1. ? 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
All peptides were synthesized with conventional FMOC solid-phase synthesis methods using 9- Fluromethylmethoxy Carbonyl (FMOC)-Gly loaded resin.
A CEM Discover System was utilized to synthesize 0.1 mmol scale of the corresponding peptide.
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.
Figure A: Sequence motif QWFGLM b6+ ion is strictly macrocyclic.
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.
Figure C : 4-Aminomethylbenzoic Acid Experiments: Demonstrates a minor oxazolone band for the b6+ fragment in the fifth position:
Figure D: Control Experiment: Poor nucleophile (aminobenzoic acid ) prevents head-to-tail macrocycle formation.
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).
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.
Proline and Aminomethylbenzoic acid (Amb) were chosen because they possess limited torsional flexibility and may impede macrocycle formation.
Overview
IRMPD Results
Results and Discussion
(A)
(B)
Sizable
Oxazolone band
Introduction
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.
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
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?
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.
Highly conjugated and stable oxazolinone struture is formed.
(D)
Control Experiment
Varying Effects of (4-AMBz) for b6 Ions
(C)
b6 Fragment Ion
Enhanced Cleavage Site
(4-AMBz)TrpPheGlyLeuMet ProGly
(4AMBz)TrpPheGlyLeuMet ?
Isomerization
Presence of QW in first and second position leads to the following mass channels.
(E)
Photofragment Mass Channels
Acknowledgments
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
University of Amsterdam: Dr. Jan van Maarseveen’s Group
Special Thanks: (MS PIRE) (OISE )
Partnership for International Research and Education
UF Office of Research for Travel Support
NSF Career CHE
Octapeptide Sequence b6
(b6-NH3)
(b6-H20)
(b5-NH3) b5
(b4-NH3) b4 b2
(b2-NH3)
(b2-H20)
(b2-H20-CO) a4
( a5-NH3) Y2 Y3
(-phe)
Unassigned
(4-AMBz)WFGLMPG
768
751
750
320
552
Q(4-AMBz) FGLMPG
710
449
281
245
391
134,106
QW(4-AMBz)GLMPG
749
601
488
297
269
173
358
QWF(4-AMBz)LMPG
839
691
298
396
120
QWFG(4-AMBz)MPG
783
766
191,244,355
(4-ABz) WFGLMPG
754
623
510
306
QPFGLMPG
674
526
413
498
(cyclo)QPFGLMPG
302,244,217
References
1 Paizs, B.; Suhai, S. MSR., 2005,24:508
2. Dongre, A.R.; Jones, J.L.; Somogyi, A.; Wysocki, V. H. JACS., 1996,118: 8365
3. Chen, X.; Yu, L.; Steill, J.D.; Oomens, J.; Polfer, N. JACS.,2009, 131:18272
4. Chen et al.; J. Mass Spectrom., 2010, 21,
5. Talaty, E. R.; Copper, T. J.; Van Stipdonk, M. J. RCMS.; 2005, 16:1305
6. Osburn, S. M.; Ochola, S. O.; Talaty, E. R.; Van Stipdonk, M. J. RCMS.; 2007, 21:3409
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)