Ultrasonication improves solid phase synthesis of

peptides specific for fibroblast Growth factor

receptor and for the protein-protein interface
RANK-TRAF6
Rúben D. M. Silva 1,† , João Franco Machado 1,2,† , Kyle Gonçalves 1 , Francisco M. Lucas 1, Salete Batista 1,

Rita Melo 1 , Tânia S. Morais 2,* and João D. G. Correia 1,3,*

Introduction

1 Why to put ultrasonication assisted SPPS among the most efficient SPPS methodologies. ?? How ultrasonication phenomenon
works ? Phenomenon of cavitation and local increment of temperature and pressure

2 What is the goal and purpose of this study . What are targeting or target
specific peptides ? Why its important to study target specific peptides
and delivery vectors . Explanation by using fibroblast growth receptor 3.
Target peptide : The FGFR , which comprises four identified subtypes (FGFR1-FGFR4) is a transmembrane tyrosine
3 kinase.

Importance of using FGFR-targeting peptides ( fibroblast growth factor receptor3 for selective drug delivery and why its
important to develop further target specific and delivery vectors with higher purity and selectivity Why its important to
4 develop fast , high yielding and high purity giving methodologies . ( all peptides can not be produced in bacterial
system )
Cont..

In this study the two methods of peptide synthesis are compared. “classical “ Fmoc-solid phase peptide synthesis (SPPS)
with ultrasound (US)-assisted SPPS.

synthesis of three peptides( target specific ): fibroblast growth factors receptors 3(FGFR3)-specific peptide pep 1
(VSPPLTLGQLLS-NH2),peptides Pep2 (RQMATADEA-NH2) and Pep3 (AAVALLPAVLLALLAPRQMATADEA-NH2

developed aimed at interfering with the intracellular protein-protein interactions (PPI)RANK-TRAF6.

ultrasonication’s improved the crude purity and reduces production time by many times as compared to “ classical” Fmoc
based solid phase peptide synthesis.
Results and discussion ( pep1 target specific drug deliver)y vector) first reaction
protocol

Solid support used: Rink Amide MBHA resin

Activating/ coupling reagents used: HBTU and DIPEA

Fmoc Deprotecting reagents: 20% piperidine in DMF ,20min for CSPPS , 4 min each for USSPPS.

Time taken for amino acid conjugation cycle: from 90 to 960 min at

room temperature for CSPPS , 5-25 min for USSPPS AT ~30degree.

Final cleavage and final deprotection: ( 95%TFA/2.5%TIS?2.5%H2O)

Purification and characterization: by RP-HPLC and ESI-MS.

Same methodology will be used in the subsequent experiments

Analysis to characterization of pep1 (14-folds faster than CPPS )

Fmoc Conjugati Crude Final Total Temp

deprotect on time Purity yield time maintaine
ion time In pep d
assembly
CSPPS 20min 90-960min 73% 42% 3515min Room
58h temp
US-SPPS 4 min 5-25min 82% 54% 250min 30C
14h approx.
Synthesis of pep2 by both CSPPS AND US-SPPS (4-folds time reduction )

Same methodology as for pep1.

Fmoc Conjugati Crude Final Total

deprotect on time Purity yield time
ion time In pep
assembly
CSPPS 25 min 5 -35 84% 32% 365min

US-SPPS 2 min 5-15 72% 49% 85min

large peptide (Pep3, AAVALLPAVLLALLAPRQMATADEA-NH2) with
high numbers of hydrophobic amino acids and homooligo-sequences.

Fmoc Conjugati Conjugati Final Final Pep3

deprotecti on cycle on cycle crude crude assembled
on time time from time from purity yield in (time)
A25 to L10 Val9 to
Ala1
2min 7min 21 min 49% 19% 347min
Conclusion

Remarkably, US-assisted SPPS led to a 14-fold (Pep1) and 4-fold time reduction (Pep2) in peptide assembly. Interestingly, in the case of Pep 1, US-assisted SPPS
yielded a crude peptide with higher purity (82%) than that obtained by “classical” SPPS (73%)

large peptide (Pep3, AAVALLPAVLLALLAPRQMATADEA-NH2) with high numbers of hydrophobic amino acids and homooligo-sequences was synthesized within a
working day with moderate purity.
Boosting Fmoc solid phase peptide synthesis by ultrasonication.

Francesco Merlino,†,∇ Stefano Tomassi,‡,∇ Ali M. Yousif,† Anna Messere,‡ Luciana Marinelli,†Paolo Grieco,† Ettore Novellino,† Sandro Cosconati,*,‡ and Salvatore Di Maro*,‡

†Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, via D. Montesano 49, 80131 Naples, Italy ‡DiSTABiF, Università degli Studi della Campania “Luigi Vanvitelli”, via A.
Vivaldi 43, 81100 Caserta, Italy
Introduction

Solid phase peptide synthesis and protecting groups strategies

Why ultrasonication ? Sonochemistry

Why not MW-SPPS

Fmoc/tBu and Boc/Bzl , protection strategies ,

Cavitation phenomenon ( local increment of temperature and pressure, leading to turbulent flow in the liquid and enhanced mass transfer ) .

Main focus in this study: effect of US on Fmoc removal and amide bond formation ( increases the number of efficient collisions )

Correcting the misconception that ultrasounds erodes solid surface contacting the fluid
 US effects om the Fmoc-removal and trends in temperature increment and 10-mer
oligo alanine : aggregation prone sequence
o Functionalized FMOC-L-Asp(OtBu)-Rink amide-AM Ps resin was treated with 20%pip/DMF solution.

o US assisted treatments 1-4 yields >99b% deprotection .

o local moderate increment of temperature depending upon

reaction time, significantly lower than conventional and

MW-SPPS reported.

o Synthesis of 10-mer oligo-alanine : Progressive gap between

USSPPS and CSPPS performances arose From the sixth alanine .

Effect of US on by products of reaction: monitoring the base related
aspartimide formation during the hector (VKDGYI) synthesis and role of
formic acid in suppressing the aspartimide formation.

o the US process did not cause the formation of any additional undesired products, but especially led to a detectable
decrement of the aspartimide byproduct .

o three different deprotection protocols were employed.

o Addition of 1% formic acid suppressed the aspartimide formation.

Testing the results of ultrasonication's on amide formation and decoupling the
roles of heat and sonication on accelerating the reaction rate

o model pentapeptide (Fmoc-KFRFD) was synthesized using a Rink amide-AM PS resin as a solid support and (HBTU)/
(HOBt) as a combination of activating/additive agents.

o Variables: stoichiometry of reactants and reaction times and

reaction trends in reaction vessels were monitored.

o ultrasonic irradiation allowed for a substantial reduction

of reagent excess and reaction time.

o Solo ultrasonication enhanced the crude purity by 25% approx.

o USSPPS allowed substantial reduction of reagents and reaction time.

Entry 7 was applied to scan the most common activating/additive agents .

ultrasonication yielded pentapeptides with a degree of purity comparable or

higher than the one achieved in the presence of HBTU/HOBt. Among them,

the combination of ultrasonication and COMU/Oxyma allowed for the synthesis

of the test peptide with a purity of 94 ± 2% (7c), and hence, it was considered as

the adopted procedure for all the subsequent experiments.

Effects of ultrasonication on amino acid racemization

Synthesis of entries 1, 3, 5 and 7. Fmoc-deprotection: procedure B (5 + 25 min); Coupling: Nα-Fmoc amino acid equiv: 4; coupling
reagents (equiv): COMU/Oxyma (4 equiv); base (equiv): DIEA (8 equiv); mechanical shaking (60 min).

Synthesis of 2, 4, 6 and 8. Fmoc-deprotection: procedure A (0.5 + 1 min); Coupling: Nα-Fmoc amino acid equiv: 2; coupling reagents
(equiv): COMU/Oxyma (2 equiv); base (equiv): DIEA (4 equiv); ultrasonic irradiation (5 min).

Eight pentapeptides were synthesized using US ultrasonication and conventional strategies . no significant increase of the
racemization for both cysteine and histidine enantiomers,

indicating that the sonication can be used without any specific

precautions also to build sequences containing amino acid

residues prone to racemization.

Synthesis of biologically relevant , long and complex peptides using US and
conditions of entry 7c

Biologically relevant sequences ranging from 10 to 44 residues , were prepared. For this study , we extended the use of US to the
synthesis of CO2H-terminal .Peptides 1, 3, 5, 7-9 (on a Rink amide resin 0.55 mmol/g; 91 mg, 0.05 mmol), 2 and 6 (on a Wang resin
0.99 mmol/g pre-loaded with the first amino acid; 50 mg, 0.05 mmol), and 4 (on a 2-CTC resin 1.60 mmol/g pre-loaded with the first
amino acid; 68 mg, 0.05 mmol).

Fmoc-deprotection: procedure A (0.5 + 1 min); Coupling: Nα-Fmoc amino acid equiv: 2; coupling reagents (equiv): COMU/Oxyma
(2 equiv); base (equiv): DIEA (4 equiv); ultrasonic irradiation (5 min)].

proving the reliability of the low-frequency sonication

on the overall synthetic process, regardless the size of the

peptides and the type of resin-bound linkers.

Two combinations of reaction times and reagent equiv were probed for assembling
peptides known as “difficult sequences “.

adopting the following reaction conditions: 20% pip/DMF, 0.5 + 1 min, for Fmoc-deprotections; and Fmoc-aa−OH (8 equiv), COMU/Oxyma (8 equiv),
DIEA (16 equiv), 10 min, for couplings. The HPLC profiles of the so-obtained Peptides, showed a significant difference in terms of crude quality,
representing strong evidence of the substantial effects of US, especially for aggregation-prone sequences.

Peptide Aib-Enk ACP65-74 JR 10-mer AB 1-42

Crude purity ~20% ~31% ~48% ~35
difference
Conclusion