3.1 Results and Discussion
3.2.2 Fmoc Solid Phase Peptide Synthesis of L-bradykinin
3.2.2.1 General Fmoc-Solid Phase Peptide Synthesis Method
See Appendix 3-III for these details
3.2.2.2 Synthesis A
BK was assembled manually on Rink resin (685 mg, 0.50 mmol, 1 equiv) in the order stated in Figure 3-2. For more detailed information on any of the following steps refer to Appendix 3-III. The resin was swollen overnight in DMF (100%). A 20% pip in
Fmoc-L-amino acids (2.00 mmol, 4 equiv) were coupled overnight (followed by a second re-coupling overnight) using TBTU (2.00 mmol, 4 equiv) and DIEA (4.00 mmol, 8 equiv) in excess as activating reagents. The Kaiser test9 was used to determine
whether the amino acids had coupled successfully. For the final deprotection, the resin was washed with a 20% pip in DMF solution (four times) and then agitated (2 x 5 minutes) in this solution. The resin was first washed with DMF and then a 50% solution of DCM/MeOH before being well suction dried. The resin bound peptide was then freeze-dried prior to cleavage (2.10 g). The resin bound peptide was deprotected and cleaved from the support using a freshly made solution of TFA/H2O/thioanisole/EDT as
per Section 3.2.2.1 to yield 1.40 g of crude product. RP-HPLC was used to purify the
parent peptide. The initial time programme used to purify the product is given in Table 3-3 however, with a faster elution at the start of the gradient and a slower gradient in the middle of the time programme (changes highlighted in Table 3-3), a better separation between the parent and other BK(Mtr) species was obtained. The parent peptide eluted at 36% B (140 mg, 26%). See Figure 3-7 for MS data. Figure 3-3 shows a RP-HPLC trace for the purification of BK using Synthesis A.
LR-MS: m/z (%, assignment) 1059.58 (100, M+H)+, 1081.56 (50, M+Na)+, 1097.54 (25, M+K)+; 1HR-MS: m/z C50H74N16O10 (M+2H)+2 530.2960, actual 530.2971 Elemental
Analysis. Calculated for C50H74N16O10 expected C: 56.70, H: 7.04, N: 21.16, actual C:
56.68, H: 6.99, N 21.00; NMR assignment in d6-DMSO conclusive with reference.11
Table 3-3 Acetonitrile stepwise gradient time programmes for the separation of the bradykinin peptide using a preparative RP-HPLC column
% Mobile phase ‘B’ Time
(min) Initial Time Programme Altered Time Programme
0 5 5 5 5 5 15 12 12 20 12 20 100 55 50 105 100 100 110 5 5 120 STOP STOP
Figure 3-7 Predicted (M+H)+ fragmentation pattern for the bradykinin (top) and actual bradykinin
obtained from Synthesis A (bottom). A similar pattern for the sodium and potassium isotope was also seen (not shown)
3.2.2.3 Synthesis B
The same procedure as per Section 3.2.2.2, was used for Synthesis B with the exception
of using resin (1 equiv), Fmoc-L-amino acids (2 equiv), coupling agents (2 equiv), and base (4 equiv). Peptide cleavage, purification, and characterisation as per Section 3.2.2.2. The yield for Synthesis B was 65 mg (12%).
3.2.2.4 Synthesis C
BK was assembled manually on Rink resin (342 mg, 0.25 mmol, 1 equiv) as per
Section 3.2.2.2 with the exception of the Arg protecting group. In this synthesis, the
Pmc protecting group (Figure 3-4) was used (instead of the Mtr group seen in Syntheses A, B, and D). Peptide cleavage, purification, and characterisation as per Section 3.2.2.2.
Crude results indicate no presence of the Pmc protected BK peptide. The pure product eluted at 36% B (5.2 mg, 2%).
3.2.2.5 Synthesis D
BK was assembled manually on Rink resin (1 equiv) as per Section 3.2.2.2. After each
successful amino acid coupling, the resin was washed well with DMF. The peptide was capped with a solution of Ac2O (4 equiv) in DMF and allowed to agitate for 10 minutes.
The resin was then washed well with DMF, deprotected, and then the next amino acid was coupled as per Section 3.2.2.2. Cleavage, purification, and characterisation as per Section 3.2.2.2. The pure product eluted at 36% B (72 mg, 27%) with elution of the BK
des-amino acid products after the elution of the parent species (c.a. 43% B). See Figure 3-6 for the RP-HPLC trace of Synthesis D.
3.2.2.6 Synthesis E
BK was assembled manually on Rink resin as per Section 3.2.2.2. The Mtr protecting
group was used to protect the first Arg in sequence, and the Pmc protecting group was used to protect the last Arg in the sequence (Figure 3-5). Peptide cleavage, purification, and characterisation as per Section 3.2.2.2. Crude results indicate no presence of the
Pmc protected BK peptide with trace amounts of the Mtr species. The pure product eluted at 35.4% B (79.5 mg, 30%).
3.2.2.7 Synthesis F
BK was assembled manually on Rink resin as per Section 3.2.2.2. The Mtr and Pmc
protecting groups were used to protect the first and last arginines respectively (Figure 3- 5). The amino acids were capped at the end of each successful coupling with Ac2O (see
Section 3.2.2.5). Peptide cleavage, purification, and characterisation as per Section 3.2.2.2. Crude results indicate no presence of the Pmc protected BK peptide with trace
3.3 Conclusion
The synthesis of the BK peptide using Fmoc-SPPS gave a useful yield of 52% (Synthesis F) using TBTU (4 equiv) and DIEA (8 equiv) as coupling reagents on Rink resin. Capping with Ac2O allowed easier purification of the parent peptide from BK
des-amino acid products, whilst exchange of the Arg9 Mtr protecting group for Pmc
allowed for a more successful synthesis with only traces amount of the BK-Mtr present in the cleaved mixture. Therefore, the reaction conditions for Synthesis F were used throughout the rest of the study for the synthesis of the BK peptide. This now leads into
Chapters 4 and 5 with the addition of peptides, both BK and other model peptides, onto
cyclodextrin and the synthesis of peptide sulfonamides, respectively.
3.4 References
1. (a) Chan, D., Gera, L., Stewart, J., Helfrich, B., Verella-Garcia, M., Johnson, G., Baron, A., Yang, J., Puck, T., and Bunn, P., Bradykinin antagonist dimer, CU201, inhibits the growth of human lung cancer cell lines by a "biased agonist" mechanism. Proceedings of the National Academy of Sciences of the United States of America 2002, 99, 4608-4613; (b) Vavrek, R. J., and Stewart, J. M.,
Competitive antagonists of bradykinin. Peptides 1985, 6, 161-164; (c) Stewart,
J. M., Bradykinin antagonists as anti-cancer agents. Current Pharmaceutical Design 2003, 9, 2036-2042; (d) Merrifield, R. B., Solid phase peptide synthesis
II. Synthesis of bradykinin. Journal of the American Chemical Society 1964, 86,
304-305; (e) Merrifield, R. B., Solid-phase peptide synthesis III. An improved synthesis of bradykinin. Biochemistry 1964, 3, 1385-1389; (f) Fridkin, M.,
Patchornik, A., and Katchalski, E., Use of polymers as chemical reagents II. Synthesis of bradykinin. Journal of the American Chemical Society 1968, 90,
2953-2957.
2. Rubina, Y. A., Bespalova, Z. D., and Bushuev, V. N., The solid phase synthesis of peptides containing an arginine residue with an unprotected guanidine group. Russian Journal of Bioorganic Chemistry 2000, 26, 235-244.
3. (a) Lange, M., Cuthbertson, A. S., Towart, R., and Fischer, P. M., Synthesis and activity of dimeric bradykinin antagonists containing diaminodicarboxylic acid bridge residues. Journal of Peptide Science 1998, 4, 282-293; (b) Chaturvedi,
D., Huelar, E., Gunthorpe, M., Gofman, M., Krapf, D. S., Apostol, E., and Lewis, W. S., Bradykinin analogs as inhibitors of angiotensin-converting enzyme. Peptide Research 1993, 6.
4. (a) Taub, J. S., Guo, R., Leeb-Lundberg, L. M., F, Madden, J. F., and Daaka, Y., Bradykinin receptor subtype 1 expression and function in prostate cancer. Cancer Research 2003, 63, 2037-2041; (b) Iyer, A. K., Khaled, G., Fang, J.,
and Maeda, H., Exploiting the enhanced permeability and retention effect for tumor targeting. Drug Discovery Today 2006, 11, 812-818; (c) Maeda H., Wu.
J., Okamoto T., Maruo K., and Akaike T., Kallikrein-kinin in infection and cancer. Immunopharmacology 1999, 43, 115-128; (d) Kastin, E. A.J., Handbook
of biologically active peptides. Elsevier: 2006; (e) Reissmann, S., Schwuchow,
C., Seyfarth, L., Pineda De Castro., L. F., Liebmann, C., Paegelow, I., Werner, H., and Stewart, J. M., Highly selective bradykinin agonists and antagonists with replacement of proline residues by N-methyl-D- and L-phenylalanine. Journal of Medicinal Chemistry 1996, 39, 929-936; (f) Chakravarty, S., Mavunkel, B.
J., Andy, R., and Kyle, D. J. Non-peptidic bradykinin receptor antagonists from
a structurally directed non-peptide library
http://www.netsci.org/Science/Combichem/feature04.html (accessed 7 January
2010).
5. Merrifield, R. B., Solid phase peptide synthesis IV. The synthesis of methionyl-lysyl-bradykinin. Journal of Organic Chemistry 1964, 29, 3100-
3102.
6. Stewart, J. M., Gera, Lajos, Chan, D. C., Bunn, P. A., York, E. J., Simkeviciene, V., and Helfrich, B.., Bradykinin-related compounds as new drugs for cancer and inflammation. Canadian Journal of Physiology and Pharmacology 2002, 80, 275-280.
7. Stewart, J. M., Bradykinin antagonists: discovery and development. Peptides (New York, NY, United States) 2004, 25, 527-532.
8. Novabiochem., Peptide Synthesis. Merck, Ed. Darmstadt, Germany, 2008/2009.
9. Chan, W. C., and White, P.D., Fmoc solid phase peptide synthesis: a practical approach. Oxford University Press: New York, 2002.
10. Kassem, T., Sabatino, D., Jia, X., Zhu, X. X., Lubell, W. D., To Rink or not to Rink amide link, that is the question to address for more economical and environmentally sound solid-phase peptide synthesis. International Journal of Peptide Research and Therapeutics 2009, 15, 211-218.
11. Mirmira, S. R., and Durani. S., Srivastava, S., and Phadke, R. S., Occurrence of
β-bends in bradykinin dissolved in DMSO-d6. Magnetic Resonance in Chemistry
Chapter Four:
Synthesis and Purification of Cyclodextrin-Peptides
4.0
Introduction
This chapter follows on from Chapter 2 where one or two linkers were attached to β-
CD and although Chapter 2 does not display any new chemistries,1 the synthetic
approach and versatility achieved allows for the addition and growth of C- and/or N- terminal peptides with the same and/or different functional properties on a solid phase support. Peptide attachment off resin in solution using stepwise or ligation synthetic protocols should also be possible. Direct coupling to unfunctionalised β-CD as desired,
was unsuccessful under all Fmoc procedures used in this study (Appendix 4-I), hence
functionalisation was essential for peptidyl attachment using SPPS. This system has great developmental potential for the transportation of drugs and/or other molecules. Owing to the large variety of cellular receptors, peptides appear to be amongst the most versatile compounds for such targeting purposes. The grafting of peptides onto CD also adds potential therapeutic dimensions.2
4.0.1 Chapter Outline
This chapter describes, for the first time, the bi-functionalisation of β-CD with a series
of model and bioactive peptides using Fmoc-SPPS. The bioactive peptide used in this study is bradykinin (BK). For synthesis and other details of the BK peptide, refer to
Chapters 1 and 3. This synthetic approach allows the functionalisation of β-CD with
one or two peptides in various combinations; e.g., the attachment of β-CD to the C-
and/or N-terminus of peptides as well as the functionalisation of β-CD with differing
ß-Cyclodextrin
X = Functional groups where X1does not equal X2
= N- and/or C-terminal peptide = Protecting group O OH OH OH O 7 O OH OH O O OH OH X1 O O OH OH OH O 5 X2 O OH OH O O OH OH X1 O O OH OH OH O 5 X2 O OH OH O O OH OH X1 O O OH OH OH O 5 X2 O OH OH O O OH OH X1 O O OH OH OH O 5 X2 (i) O OH OH O O OH OH X1 O O OH OH HO O X2 5 O OH OH O O OH OH X1 O O OH OH OH O 5 X2 (ii)
Figure 4-1 Derivatisation of β-CD using solid phase peptide synthesis. (i) Synthesis of bi-functional β-
CD to enable selective peptidyl attachment in SPPS. (ii) Attachment of peptides to β-CD using Fmoc-
This Chapter details the synthesis, purification, and characterisation of peptide addition to mono-6A-succinylamino-β-cyclodextrin (4, synthesis details outlined in Chapter 2, Sections 2.4.2.3) and mono-6A-fluorenylmethyloxycarbonylamino-mono-6X-succinyl- β-cyclodextrin (12, synthesis details outlined in Chapter 2, Section 2.4.4.4) using
Fmoc SPPS.
Model peptides were first used in this study to show proof-of-concept and to obtain reaction conditions that gave acceptable yields. The model peptides used include the di- peptide, Gly-Ala, and the tri peptides, tri-glycine (Gly-Gly-Gly) and Val-Gly-Ala. These peptides were chosen due to availability and cost enabling a number of small preliminary studies to be undertaken, to ensure successful syntheses. This was important when it came to the addition of the bioactive peptide which was much longer, and much more complicated in structure to make (see Chapter 3 for specific details).
Figure 4-2 outlines the layout of this chapter. Part a details the synthesis of mono- peptidyl-CD using mono-6A-succinylamino-β-cyclodextrin (4) where the peptide is a
model or BK. Part bandPart c describe the addition of the model and BK peptides to mono-6A-fluorenylmethyloxycarbonylamino-mono-6X-succinyl-β-cyclodextrin (12) in
either N- or C-terminal combinations, respectively. Part d details the addition of a peptide to both the N- and C-terminus of 12, whilst Part e shows the addition of 12 to
both ends of the peptide (model peptide or BK) creating a CD-peptide-CD moiety. In Part f, spacers are added between the resin and/or β-CD and/or peptides in varying
combinations. The spacer employed is ε-aminocaproic acid. This was done for bioassay
purposes (Chapter 6) to see what effect of the spacer had on the activity of the
bioactive peptide in relation to the non-spacer molecules (Parts a and b). Yield studies were also looked into for these and compared against non-spacer experiments. It was thought that the longer spacer between the resin and β-CD or peptide may improve its
coupling by decreasing steric factors. See Chapter 2, Section 2.4.4.4 for more details
Figure 4-2 Outline for the layout of Chapter 4
Part a N-terminal peptide addition to β-CD (4)
Part b C-terminal peptide addition to β-CD (12)
Part c N-terminal peptide addition to β-CD (12)
Part d C- and N-terminal peptide addition to β-CD (12)
Part e C- and N-terminal β-CD (12) addition to peptide
Part f Spacer addition to the peptide β-CD (12) moiety
Mono-functionalisation of mono-6A-
succinylamino-β-cyclodextrin (4) with
peptides
Bi-functionalisation of mono-6A-
fluorenylmethyloxycarbonylamino-
mono-6X-succinyl-β-cyclodextrin (12)
with peptides
11