Yazarlar TEM Connecticut Devlet kolej ve üniversite (CSCU) Merkezi nanoteknoloji ve Dr Ishita Mukerji Wesleyan Üniversitesi'nde için erişim için onu CD kullanarak yardım ve Dr. B. Ellen Scanley onun teknik eğitim için teşekkür etmek istiyorum Spektrofotometre. Raporlanan çalışma kısmen Bilim Enstitüsü Üniversitesi Fairfield, NASA Connecticut alan Grant Konsorsiyumu tarafından ve Ulusal Bilim Vakfı Hibe numarası CHE-1624774 altında tarafından desteklenmiştir.

1. sentez ve 1,2-Dithiolane arınma peptid modifiye
<ol><li>Sentezi dithiolane habercisi, 3-(acetylthio) -2-(acetylthiomethyl) propanoic asit19.
	<ol>
<li>3 1 g ekleyin-bromo - 2-(bromomethyl) propiyonik asit (1 eşdeğeri) 1 M NaOH (yaklaşık 4 mL) 55 ° C'de karıştırma ile 25 mL yuvarlak alt tepki şişesi için en az miktarda çözünmüş Reaksiyon şişeye bir septa ile mühür ve azot atmosferi altında yerleştirin.</li>
		<li>1,49 g potasyum thioacetate içeren bir çözüm hazırlamak (3.2 eşdeğeri) 4 ml deiyonize su ve 2 M sülfürik asit 3 mL (H2SO4) Tioasetik asit in situoluşturmak için.</li>
		<li>Tioasetik asit solüsyonu çek plastik tek kullanımlık 10 mL şırınga ve iğne şırıngayı yerleştirin. Karışımı dropwise septa iğneyle delme tarafından tepki şişeye ekleyin. Reaksiyon gecede 55 ° C'de devam</li>
		<li>Metanol ve diklorometan (1:9) karışımı kullanarak silika jel 60 F254 tabaklarda ince tabaka Kromatografi (TLC) tarafından tepki izlemek. Bromocresol yeşil leke tarafından reaksiyon ilerleme görselleştirin. Ürün bir Rf vardır 0,57 =.</li>
		<li>Tepki tam ve soğutmalı için oda sıcaklığında sonra pH 1 2 M H2karışıma çok acidify4. Sarı bir yağ dışarı çözüm ayırır.</li>
		<li>Soğuk kloroform (40 mL x 3) ile ürün ayıklayın. Organik katmanları birleştirmek ve magnezyum sülfat üzerinde kuru. Düşük basınç altında kloroform kaldırmak.</li>
		<li>İzole ürün, 1, kimliğini rakamlar 1B ve Cgösterildiği gibi nükleer manyetik rezonans (NMR) spektroskopisi ile onaylayın. Aşağıdaki sonuçlar bekliyoruz: 1H NMR, CDCl3, 300 MHz: d = 10.1 (b, 1 H), 3.2 (m, 4 H), (m, 1 H) 2,9, 2.4 (s, 6 H); 13 C NMR, CDCl3, 75 MHz: d = 195.1 (CH3COS-), 177.6 (-COOH), 45.1 (-CH2S-), 30.5 (CH), 29,2 (-SCOCH3). Not: Ürün sarı yağ ve % 83 genel bir verim vardır. Olmadan daha fazla arıtma ürün kullanın.</li>
	</ol></li>
	<li>SPP'ler ve Dithiolane habercisi-reçine kaplin Not: aşağıda açıklanan katı fazlı peptid sentez önerilen üreticinin iletişim kurallarını izleyen bir otomatik peptid synthesizer üzerinde yapılmıştır. Ayarları ve Kimyasalları uzman amino asitler kullanırken veya diğer ticari araçlar için adapte olabilir.<ol>
<li>Rink Amid 4-methylbenzhydrylamine (MBHA) reçine (0,1 mmol) 0,156 g tartmak ve bir reaksiyon gemi yerleştirin. Dimethylformamide (DMF) reçine en az 15 dk önce sentez başlangıcı için güzel.</li>
		<li>Her fluorenylmethyloxycarbonyl (Fmoc) 4 karşılıkları dışarı tartmak korumalı amino asit (0.4 mmol) dizisi ve 0,152 g N, N, N', N'- tetramethyl-O-(1H- benotriazol-1-yl) uranyum hexafluorophosphate (0.4 mmol, HBTU) için dizideki her amino asit. Her kartuş Fmoc korumalı amino asit ve HBTU içerir.</li>
		<li>Tüm ön sentez denetler synthesizer üzerinde çalıştırdıktan sonra (reaktifler, dolum reaktif satırları temizlenen ve tüm reaktif şişe basınçlı), c atlıkarınca amino asit kartuşları yerleştirin ' n ' terminus yön. Son amino asit pozisyon için son N-terminal Fmoc deprotection adım sonra bir boş kartuşu yerleştirin.</li>
		<li>Standart önerilen ayarlar kullanıldığında peptid sentez.<ol>
<li>Reçine ile 5 mL Fmoc grubundan, % 20 Piperodin DMF (5 dk x 2) içinde deprotect.</li>
			<li>DMF (6 x 5 mL) ile reçine kaplin adım önce yıkayın.</li>
			<li>Tek kaplin adım için 0.4 M N-methylmorpholine 4 mL DMF içinde Fmoc korumalı amino asit ve HBTU ekleyin. 30 amino asit Fmoc korumalı çözümü etkinleştirin çözüm tepki taşıyıcıyı aktarmadan önce s. Not: Otomatik peptid synthesizer reçine ve çözüm kabarcıklanma N2 gaz tarafından her 30 karışımları için 20 dk kaplin reaksiyon sırasında yer alır. El ile peptid sentez için tepki gemi bir orbital çalkalayıcı düşük hızda bağlantı adım süresince yerleştirin.</li>
			<li>Çözüm drenaj ve reçine DMF (3 x 5 mL) ile yıkayın.</li>
			<li>Her Fmoc korumalı amino asit c için adımları 1.2.4.1 1.2.4.4 aracılığıyla yineleyin ' n '-peptid ilgi sentezlemek için terminus yön.</li>
		</ol>
</li>
		<li>Son N-terminal deprotection adım sonra reçine tek kullanımlık fritted enjektör aktarın. Reçine DMF (3 x 5 mL) ve diklorometan (DCM, 3 x 5 mL) ile yıkayın. Not: Bir vakum desiccator yıkama DCM sonra reçine depolanabilir. Reçine kaplin önceki depolanmışsa, DMF reçine kaplin reaksiyon önce şişmeye emin olun.</li>
		<li>Dithiolane öncül (1)-reçine peptid N-terminus 1, 5 mL DMF, de HBTU 4 karşılıkları ve n, N-diisopropylethylamine (DIPEA) 10 eşdeğerleri 4 karşılıkları ekleyerek birkaç. 10 dk kaplin karışımı fritted şırınga içeren reçine eklemeden önce önceden etkinleştirin.</li>
		<li>Kaplin reaksiyon 2s için salla. 2 h sonra reçine DMF (3 x 5 mL) ile yıkayın ve bir gecede sallayarak ile kaplin tepki tekrarlayın.</li>
		<li>Sonra ikinci bağlantı, reçine DMF (3 x 5 mL) ve DCM (3 x 5 mL) ile yıkayın. Not: Reçine vakum altında bu noktada bölünme kadar saklanabilir.</li>
	</ol></li>
	<li>Thioacetate Deprotection ve peptid bölünme reçine üzerinden
	<ol>
<li>N-terminus dithiolane habercisi thioacetate grubundan deprotect için kuru reçine bir 10 mL mikrodalga tepki tüp aktarmak ve DMF 2 mL ekleyin. Reçine şişmeye, gemi küçük manyetik heyecan çubuğu eklemek ve bir alçak hız-in manyetik karıştırarak 15 dakika ile yeniden askıya sağlar.</li>
		<li>2 mL konsantre amonyum hidroksit ekleyin, reaksiyon teknesi ile silikon septa kap ve reaksiyon taşıyıcıyı karıştırma ile 45 dk için 75 ° C mikrodalga ayarlarını kullanarak bir mikrodalga reaktör içine koyun.</li>
		<li>Mikrodalga Reaksiyon tamamlandıktan sonra reçine temiz tek kullanımlık fritted enjektör aktarın. DMF (2 x 5 mL) ve metanol (MeOH, 2 x 5 mL) ile yıkayın.</li>
		<li>Metanol (1:4), konsantre amonyum hidroksit çözeltisi toplam hacmi 5 mL için ekleyin. Dithiolane ringde disülfür bağ intramolecular oksidasyon artırmak için gecede sallamak için bırakın.</li>
		<li>Reçine MeOH (2 x 5 mL) ve DCM (3 x 5 mL) ile yıkayın. Not: Kurutulmuş reçine bir vakum desiccator bu noktada depolanabilir.</li>
		<li>Bölünme kokteyl ile 1,5 saat için hafifçe sallayarak şırınga içeren reçine ekleyin. Göğüs arası kokteyl kullanılan % 95 trifluoroacetic asit (TFA), % 2.5 triisopropylsilane (ipuçları) ve toplam hacmi 5 mL % 2.5 su var. Dikkat: sadece kimyasal duman başlık altında çalışır. TFA uçucu ve aşındırıcı. Not: Yukarıdaki bölünme kokteyl çözüm peptid dizileri ve amino asit sidechain grupları korumak çoğunluğu için yeterlidir; Ancak, alternatif bölünme kokteyller için belirli amino asit yan zinciri gruplarında (belirli peptidler Cys, Met, Trp ve Arg içeren) veya diğer reçine Kimya20korunması gerekli olabilir.</li>
		<li>Dropwise ek fritted şırıngadan tarafından 25 mL 50 mL konik tüp içinde soğuk dietil eter içine ham peptid çökelti. Peptid beyaz katı precipitates. Pelet peptid Santrifüjü 1300 x g 10 dakika süreyle de tarafından özenle dikkatle boşaltmak dietil eter atık toplama için ayrı bir kaba.</li>
		<li>Başka bir 25 mL dietil eter konik tüp ekleyin ve çökelti vortexing tarafından yeniden askıya alma. Santrifüjü 1300 x g de 10 dakikadır tekrar ve tekrar dietil eter dikkatle boşaltmak. Pelet vakum altında kuru.</li>
	</ol></li>
	<li>1,2-Dithiolane arınma peptid modifiye Not: ham peptid ters faz-HPLC tarafından arındırmak. Toplamak ve peptid doruklarına birleştirmek ve kitle tarafından MALDI-TOF Kütle spektrometresi onaylayın.<ol>
<li>Asetonitril % 0.1 ile en az miktarda ham peptid Pelet dağıtılması TFA. Peptid'ın hydrophobicity ve toplama eğilimi nedeniyle örnek 40 ° C'de çözünürlük içinde yardım etmek için ısı yavaşça. Not: potansiyel disülfür Satım reaksiyonlar21,22,23önlemek için daha yüksek sıcaklık ve peptid sonication kaçının.</li>
		<li>Ham peptid 1 mL HPLC arıtma için hazırlamak için konsantre peptid stokunun 400 μL Asetonitril içinde H2O %0.1 TFA ve filtre ile 600 μL 22 mikron şırınga filtreden HPLC şişe ekleyin. Ek bir % 5 isopropanol peptid toplama ve yağış önlemek için ilave edilebilir.</li>
		<li>3 mL/dk 15-%55 Asetonitril 20 dak Set doğrusal bir gradyan üzerinde bir akış hızı ile C-18 yarı partiye hazırlık sütun kullanarak peptid 222 için UV dedektörleri arındırmak nm (Amid omurga) ve 330 nm (disülfür bağ). Toplamak ve faiz (Şekil 2A) doruklarına birleştirmek.</li>
		<li>Peptid ürün kitle tarafından MALDI-TOF Kütle Spektrometre reflektron modunda (Şekil 2B) onaylayın. Analiz için 0.5 μL maldı plaka üzerinde toplanan Peak 2,5-dihidroksibenzoik asit (DHB) matris 0.5 μL ile karıştırın (10 mg/mL DHB % 50 Asetonitril, % 0,1 TFA). Not: Ortak adducts MALDI-TOF Kütle spektrometresi dahil sodyum ve potasyum tuzu adduct ([M + Na]+ ve [M + K]+) tepeler. Örnek analiz önce desalting tavsiye edilir Eğer tuz adduct doruklarına ana [M + H]+ en yüksek sinyal bastırmak. Ayrıca, [M + O]+ oksitlenmiş bir zirve da tespit 1,2-dithiolane değiştiren peptid. Lazer indüklenen oksidasyon DHB matrisi kullanarak maldı iyonlaşma gelen raporda örnek toplama, solvent ve lazer şiddeti gibi faktörlere bağlı maldı oksidasyon artifakı24sınırlamak için değiştirilebilir öneriyor.</li>
		<li>Onay alındıktan sonra MALDI-TOF'doğru kütlesinin peptid sonra flaş dondurma lyophilize. Peptid liyofilize toz vakum altında derleme kadar tutun.</li>
	</ol></li>
</ol>2. karakterizasyonu Supramolecular kendinden montajlı yapıları
<ol><li>
Amiloid lifleri oluşumu
	<ol>
<li>Kendinden montajlı çözüm hazırlamak için bir analitik denge kullanarak peptid toz 1 mg tartın. (PH 7.5) % 20 Asetonitril ve karışımı 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic asit (HEPES) son konsantrasyonu 1 mg/mL peptid derleme karışımı bir 1.5 mL microcentrifuge tüp. içine dağıtılması Girdap derleme çözüm ve oda sıcaklığında bir araya bırakın.</li>
	</ol>
</li>
	<li>
Amiloid liflerinin spektroskopik karakterizasyonu
	<ol>
<li>Peptid derleme işlemi birkaç günde Fourier dönüşümü kızılötesi (FTIR) spektroskopisi ile izleyin. Örnek17unassembled peptidler kaynaklanan IR imza, 1670 cm-1 merkezli bir geniş tepedir. Peptid montaj örnekleri genellikle kaybolur ve olgunlaşma ulaşmak geniş unassembled zirve için bir iki hafta sürebilir.<ol>
<li>ATR elmas kristal üzerinde ince bir film olarak 8-10 μL derleme çözümün bir aliquot kuru. 1640 1630 cm-1 bir büyük ve geniş su tepe kaybolması Kuru film formları olarak izlemek.</li>
			<li>IR spectra 2 cm-1 çözünürlük 50 taramaları ortalama 1500-1800 cm-1 üzerinden elde etmek. Elde etmek ve arka plan inceden inceye gözden geçirmek her örnek taramadan önce çıkarma. Cm-1 (Şekil 3A)25,261625 ve 1635 arasında keskin bir tepe β-yapraklık derleme için IR imzadır.</li>
		</ol>
</li>
		<li>β-yapraklık zengin supramolecular yapıları peptid derlemesini dairesel dichroism (CD) tarafından karakterize. CD spectropolarimeter ile Peltier bir sıcaklık kontrol sistemi kullanarak spectra kaydedin.<ol>
<li>0,1 mm yol uzunluğu microcuvette içinde derleme çözümün 30 μL pipet. Not: Bir hücre tutucu klamp ve araç kısa yol uzunluğu hücreye yerleştirin için gereklidir.</li>
			<li>Her spektrum için CD enstrüman için aşağıdaki parametreleri ayarlayın: 300 dalga boylarında tarama nm 180 nm, tarama hızı 100/dk, bant genişliği 1 nm, 25 ° C, ortalama üç inceden inceye gözden geçirmek için.</li>
			<li>Arabellek (% 20 Asetonitril/10 mM HEPES, pH 7.5) bir spektrum toplamak ve her örnek tarama denetimi olarak değerden çıkarmak. β-yaprak CD imzası şöyledir: bir ellipticity en az yaklaşık 220 nm (Şekil 3B)27merkezli.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Amiloid liflerinin mikroskobu
	<ol>
<li>2-3 hafta β-yapraklık zengin supramolecular yapılarına olgunlaşmaya peptid örnekleri için izin. Not: Derlemelerin derleme işlemi de daha önceki aşamalarında transmisyon elektron mikroskobu (TEM) kullanarak yansıma.<ol>
<li>Peptid derleme çözüm TEM karbon kılavuz yüzeyinin 10 μL pipet. Not: pipet ucu ızgara yüzeyine dokunmamaya dikkat çekmek. Yüksek hassasiyetli, kendi kendine kapanış cımbız TEM kılavuz hazırlık sırasında tutmak için kullanılır.</li>
			<li>Derlemeler ızgara yüzeyine absorbe izin vermek için 1-2 dakika bekleyin. Aşırı örnek filtre kağıdı kılavuz kenarına dokunarak kaldırın.</li>
			<li>% 2 uranyl asetat leke piyasada bulunan %4 uranyl asetat eriyik-e doğru 100 μL deiyonize su ekleyerek hazırlayın. % 2 uranyl asetat leke kılavuz yüzeyine 10 μL pipet ve 2-3 dakika için kuluçkaya. Kuluçka sonra fazla leke kılavuz kenarına filtre kağıdı dokunarak kaldırın.</li>
			<li>TEM Izgaralar bir vakum desiccator gecede yerleştirin. Vakum altında görüntüleme kadar saklayın.</li>
			<li>TEM (Şekil 3 c) ile hazırlanmış örnekler görüntü. Mikroskopi için tipik parametreleri aşağıdaki gibidir: 9.300 X 23 için değişen büyüklüklerde görüntülere 000 X, tungsten filaman 120 hızlanan bir gerilim ile kV. Not: ImageJ edinsel TEM görüntüleri28supramolecular yapılarının ortalama lif genişliğini ölçmek için kullanılabilir. Dikkat: Tüm ilgili güvenlik bilgi formları (SDS) kullanmadan önce lütfen danışın. Çeşitli kimyasalların sentezinde, arıtma, kullanılan ve açıklanan 1,2-dithiolane değiştiren Self montaj peptidler karakterizasyonu aşındırıcı veya toksik ve yalnızca bir kimyasal duman başlık altında kullanılmalıdır. Her zaman uygun kişisel koruyucu ekipman (koruyucu gözlük, laboratuvar önlüğü, tam uzunlukta pantolon, kapalı-toe Ayakkabı dahil olmak üzere) kullan laboratuvar olarak çalışırken.</li>
		</ol>
</li>
	</ol>
</li>
</ol>

<ol>
	<li>Wang, J., Liu, K., Xing, R., Yan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+self-assembly:+Thermodynamics+and+kinetics.">Peptide self-assembly: Thermodynamics and kinetics.</a> Chemical Society Reviews. 45, 5589-5604 (2016).</li><li>Dong, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+supramolecular+polymers+for+biomedical+applications.">Functional supramolecular polymers for biomedical applications.</a> Advanced Materials. 27, 498-526 (2015).</li><li>Edwards-Gayle, C. J. C., Hamley, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+bioactive+peptides,+peptide+conjugates,+and+peptide+mimetic+materials.">Self-assembly of bioactive peptides, peptide conjugates, and peptide mimetic materials.</a> Organic and Biomolecular Chemistry. 15, 5867-5876 (2017).</li><li>Goor, O. J. G. M., Hendrikse, S. I. S., Dankers, P. Y. W., Meijer, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+supramolecular+polymers+to+multi-component+biomaterials.">From supramolecular polymers to multi-component biomaterials.</a> Chemical Society Reviews. 46, 6621-6637 (2017).</li><li>DiMaio, J. T. M., Doran, T. M., Ryan, D. M., Raymond, D. M., Nilsson, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+supramolecular+peptide+hydrogel+viscoelasticity+using+biomolecular+recognition.">Modulating supramolecular peptide hydrogel viscoelasticity using biomolecular recognition.</a> Biomacromolecules. 18, 3591-3599 (2017).</li><li>DiMaio, J. T. M., Raymond, D. M., Nilsson, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Display+of+functional+proteins+on+supramolecular+peptide+nanofibrils+using+a+split-protein+strategy.">Display of functional proteins on supramolecular peptide nanofibrils using a split-protein strategy.</a> Organic and Biomolecular Chemistry. 15, 5279-5283 (2017).</li><li>Mahmoud, Z. N., Gunnoo, S. B., Thomson, A. R., Fletcher, J. M., Woolfson, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+dual+functionalization+of+self-assembling+peptide+fibers.">Bioorthogonal dual functionalization of self-assembling peptide fibers.</a> Biomaterials. 32, 3712-3720 (2011).</li><li>Petkau-Milroy, K., Uhlenheuer, D. A., Spiering, A. J. H., Vekemans, J. A. J. M., Brunsveld, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+and+bio-orthogonal+protein+assembly+along+a+supramolecular+polymer.">Dynamic and bio-orthogonal protein assembly along a supramolecular polymer.</a> Chemical Science. 4, 2886-2891 (2013).</li><li>Li, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibrillar+tangle+surrogates:+Histone+H1+binding+to+patterned+phosphotyrosine+peptide+nanotubes.">Neurofibrillar tangle surrogates: Histone H1 binding to patterned phosphotyrosine peptide nanotubes.</a> Biochemistry. 53, 4225-4227 (2014).</li><li>Sadownik, A., Stefely, J., Regen, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerized+liposomes+formed+under+extremely+mild+conditions.">Polymerized liposomes formed under extremely mild conditions.</a> Journal of the American Chemical Society. 108, 7789-7791 (1986).</li><li>Zhang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATN-161+Peptide+functionalized+reversibly+cross-linked+polymersomes+mediate+targeted+doxorubicin+delivery+into+melanoma-bearing+C57BL/6+mice.">ATN-161 Peptide functionalized reversibly cross-linked polymersomes mediate targeted doxorubicin delivery into melanoma-bearing C57BL/6 mice.</a> Molecular Pharmaceutics. 14, 2538-2547 (2017).</li><li>Margulis, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+polymeric+nanocubes+by+self-assembly+and+crystallization+of+dithiolane-containing+triblock+copolymers.">Formation of polymeric nanocubes by self-assembly and crystallization of dithiolane-containing triblock copolymers.</a> Angewandte Chemie International Edition. 56, 16357-16362 (2017).</li><li>Zhang, X., Waymouth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1,2-Dithiolane-Derived+Dynamic,+Covalent+Materials:+Cooperative+Self-Assembly+and+Reversible+Cross-Linking.">1,2-Dithiolane-Derived Dynamic, Covalent Materials: Cooperative Self-Assembly and Reversible Cross-Linking.</a> Journal of the American Chemical Society. 139, 3822-3833 (2017).</li><li>Sakia, N., Matile, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stack+exchange+strategies+for+the+synthesis+of+covalent+double-channel+photosystems+by+self-organizing+surface-initiated+polymerization.">Stack exchange strategies for the synthesis of covalent double-channel photosystems by self-organizing surface-initiated polymerization.</a> Journal of the American Chemical Society. 133, 18542-18545 (2011).</li><li>Uji, H., Morita, T., Kimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+direction+dependence+of+single-molecule+conductance+of+a+helical+peptide+in+molecular+junction.">Molecular direction dependence of single-molecule conductance of a helical peptide in molecular junction.</a> Physical Chemistry Chemical Physics. 15, 757-760 (2013).</li><li>Liang, C., Ni, R., Smith, J. E., Childers, W. S., Mehta, A. K., Lynn, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+intermediates+in+amyloid+assembly.">Kinetic intermediates in amyloid assembly.</a> Journal of the American Chemical Society. 136, 15116-15149 (2014).</li><li>Smith, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+dynamic+conformational+network+of+cross-&#946;+peptide+assembly.">Defining the dynamic conformational network of cross-&#946; peptide assembly.</a> Israel Journal of Chemistry. 55, 763-769 (2015).</li><li>Black, S. P., Sanders, J. K. M., Stefankiewicz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+exchange:+Exposing+supramolecular+reactivity+through+dynamic+covalent+chemistry.">Disulfide exchange: Exposing supramolecular reactivity through dynamic covalent chemistry.</a> Chemical Society Reviews. 43, 1861-1872 (2014).</li><li>Vendetti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dihydroasparagusic+acid:+Antioxidant+and+tyrosinase+inhibitory+activities+and+improved+synthesis.">Dihydroasparagusic acid: Antioxidant and tyrosinase inhibitory activities and improved synthesis.</a> Journal of Agricultural and Food Chemistry. 61, 6848-6855 (2013).</li><li>Stawikowski, M., Fields, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+peptide+synthesis.">Introduction to peptide synthesis.</a> Current Protocols in Protein Science. 26, (2002).</li><li>Canadell, J., Goossens, H., Klumperman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-healing+materials+based+on+disulfide+links.">Self-healing materials based on disulfide links.</a> Macromolecules. 44, 2536-2541 (2011).</li><li>Lafont, U., van Zeijl, H., van der Zwaag, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cross-linkers+on+the+cohesive+and+adhesive+self-healing+ability+of+polydisulfide-based+thermosets.">Influence of cross-linkers on the cohesive and adhesive self-healing ability of polydisulfide-based thermosets.</a> ACS Applied Materials and Interfaces. 4, 6280-6288 (2012).</li><li>Komaromy, D., Stuart, M. C. A., Santiago, G. M., Tezcan, M., Krasnikov, V. V., Otto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+can+direct+dynamic+covalent+bond+formation+toward+diversity+or+specificity.">Self-assembly can direct dynamic covalent bond formation toward diversity or specificity.</a> Journal of the American Chemical Society. 139, 6234-6241 (2017).</li><li>McAvery, K. M., Guan, B., Fortier, C. A., Tarr, M. A., Cole, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+oxidation+of+cholesterol+observed+during+MALDI-TOF+mass+spectrometry.">Laser-induced oxidation of cholesterol observed during MALDI-TOF mass spectrometry.</a> Journal of the American Society for Mass Spectrometry. 22, 659-669 (2011).</li><li>Krimm, S., Bandekar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrational+spectroscopy+and+conformation+of+peptides,+polypeptides,+and+proteins.">Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins.</a> Advances in Protein Chemistry. 38, 181-364 (1986).</li><li>Halverson, K. J., Sucholeiki, I., Ashburn, T. T., Lansbury, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+&#946;-sheet-forming+sequences+in+amyloid+proteins+by+FTIR.">Location of &#946;-sheet-forming sequences in amyloid proteins by FTIR.</a> Journal of the American Chemical Society. 113, 6701-6703 (1991).</li><li>Greenfield, N., Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computed+circular+dichroism+spectra+for+the+evaluation+of+protein+confirmation.">Computed circular dichroism spectra for the evaluation of protein confirmation.</a> Biochemistry. 8, 4108-4116 (1969).</li><li>. ImageJ Available from: <a target="_blank" href="https://imagej.nih.gov/ij">https://imagej.nih.gov/ij</a> (2016)</li><li>Roy, S., Shinde, S., Hamilton, G. A., Hartnett, H. E., Jones, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+[FeFe]-hydrogenase:+On+resin+modification+of+an+amino+acid+to+anchor+a+hexacarbonyldiiron+cluster+in+a+peptide+framework.">Artificial [FeFe]-hydrogenase: On resin modification of an amino acid to anchor a hexacarbonyldiiron cluster in a peptide framework.</a> European Journal of Inorganic Chemistry. 2011, 1050-1055 (2011).</li><li>Van Duinen, S. G., Castano, E. M., Prelli, F., Bots, G. T. A. B., Luyendijk, W., Frangione, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hereditary+cerebral+hemorrhage+with+amyloidosis+in+patients+of+Dutch+origin+is+related+to+Alzheimer+disease.">Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 5991-5994 (1987).</li><li>Barth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+infrared+absorption+of+amino+acid+sidechains.">The infrared absorption of amino acid sidechains.</a> Progress in Biophysics and Molecular Biology. 74, 141-173 (2000).</li><li>Jayaraman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+amyloid+nucleation+via+&#945;-helix-rich+oligomeric+intermediates+in+short+polyglutamine-containing+Huntingtin+fragments.">Slow amyloid nucleation via &#945;-helix-rich oligomeric intermediates in short polyglutamine-containing Huntingtin fragments.</a> Journal of Molecular Biology. 415, 881-899 (2012).</li></ol>

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Bu makalede bir N-terminal 1,2-dithiolane değiştiren Self montaj peptid saflaştırılması ve karakterizasyonu elde edilen supramolecular yapıları ve hem sentezi ayrıntılarını anlatılır. Rapor burada 1,2-dithiolane peptid sentez 3-(acetylthio) -2 dithiolane habercisi üretmek için bir tek adımlı sentez de dahil olmak üzere avantajı vardır-(acetylthiomethyl) propanoic asit ve üzerinde reçine mikrodalga deprotection tepki Grup olarak toksik hidrazin deprotection için daha güvenli bir alternatif daha ö...

Kimya şekillendirme sağlam peptit bağı katı fazlı peptid sentezinde yer ve sıra uzunluğu ve kompozisyon kontrol yeteneği yapmak supramolecular yapılarına ağır araştırılmış bir alanı kendi kendine araya peptidler. Kontrol ve yan zincir steric dahil olmak üzere peptid kendi kendine monte yapılar ve elektrostatik etkileşimler, hidrojen bağı ve hidrofobik etkileri1, stabilize faktörler tasarım kuralları kümesi olarak hizmet vermektedir. Bu temel tasarım kuralları içine araştırma ilerlemeye devam ediyor gibi peptid mantıksal sonraki adımda kendinden montajlı çeşitliliği peptid temel yapıları ve işlevleri genişletme kapsar. Kendi kendine peptidler montaj sırasında birçok biyomedikal uygulamalarda peptit dizisi veya derleme koşullar2,3,4, için strateji geliştirme ayarlama tarafından kullanılan çok yönlü bir biomaterial vardır peptid nanofibers5,6,7,8,9 sonrası derleme değişiklikler nispeten keşfedilmemiş bir alan kalır.
Dinamik disülfür exchange ve thiol Kimya supramolecular yapıları yüzeyde yeni ve işlevsel Biyomalzeme verim potansiyeline sahip bir alandır. 1,2-dithiolane moieties (genellikle bir türevi lipoic asit (la) veya asparagusic asit (aa)) birleşme, lipozom sistemleri10,11, blok kopolimerler12,13ve olarak bildirilmiştir çapa yüzeyler14,15düzenleme. Burada, biz sentezi ve karakterizasyonu, N-terminus bir 1,2-dithiolane fonksiyonel grup16ile değiştirilir Alzheimer Hastaligi ile ilişkili Aβ peptid nucleating çekirdeğinden elde edilen bir kendi kendine montaj peptid raporu, 17. Elde edilen supramolecular lifleri şimdi amiloid lifleri18supramolecular yüzeyde disülfür-Satım ve thiol reaktivite çalışmaya deneysel bir platform olarak hizmet vermektedir.

<table><tbody><tr><td>Rink amide MBHA resin, high load</td><td>Gyros Protein Technologies</td><td>RAM-5-HL</td><td>Avoid contact with skin and eyes; do not inhale</td></tr><tr><td>N,N-Dimethylformamide</td><td>Fisher Scientific</td><td>D119-4</td><td>Flammable liquid and vapor; irritating to eyes and skin; Use personal protective equipment; keep away from open flame</td></tr><tr><td>Fmoc-L-Val-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-V</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>Fmoc-L-Leu-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-L</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>Fmoc-L-Lys(Boc)-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-KBC</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>Fmoc-L-Phe-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-F</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>Fmoc-L-Ala-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-A</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>Fmoc-L-Gln(Trt)-OH</td><td>Gyros Protein Technologies</td><td>FLA-25-QT</td><td>Wear personal protective equipment; do not inhale</td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime;-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate</td><td>Gyros Protein Technologies</td><td>26432</td><td>Causes skin, eye and respiratory irritation; do not inhale; use under hood or in well ventilated area</td></tr><tr><td>0.4 M N-methylmorpholine in DMF</td><td>Gyros Protein Technologies</td><td>PS3-MM-L</td><td>highly flammable; wear personal protective equipment; keep away from heat and keep container tightly closed; do not inhale or swallow; wash skin thoroughly after handling</td></tr><tr><td>20% piperidine in DMF</td><td>Gyros Protein Technologies</td><td>PS3-PPR-L</td><td>Causes severe eye and skin burns; Flammable Liquid and vapor; Do not inhale</td></tr><tr><td>dichloromethane</td><td>Fisher Scientific</td><td>D37-4</td><td>May cause cancer; Do not inhale; Wear personal protective equipment; use under hood only; if contacted rise with water for at least 15 minutes and obtain medical attention</td></tr><tr><td>acetonitrile</td><td>Fisher Scientific</td><td>A998-4</td><td>Flammable; irritating to eyes; Use personal protective equipment; Use only under a fume hood; keep away from open flame or hot surface; if contacted rinse wiith water for at least 15 minutes and obtain medical attention</td></tr><tr><td>trifluoroacetic acid</td><td>Fisher Scientific</td><td>A116-50</td><td>Causes severe burns; do not inhale; harmful to aquatic life; use personal protective equipment; use only under fume hood; if contacted rinse with water for at least 15 minutes and obain immediate medical attention</td></tr><tr><td>4% uranyl acetate</td><td>Electron Microscopy Sciences</td><td>22400-4</td><td>Do not inhale; harmful to aquatic life</td></tr><tr><td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid</td><td>Acros Organics</td><td>AC172571000</td><td>Do not inhale; use outdoors or in well-ventilated area</td></tr><tr><td>nitrogen Gas</td><td>TechAir</td><td></td><td>Contents under pressure, may explode if heated</td></tr><tr><td>3-bromo-2-(bromomethyl)propionic acid</td><td>Alfa Aesar</td><td>AAA1963014</td><td>Do not inhale; causes irritation to skin and eyes; corrosive</td></tr><tr><td>sodium hydroxide</td><td>Fisher Scientific</td><td>S318-100</td><td>Use personal protective equipment; use only under fume hood; if contact rinse area for at least 15 minutes and obtain medical attention</td></tr><tr><td>potassium thioacetate</td><td>Acros Organics</td><td>AC221300250</td><td>Causes skin and eye irritation; do not inhale; use personal protective equipment</td></tr><tr><td>sulfuric acid</td><td>Fisher Scientific</td><td>SA213</td><td>Causes burns; keep away from water; keep away from combustible material; do not inhale; use personal protective equipment; if contact rinse area for at least 15 minutes and obtain medical attention</td></tr><tr><td>chloroform-d</td><td>Acros Organics</td><td>AC320690075</td><td>Possible cancer hazard; irritating to skin and eyes; do not inhale; Use personal protective equipment; use only under fume hood; If contact rinse area for at least 15 minutes and obtain medical attention</td></tr><tr><td>chloroform</td><td>Fisher Scientific</td><td>C298-4</td><td>Possible cancer hazard; irritating to skin and eyes; do not inhale; Use personal protective equipment; use only under fume hood; If contact rinse area for at least 15 minutes and obtain medical attention</td></tr><tr><td>N,N-diisopropylethylamine</td><td>Acros Organics</td><td>AC367841000</td><td>Highly flammable; harmful to aquatic life; wear personal protective equipment; do not swallow</td></tr><tr><td>ammonium hydroxide</td><td>Fisher Scientific</td><td>A669S-500</td><td>Corrosive; do not inhale</td></tr><tr><td>methanol</td><td>Fisher Scientific</td><td>A452-4</td><td>Flammable liquid and vapor; use personal protective equipment; do not inhale; If contact rinse area for at least 15 minutes and obtain medical attention</td></tr><tr><td>triisopropylsilane</td><td>Sigma Aldrich</td><td>233781</td><td>Flammable; use personal proctective safety equipment; keep container tightly closed</td></tr><tr><td>diethyl ether</td><td>Fisher Scientific</td><td>E138-1</td><td>Extremely flammable; Irritating to skin and eyes; Use personal protective equipment</td></tr><tr><td>2,5-dihydroxybenzoic acid</td><td>Sigma Aldrich</td><td>39319-10x10MG-F</td><td>do not inhale; irritating to skin and eyes</td></tr><tr><td>alpha-cyano-4-hydroxycinnamic acid</td><td>Alfa Aesar</td><td>AAJ67635EXK</td><td></td></tr><tr><td>c18 zip-tip</td><td>Millipore</td><td>ZTC18S096</td><td></td></tr><tr><td>tris(2-carboxyethyl) phospine hydrochloride</td><td>Thermo Scientific</td><td>PI20490</td><td></td></tr><tr><td>silica gel 60 F254 coated aluminum-backed TLC sheets</td><td>EMD Millipore</td><td>1.05549.0001</td><td></td></tr><tr><td>Thin walled Precision NMR tubes</td><td>Bel-Art</td><td>663000585</td><td>5mm O.D.</td></tr><tr><td>All-plastic Norm-Ject syringes</td><td>Air Tite</td><td>AL10</td><td></td></tr><tr><td>single-use needle</td><td>BD PrecisionGlide</td><td>BD 305185</td><td>used needles get disposed on in sharps waste container</td></tr><tr><td>disposable fritted syringe</td><td>Torviq</td><td>SF1000LL</td><td>10mL fritted syringes were used in the report, but larger syringes are avaibale if needed for larger scale synthesis.</td></tr><tr><td>carbon grid</td><td>Ted Pella, Inc.</td><td>CF200-CU</td><td>Make sure to prepare samples and staining on the carbon grid side, not the shiny copper side of grid</td></tr><tr><td>self-closing tweezers</td><td>Electron Microscopy Sciences</td><td>78318-3X</td><td>very sharp tips, length: 120 mm</td></tr><tr><td>0.1 mm short path length cell</td><td>Starna Cells, Inc.</td><td>20/C-Q-0.1</td><td>Fragile</td></tr><tr><td>10mL Vessel Caps</td><td>CEM</td><td>909210</td><td></td></tr><tr><td>10mL Pressure Vessels</td><td>CEM</td><td>908035</td><td></td></tr><tr><td>Aeris Semi-Prep HPLC column</td><td>Phenomenex</td><td>00F-4632-N0</td><td>150 x 10mm</td></tr><tr><td>cell holder</td><td>Starna Cells, Inc.</td><td>CH-2049</td><td>Needed when using short pathlength cells</td></tr><tr><td>PS3 peptide synthesizer</td><td>Gyros Protein Technologies</td><td></td><td></td></tr><tr><td>DiscoverSP Microwave Reactor</td><td>CEM</td><td></td><td></td></tr><tr><td>centrifuge</td><td>HERMLE</td><td>Z 206 A</td><td>used a fixed 6x50 mL rotor</td></tr><tr><td>HPLC</td><td>Shimadzu</td><td></td><td>UV Detector</td></tr><tr><td>nuclear magnetic resonance spectrometer</td><td>Avance, Bruker</td><td></td><td>300 MHz</td></tr><tr><td>MALDI-TOF mass spectrometer</td><td>Axima Confidence, Shimadzu</td><td></td><td></td></tr><tr><td>lyophilizer</td><td>Millrock Technology</td><td>BT85A</td><td></td></tr><tr><td>Fourier-Transform Infrared Spectrometer</td><td>Alpha Tensor, Bruker</td><td></td><td></td></tr><tr><td>Transmission Electron Microscope</td><td>Tecnai Spirit, FEI</td><td></td><td>Used with Gatan Orius Fiberoptic CCD digital camera. Accessed at CSCU Center for Nanotechnology</td></tr><tr><td>Circular Dichroism Spectropolarimeter</td><td>J-810, JASCO</td><td></td><td>Used with a six-cell Peltier temperature controller. Accessed at Wesleyan University.</td></tr></tbody></table>

synthesis and characterization of 1,2-dithiolane modified self-assembling peptides

Kendi kendine montaj peptid ve sonucu olan karakterizasyonu bir N-terminus 1,2-dithiolane sentezi üzerinde duruluyor değiştiren bu rapor supramolecular yapıları kendi kendine monte. Katı faz peptid sentez dithiolane habercisi molekülü 3-(acetylthio) -2-reçine MANŞONLA sentetik rota yararlanır-(acetylthiomethyl) propanoic asit ve mikrodalga destekli thioacetate deprotection peptid N-terminus reçine üzerinden son bölünme önce 1,2-dithiolane vermeye peptid değiştiren. Alzheimer hastalığı, ilişkili Aβ peptid nucleating çekirdeğinden elde edilen 1,2-dithiolane peptid, yüksek performanslı sıvı kromatografi (HPLC) arıtma sonra peptid çapraz-β amiloid lifleri kendi kendine montajı gösterilmiştir. Fourier dönüşümü kızılötesi spektroskopi (FT-IR), dairesel dichroism spektroskopisi (CD) ve transmisyon elektron mikroskobu (TEM) amiloid lifler karakterize etmek için protokolleri mevcuttur. N-terminal geliştirmeleriyle 1,2-dithiolane yan iyi karakterize kendi kendine montaj peptidler yöntemleri şimdi sonrası derleme değişiklik stratejiler geliştirmek ve dinamik kovalent kimya üzerinde keşfetmek için model sistemleri olarak keşfedilmeyi olabilir Supramolecular peptid nanofiber yüzeyler.

Dithiolane öncül molekül ilk tek adımlı sentezi kenara 1,2-dithiolane değiştiren peptid sentez geri kalanı sağlam destek (Şekil 1A) oluşur. 3 dönüşüm-bromo - 2-(bromomethyl) propiyonik asit 3-(acetylthio) -2-(acetylthiomethyl) propanoic asit, dithiolane habercisi 1tarafından H doğruladı ve 13C NMR (Şekil 1B ve C) daha önce ücretsiz için birleştiğinde Bir peptid hala-r...

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Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

1,2-dithiolane sentezi için bir protokol peptid ve peptid kendinden montajlı kaynaklanan supramolecular yapıları karakterizasyonu değiştiren.

1,2-Dithiolane malzemelerin ve kendi kendine montaj peptidler değiştirilme tarihi

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

synthesis-characterization-12-dithiolane-modified-self-assembling

Universidad San Sebastian

Research

JoVE Journal

Chemistry

7.1K Görüntüleme.  Fairfield University. 1,2-dithiolane sentezi için bir protokol peptid ve peptid kendinden montajlı kaynaklanan supramolecular yapıları karakterizasyonu değiştiren.

Video: Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

Kısa Peptidlerinin Self-meclisi tarafından Sıralı Biyomoleküler Yapıların Oluşumu

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Kimya

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Split-ve-havuz Sentezi ve Peptit Tersiyer Amid Kütüphanesi Karakterizasyonu

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

Nükleosid trifosfatlar - sentezinden Biyokimyasal Karakterizasyonu için

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Yüksek Sıralı Nanosheets Peptoid Polimerler ve Self-Meclis Solid-faz Submonomer Sentezi

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Biyomühendislik

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Thioether/vinil sülfür-gergin helisel peptidler fotoğraf yolu ile indüklenen Thiol-KD/yne Hydrothiolation oluşturmak

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

Bilgi taşıyan Peptoidlerin sentezi ve Dizi yönelimli Dinamik Kovalent Öz-montaj

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

On-Tether Sülfonyum Merkezi Kullanarak Döngüsel Peptitlerin Oluşturulması

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

<meta charset="utf8"></head><body> Peptit Hidrojeller: ECF-5'in Çevreye Duyarlı Jelasyonu için Yöntemler

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking networks. Their morphology closely resembles that of the extracellular matrix, allowing them to exhibit both the biological functions of peptides and responsive gelation properties. These unique characteristics have led to their extensive application in tissue engineering, three-dimensional cell culture, cancer therapy, regenerative medicine, and other biomedical fields. This article describes three methods for preparing ECF-5 peptide hydrogels using self-assembling peptides with environmentally responsive gelation processes: (1) pH-responsive gelation: varying pH levels induce the protonation or deprotonation of amino acid residues, altering electrostatic interactions between peptide molecules and promoting their self-assembly into hydrogels; (2) Metal ion addition: polyvalent metal ions chelate with negatively charged amino acid residues, acting as bridges between peptides to form a network hydrogel; (3) Solvent exchange: hydrophobic peptides are initially dissolved in non-polar organic solvents and subsequently induce self-assembly into hydrogels upon transitioning to a polar aqueous environment. These methods utilize conventional experimental procedures to facilitate peptide self-assembly into hydrogels. By designing peptide sequences to align with specific gelation-inducing conditions, it is possible to achieve finely tuned micro/nanostructures and biological functions, highlighting the significant potential of peptide hydrogels in the biomedical domain.

<meta charset="utf8"></head><body>Yazar Spot Işığı: Üstün Biyouyumluluk için Kendi Kendine Birleşen Liflerin ve Peptit Hidrojellerin Üretiminin İyileştirilmesi

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward conditions.

Mekanik Olarak Kararlı Kendinden Montajlı Peptitler Hidrojellerin Hazırlanması

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking ...

1,2-Dithiolane malzemelerin ve kendi kendine montaj peptidler değiştirilme tarihi

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Bu videodaki bölümler

Kısa Peptidlerinin Self-meclisi tarafından Sıralı Biyomoleküler Yapıların Oluşumu

Split-ve-havuz Sentezi ve Peptit Tersiyer Amid Kütüphanesi Karakterizasyonu

Nükleosid trifosfatlar - sentezinden Biyokimyasal Karakterizasyonu için

Yüksek Sıralı Nanosheets Peptoid Polimerler ve Self-Meclis Solid-faz Submonomer Sentezi

Thioether/vinil sülfür-gergin helisel peptidler fotoğraf yolu ile indüklenen Thiol-KD/yne Hydrothiolation oluşturmak

Bilgi taşıyan Peptoidlerin sentezi ve Dizi yönelimli Dinamik Kovalent Öz-montaj

On-Tether Sülfonyum Merkezi Kullanarak Döngüsel Peptitlerin Oluşturulması

Mekanik Olarak Kararlı Kendinden Montajlı Peptitler Hidrojellerin Hazırlanması

Mekanik Olarak Kararlı Kendinden Montajlı Peptitler Hidrojellerin Hazırlanması

Mekanik Olarak Kararlı Kendinden Montajlı Peptitler Hidrojellerin Hazırlanması