of oxazolone and macrocycle structures in the
gas phase.3,4
Figure C : 4-Aminomethylbenzoic Acid Experiments: Demonstrates a minor oxazolone band
for the b6+ fragment in the fifth position:
Can some residue favor Oxazolone?
0.0
1200
 This raises the question: Is macrocycle formation a general feature in b ion formation, or
are the peptide systems chosen thus far exceptions to the general rule?
1200
1400
1600
1400
1800
2000
-1
Wavenumber/cm
-1
(C)
1600
1800
Varying Effects of (4-AMBz) for b6 Ions
 Figure D: Control Experiment: Poor nucleophile (aminobenzoic acid ) prevents head-to-tail
macrocycle formation.
(4-ABz)WFGLMPG
(4-AMBz)WFGLMPG
Highly conjugated and stable
oxazolinone struture is formed.
(D)
Control Experiment
Wavenumber/cm
 The central hypothesis ,in this study, is depicted in the scheme below :
Proline and Aminomethylbenzoic acid5,6 were chosen because they possess limited
torsional flexibility and may impede macrocycle formation.
 The presence of oxazolone vs. macrocycle b6 fragment structure was validated by infrared
multiple-photon dissociation (IRMPD) spectroscopy, using the free electron laser FELIX coupled
to a home-built 4.7T FTICR instrument.
Sizable
Oxazolone band
0.5
Introduction
 All peptides were synthesized with conventional FMOC solid-phase synthesis methods using 9Fluromethylmethoxy Carbonyl (FMOC)-Gly loaded resin.
 Figure E: Photofragments Table from IRMPD Spectra: Evidence of Scrambling is observed in
(4-AMBz)WFGLMPG , Q(4-AMBz)FGLMPG .
QWF(4AMBz)MLPG exhibits two competing mass channels , m/z 297 (b2-NH3) and
m/z 298 (b2-H20).
(4-AMBz)TrpPheGlyLeuMet ProGly
Enhanced
Cleavage Site
Presence of QW in first and second position
leads to the following mass channels.
b6 Fragment Ion
(4AMBz)TrpPheGlyLeuMet
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1400
1600
1800
2000
1200
1400
Wavenumber/cm-1
(E)
1600
1800
2000
Wavenumber/cm-1
Acknowledgments
Octapeptide Sequence
b6
(b6-NH3)
(b6-H20)
(b5-NH3)
b5
(b4-NH3)
(4-AMBz)WFGLMPG
768
751
750
Q(4-AMBz) FGLMPG
710
QW(4-AMBz)GLMPG
749
601
QWF(4-AMBz)LMPG
839
691
QWFG(4-AMBz)MPG
783
(4-ABz) WFGLMPG
754
QPFGLMPG
674
526
413
(cyclo)QPFGLMPG
674
526
413
b4
b2
(b2-NH3)
(b2-H20)
(b2-H20-CO)
320
449
281
245
Y3
(-phe)
Unassigned
 Dr. John Eyler , Dr. David Powell
 Polfer Group Members: Especially Dr. Kerim Gulyuz
 FOM Institute in The Netherlands: Dr. Britta Redlich and Dr. Lex van der Meer
134,106
269
173
297
297
510
Y2
391
297
298
623
( a5-NH3)
552
488
766
a4
396
269
306
 University of Amsterdam: Dr. Jan van Maarseveen’s Group
358
 Special Thanks: (MS PIRE) (OISE-0730072)
120
488
Partnership for International Research and Education http://rodgers.chem.wayne.edu/pire
120
191,244,355
 UF Office of Research for Travel Support
 NSF Career CHE-0845450
120
498
References
302,244,217
Scrambled Sequences:
(4-AMBz)WFGLMPG (b6)
a4 (4-AMBz)WFLGoxa) (m/z 552)
Q(4-AMBz)FGLMPG (b6)
b2 (4-AMBz)FQGLM(oxa) (m/z 281)
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